US20050183165A1 - Grapevine leafroll virus (type 2) proteins and their uses - Google Patents

Grapevine leafroll virus (type 2) proteins and their uses Download PDF

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US20050183165A1
US20050183165A1 US11/008,710 US871004A US2005183165A1 US 20050183165 A1 US20050183165 A1 US 20050183165A1 US 871004 A US871004 A US 871004A US 2005183165 A1 US2005183165 A1 US 2005183165A1
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Hai-Ying Zhu
Kai-Shu Ling
Dennis Gonsalves
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Cornell Research Foundation Inc
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    • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • C12N2770/00022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses

Definitions

  • the present invention relates to grapevine leafroll virus (type 2) proteins, DNA molecules encoding these proteins, and their uses.
  • the major virus diseases are grouped into: (1) the grapevine degeneration caused by the fanleaf nepovirus , other European nepoviruses, and American nepoviruses, (2) the leafroll complex, and (3) the rugose wood complex (Martelli, ed., Graft Transmissible Diseases of Grapevines, Handbook for Detection and Diagnosis , FAO, UN, Rome, Italy (1993)).
  • grapevine leafroll complex is the most widely distributed throughout the world. According to Goheen (“Grape Leafroll,” in Frazier et al., eds., Virus Diseases of Small Fruits and Grapevines ( A Handbook ), University of California, Division of Agricultural Sciences, Berkeley, Calif., USA, pp. 209-212 (1970) (“Goheen (1970)”), grapevine leafroll-like disease was described as early as the 1850s in German and French literature. However, the viral nature of the disease was first demonstrated by Scheu (Scheu, “Die Rollkrankheit des Rebstockes (Leafroll of grapevine),” D. D. Weinbau 14: 222-358 (1935) (“Scheu (1935)”)).
  • Leafroll is a serious viral disease of grapes and occurs wherever grapes are grown. This wide distribution of the disease has come about through the propagation of diseased vines. It affects almost all cultivated and rootstock varieties of Vitis . Although the disease is not lethal, it causes yield losses and reduction of sugar content. Scheu estimated in 1936 that 80 percent of all grapevines planted in Germany were infected (Scheu, Mein Winzerbuch , Berlin:Reichsnahrstand-Verlags (1936)). In many California wine grape vineyards, the incidence of leafroll (based on a survey of field symptoms conducted in 1959) agrees with Scheu's initial observation in German vineyards (Goheen et al., “Studies of Grape Leafroll in California,” Amer. J. Enol.
  • Leafroll disease Symptoms of leafroll disease vary considerably depending upon the cultivar, environment, and time of the year. On red or dark-colored fruit varieties, the typical downward rolling and interveinal reddening of basal, mature leaves is the most prevalent in autumn; but not in spring or early summer. On light-colored fruit varieties however, symptoms are less conspicuous, usually with downward rolling accompanied by interveinal chlorosis. Moreover, many infected rootstock cultivars do not develop symptoms. In these cases, the disease is usually diagnosed with a woody indicator indexing assay using Vitis vivifera cv. Carbernet Franc (Goheen (1988)).
  • GLRaV grapevine leafroll associated viruses
  • Chasselas 8/22 is the same as the GLRaV-2 isolate from France (Zimmermann (1990)) which also include the isolates of grapevine corky bark associated closteroviruses from Italy (GCBaV-BA) (Boscia (1995)) and from the United States (GCBaV-NY) (Namba et al., “Purification and Properties of Closterovirus -like Particles Associated with Grapevine Corky Bark Disease,” Phytopathology, 81: 964-970 (1991) (“Namba (1991)”)).
  • the Ia component of cv. Chasselas 8/22 was given the provisional name of grapevine leafroll associated virus 6 (GLRaV-6).
  • Virions of GLRaV-2 are flexuous, filamentous particles about 1,400-1,800 nm in length (Gugerli et al., “L'enroulement de la Vigne: Mise en Evidence de Particles Virales et Development d'une Methode Immuno-enzymatique Pour le Diagnostic Rapide (Grapevine Leafroll: Presence of Virus Particles and Development of an Immuno-enzyme Method for Diagnosis and Detection),” Rev. foundedizat. Arboricult. Horticult. 16: 299-304 (1984)).
  • dsRNA double-stranded RNA
  • the coat protein of GLRaV-2 is ca 22 ⁇ 26 kDa (Zimmermann et al., “Characterization and Serological Detection of Four Closterovirus -like Particles Associated with Leafroll Disease on Grapevine,” J.
  • GLRaV-2 has been classififed as a member of the genus Closterovirus based on particle morphology and cytopathology (Martelli, Circular of ICTV-Plant Virus Subcommittee Study Group on Closterolike Viruses” (1996)), its molecular and biochernical properties are not well characterized.
  • BYV beet yellows virus
  • Virology 79(5): 1289-1301 revealed several common features of the closteroviruses, including the presence of HSP70 chaperone heat shock protein and a duplicate of the coat protein gene (Agranovsky “Principles of Molecular Organization, Expression, and Evolution of Closteroviruses: Over the Barriers,” Adv. in Virus Res. 47: 119-218 (1996); Dolja et al. “Molecular Biology and Evolution of Closteroviruses: Sophisticated Build-up of Large RNA Genomes,” Annual Rev. Photopathology 32: 261-85 (1994); Boyko et al.,. “Coat Protein Gene Duplication in a Filamentous RNA Virus of Plants,” Proc. Nat. Acad. Sci. USA 89: 9156-60 (1992)). Characterization of the genome organization of GLRaVs would provide molecular information on the serologically distinct closteroviruses that cause similar leafroll symptoms in grapevine.
  • GVA grapevine virus A
  • GVB grapevine virus B
  • leafroll symptoms may be induced by more than one virus or they may be simply a general plant physiological response to invasion by an array of phloem-inhabiting viruses.
  • Grapevine leafroll is transmitted primarily by contaminated scions and rootstocks.
  • several species of mealybugs have been shown to be the vector of leafroll (Engelbrecht et al., “Transmission of Grapevine Leafroll Disease and Associated Closteroviruses by the Vine Mealybug Planococcus - ficus,” Phytophylactica, 22: 341-346 (1990), Rosciglione, et al., “Transmission of Grapevine Leafroll Disease and an Associated Closterovirus to Healthy Grapevine by the Mealybug Planococcus ficus ,” (Abstract), Phytoparasitica, 17: 63-63 (1989), and Tanne, “Evidence for the Transmission by Mealybugs to Healthy Grapevines of a Closter-like Particle Associated with Grapevine Leafroll Disease,” Phytoparasitica, 16: 288 (1988)).
  • the present invention is directed to overcoming this deficiency in the art.
  • the present invention relates to an isolated protein or polypeptide corresponding to a protein or polypeptide of a grapevine leafroll virus (type 2).
  • the encoding RNA and DNA molecules in either isolated form or incorporated in an expression system, a host cell, a transgenic Vitis or citrus scion or rootstock cultivar, or a transgenic Nicotiana plant or beet plant are also disclosed.
  • Another aspect of the present invention relates to a method of imparting grapevine leafroll virus (type 2) resistance to Vitis scion or rootstock cultivars or Nicotiana plants by transforming them with a DNA molecule encoding the protein or polypeptide corresponding to a protein or polypeptide of a grapevine leafroll virus (type 2).
  • Other aspects of the present invention relate to a method of imparting beet yellows virus resistance to beet plants and a method of imparting tristeza virus resistance to citrus scion or rootstock cultivars, both by transforming the plants or cultivars with a DNA molecule encoding the protein or polypeptide corresponding to a protein or polypeptide of a grapevine leafroll virus (type 2).
  • the present invention also relates to an antibody or binding portion thereof or probe which recognizes the protein or polypeptide.
  • Grapevine leafroll virus resistant transgenic variants of the current commercial grape cultivars and rootstocks allows for more complete control of the virus, while retaining the varietal characteristics of specific cultivars. Furthermore, these variants permit control of GLRaV-2 transmitted either by contaminated scions or rootstocks or by a presently uncharacterized insect vector. With respect to the latter mode of transmission, the present invention circumvents increased restriction of pesticide use which has made chemical control of insect infestation increasingly difficult. In this manner, the interests of the environment and the economics of grape cultivation and wine making are all furthered by the present invention.
  • FIGS. 1A and 1B are a comparison of a double-stranded RNA (dsRNA) profile ( FIG. 1A ) of GLRaV-2 and its Northern hybridization analysis ( FIG. 1B ).
  • dsRNA double-stranded RNA
  • FIG. 1A lane M, lambda Hind III DNA marker; and lane 1, dsRNA pattern in 1% agarose gel stained with ethidium bromide.
  • FIG. 1B is a northern hybridization of isolated high molecular weight dsRNA of GLRaV-2 with a probe prepared with 32 P [ ⁇ -dATP] labeled cDNA insert from GLRaV-2 specific cDNA clone TC-1.
  • Lane 1 high molecular weight dsRNA of GLRaV-2.
  • Lane 2 total RNA extracted from healthy grapevine.
  • FIG. 2 displays the genome organization of GLRaV-2 and its sequencing strategy. Boxes represent ORFs encoded by deduced amino acid sequences of GLRaV-2, numbered lines represent nucleotide coordinates, beginning from 5′-terminal of RNA in kilobases (kb). The lines below GLRaV-2 RNA genome represent the cDNA clones used to determine the nucleotide sequences.
  • FIG. 3A-3D are comparisons between ORF1a/ORF1b of GLRaV-2 and BYV.
  • FIG. 3A-3D show the conserved domains of two papain-like proteases (P-PRO), methyltransferase (MT/MTR), helicase (HEL), and RNA-dependent RNA polymerase (RdRP), respectively. Exclamation marks indicate the predicted catalytic residues of the leader papain-like protease; slashes indicate the predicted cleavage sites.
  • P-PRO papain-like proteases
  • MT/MTR methyltransferase
  • HEL helicase
  • RdRP RNA-dependent RNA polymerase
  • FIGS. 4A and 4B are alignments of the nucleotide ( FIG. 4A ) and deduced amino acid ( FIG. 4B ) sequences of ORF1a/ORF1b overlapping region of GLRaV-2, BYV, BYSV, and CTV. Identical nucleotides and amino acids are shown in consensus. GLRaV-2 putative +1 frameshift site (TAGC) and its corresponding sites of BYV (TAGC) and BYSV (TAGC) and CTV (CGGC) at nucleotide and amino acid sequences are highlighted with underlines.
  • TAGC +1 frameshift site
  • TAGC BYV
  • TAGC BYSV
  • CTV CTV
  • FIG. 5 is an alignment of the amino acid sequence of HSP70 protein of GLRaV-2 and BYV.
  • the conserved motifs (A to H) are indicated with overlines and marked with respective letters.
  • the alignment was conducted with the MegAlign program of DNASTAR.
  • FIG. 6A is a comparison of the coat protein (CP) and coat protein duplicate (CPd) of GLRaV-2 with other closteroviruses.
  • the amino acid sequence of the GLRaV-2 CP and CPd are aligned with the CP and CPd of BYV, BYSV, and CTV. The conserved amino acid residues are in bold and the consensus sequences are indicated. Sequence alignment and phylogenetic tree were constructed by Clustal Method in the MegAlign Program of DNASTAR.
  • FIG. 6B is a tentative phylogenetic tree of the CP and CPd of GLRaV-2 with BYV, BYSV, CTV, LIYV, LChV, and GLRaV-3.
  • FIG. 7 is a comparison of the genome organization of GLRaV-2, BYV, BYSV, CTV, LIYV, LChV, and GLRaV-3.
  • P PRO, papain-like protease; MT/MTR, methyltransferase; HEL, helicase; RdRP, RNA-dependent RNA polymerase; HSP70, heat shock protein 70; CP, coat protein; CPd, coat protein duplicate.
  • FIG. 8 is a tentative phylogenetic tree showing the relationship of RdRP of GLRaV-2 with respect to BYV, BYSV, CTV, and LIYV.
  • the phylogenetic tree was constructed using the Clustal method with the MegAlign program in DNASTAR.
  • FIG. 9 is an alignment of the amino acid sequence of HSP90 protein of GLRaV-2 with respect to other closteroviruses, BYS, BYSV, and CTV. The most conserved motifs (I to II) are indicated with the highlighted lines and marked with respective letters.
  • FIG. 10 is an alignment of the nucleotide sequence of 3′-terminal untranslated region of GLRaV-2 with respect to the closteroviruses BYV (Agranovsky et al., “Beet Yellows Closterovirus : Complete Genome Structure and Identification of a Papain-like Thiol Protease,” Virology 198: 311-24 (1994), which is hereby incorporated by reference), BYSV (Karasev et al., Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221: 199-207 (1996), which is hereby incorporated by reference), and CTV (Karasev et al., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,” Virology 208: 511-20 (1995), which is hereby incorporated by reference). The consensus sequences are shown, and the distance to the 3′-end is indicated. A complementary
  • FIGS. 11A and 11B are genetic maps of the transformation vectors pGA482GG/EPT8CP-GLRaV-2 and pGA482G/EPT8CP-GLRaV-2, respectively.
  • the plant expression cassette EPT8CP-GLRaV-2
  • the plant expression cassette which consists of a double cauliflower mosaic virus (CaMV) 35S-enhancer, a CaMV 35S-promoter, an alfalfa mosaic virus (ALMV) RNA4 5′ leader sequence, a coat protein gene of GLRaV-2 (CP-GLRaV-2), and a CaMV 35S 3′ untranslated region as a terminator, was cloned into the transformation vector by EcoR I restriction site.
  • the CP of GLRaV-2 was cloned into the plant expression vector by Nco I restriction site.
  • FIG. 12 is a PCR analysis of DNA molecules extracted from the leaves of putative transgenic plants using both the CP gene of GLRaV-2 and NPT II gene specific primers.
  • An ethidium bromide-stained gel shows a 720 bp amplified DNA fragment for NPT II gene, and a 653 bp DNA fragment for the entire coding sequence of the CP gene.
  • FIG. 13 is a comparison of resistant (right side 3 plants) and susceptible (left side 3 plants) transgenic Nicotiana benthamiana plants. Plants are shown 48 days after inoculation with GLRaV-2.
  • FIG. 14 is a northern blot analysis of transgenic Nicotiana benthamiana plants. An aliquot of 10 g of total RNA extracted from putative transgenic plants was denatured and loaded onto 1% agarose gel containing formaldehyde. The separated RNAs were transferred to Gene Screen Plus membrane and hybridized with a 32 P-labeled DNA probe containing the 3′ one third CP gene sequence. Lanes 1, 3, and 4 represent nontransformed control plants without RNA expression. The remaining lanes represent transgenic plants from different lines: lanes 2, 14-17, and 22-27 represent plants with high RNA expression level which are susceptible to GLRaV-2; all other lanes represent plants with undetectable or low RNA expression level which are resistant to GLRaV-2.
  • the present invention relates to isolated DNA molecules encoding for the proteins or polypeptides of a grapevine leafroll virus (type 2).
  • a substantial portion of the grapevine leafroll virus (type-2) (“GLRaV-2”) genome has been sequenced. Within the genome are a plurality of open reading frames (“ORFs”) and a 3′ untranscribed region (“UTR”), each containing DNA molecules in accordance with the present invention.
  • the DNA molecule which constitutes a substantial portion of the GLRaV-2 genome comprises the nucleotide sequence corresponding to SEQ. ID. No.
  • Another DNA molecule of the present invention includes nucleotides 4-7923 of SEQ. ID. No. 1 and is believed to code for a large, grapevine leafroll virus polyprotein containing the conserved domains characteristic of two papain-like proteases, a methyltransferase, and a helicase.
  • This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No.
  • the large polyprotein (papain-like proteases, methyltransferase, and helicase) has an amino acid sequence corresponding to SEQ. ID. No. 3 as follows: Thr Leu Arg Glu Asn Pro Ile Ser Val Ser Gly Val Asn Leu Gly Arg 1 5 10 15 Ser Ala Ala Ala Gln Val Ile Tyr Phe Gly Ser Phe Thr Gln Pro Phe 20 25 30 Ala Leu Tyr Pro Arg Gln Glu Ser Ala Ile Val Lys Thr Gln Leu Pro 35 40 45 Pro Val Ser Val Val Lys Val Glu Cys Val Ala Ala Glu Val Ala Pro 50 55 60 Asp Arg Gly Val Val Asp Lys Lys Pro Thr Ser Val Gly Val Pro Pro 65 70 75 80 Gln Arg Gly Val Leu Ser Phe Pro Thr Val Val Arg Asn Arg Gly Asp 85 90 95 Val Ile Ile Thr Gly Val Val His Glu Ala Leu Lys Lys Ile Lys Asp
  • Another such DNA molecule includes nucleotides 7922-9301 of SEQ. ID. No. 1 and codes for a grapevine leafroll virus RNA-dependent RNA polymerase (RdRP). This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No.
  • GLRAV-2 ORF2 Another such DNA molecule (GLRAV-2 ORF2) includes nucleotides 9365-9535 of SEQ. ID. No. 1 and codes for a small, grapevine leafroll virus hydrophobic protein or polypeptide.
  • This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 6 as follows: ATGAATCAGG TTTTGCAGTT TGAATGTTTG TTTCTGCTGA ATCTCGCGGT TTTTGCTGTG 60 ACTTTCATTT TCATTCTTCT GGTCTTCCGC GTGATTAAGT CTTTTCGCCA GAAGGGTCAC 120 GAAGGACCTG TTCCCGTTGT TCGTGGCGGG GGTTTTTCAA CCGTAGTGTA G 171
  • the small hydrophobic protein or polypeptide has an amino acid sequence corresponding to SEQ. ID. No. 7 as follows: Met Asn Gln Val Leu Gln Phe Glu Cys Leu Phe Leu Leu Asn Leu Ala 1 5 10 15 Val Phe Ala Val Thr Phe Ile Phe Ile Leu Leu Val Phe Arg Val Ile 20 25 30 Lys Ser Phe Arg Gln Lys Gly His Glu Ala Pro Val Pro Val Val Arg 35 40 45 Gly Gly Gly Phe Ser Thr Val Val 50 55 and a molecular weight from about 5 to about 7 kDa, preferably about 6 kDa.
  • Another such DNA molecule includes nucleotides 9551-11350 of SEQ. ID. No. 1 and encodes for a grapevine leafroll virus heat shock 70 protein. This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No.
  • the heat shock 70 protein is believed to function as a chaperone protein and has an amino acid sequence corresponding to SEQ. ID. No. 9 as follows: Met Val Val Phe Gly Leu Asp Phe Gly Thr Thr Phe Ser Thr Val Cys 1 5 10 15 Val Tyr Lys Asp Gly Arg Val Phe Ser Phe Lys Gln Asn Asn Ser Ala 20 25 30 Tyr Ile Pro Thr Tyr Leu Tyr Leu Phe Ser Asp Ser Asn His Met Thr 35 40 45 Phe Gly Tyr Glu Ala Glu Ser Leu Met Ser Asn Leu Lys Val Lys Gly 50 55 60 Ser Phe Tyr Arg Asp Leu Lys Arg Trp Val Gly Cys Asp Ser Ser Asn 65 70 75 80 Leu Asp Ala Tyr Leu Asp Arg Leu Lys Pro His Tyr Ser Val Arg Leu 85 90 95 Val Lys Ile Gly Ser Gly Leu Asn Glu Thr Val Ser Ile Gly Asn Phe 100 105 110 Gly Gly Thr Val
  • Another such DNA molecule includes nucleotides 11277-12932 of SEQ. ID. No. 1 and codes for a putative grapevine leafroll virus heat shock 90 protein.
  • This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the heat shock 90 protein has an amino acid sequence corresponding to SEQ. ID. No. 11 as follows: Met Ser Asn Tyr Ser Trp Glu Ser Leu Phe Lys Lys Phe Tyr Gly Glu 1 5 10 15 Ala Asp Trp Lys Lys Tyr Leu Ser Arg Ser Ile Ala Ala His Ser Ser 20 25 30 Glu Ile Lys Thr Leu Pro Asp Ile Arg Leu Tyr Gly Gly Arg Val Val 35 40 45 Lys Lys Ser Glu Phe Glu Ser Ala Leu Pro Asn Ser Phe Glu Gln Glu 50 55 60 Leu Gly Leu Phe Ile Leu Ser Glu Arg Glu Val Gly Trp Ser Lys Leu 65 70 75 80 Cys Gly Ile Thr Val Glu Glu Ala Ala Tyr Asp Leu Thr Asn Pro Lys 85 90 95 Ala Tyr Lys Phe Thr Ala Glu Thr Cys Ser Pro Asp Val Lys Gly Glu 100 105 110 Gly Gln Lys Tyr Ser Met
  • DNA molecule of the present invention includes nucleotides 12844-13515 of SEQ. ID. No. 1 and codes for a diverged coat protein. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the diverged coat protein has an amino acid sequence corresponding to SEQ. ID. No. 13 as follows: Met Ser Ser Asn Thr Ser Val Pro Val Gly Gly Leu Glu Ala Leu Glu 1 5 10 15 Thr Ser Gly Val Val Leu Thr Thr Arg Lys Glu Ala Val Asp Lys Phe 20 25 30 Phe Asn Glu Leu Lys Asn Glu Asn Tyr Ser Ser Val Asp Ser Ser Arg 35 40 45 Leu Ser Asp Ser Glu Val Lys Glu Val Leu Glu Lys Ser Lys Glu Ser 50 55 60 Phe Lys Ser Glu Leu Ala Ser Thr Asp Glu His Phe Val Tyr His Ile 65 70 75 80 Ile Phe Phe Leu Ile Arg Cys Ala Lys Ile Ser Thr Ser Glu Lys Val 85 90 95 Lys Tyr Val Gly Ser His Thr Tyr Val Val Asp Gly Lys Thr Tyr Thr 100 105 110 Val Leu Asp Ala Trp Val Phe Asn Met Met Lys Ser Leu
  • Another such DNA molecule includes nucleotides 13584-14180 of SEQ. ID. No. 1 and codes for a grapevine leafroll virus coat protein. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the coat protein has an amino acid sequence corresponding to SEQ. ID. No. 15 as follows: Met Glu Leu Met Ser Asp Ser Asn Leu Ser Asn Leu Val Ile Thr Asp 1 5 10 15 Ala Ser Ser Leu Asn Gly Val Asp Lys Lys Leu Leu Ser Ala Glu Val 20 25 30 Glu Lys Met Leu Val Gln Lys Gly Ala Pro Asn Glu Gly Ile Glu Val 35 40 45 Val Phe Gly Leu Leu Leu Tyr Ala Leu Ala Ala Arg Thr Thr Ser Pro 50 55 60 Lys Val Gln Arg Ala Asp Ser Asp Val Ile Phe Ser Asn Ser Phe Gly 65 70 75 80 Glu Arg Asn Val Val Val Val Thr Glu Gly Asp Leu Lys Lys Val Leu Asp 85 90 95 Gly Cys Ala Pro Leu Thr Arg Phe Thr Asn Lys Leu Arg Thr Phe Gly 100 105 110 Arg Thr Phe Thr Glu
  • Another such DNA molecule includes nucleotides 14180-14665 of SEQ. ID. No. 1 and codes for a second undefined grapevine leafroll virus protein or polypeptide.
  • This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the second undefined grapevine leafroll virus protein or polypeptide has a deduced amino acid sequence corresponding to SEQ. ID. No. 17 as follows: Met Glu Asp Tyr Glu Glu Lys Ser Glu Ser Leu Ile Leu Leu Arg Thr 1 5 10 15 Asn Leu Asn Thr Met Leu Leu Val Val Lys Ser Asp Ala Ser Val Glu 20 25 30 Leu Pro Lys Leu Leu Ile Cys Gly Tyr Leu Arg Val Ser Gly Arg Gly 35 40 45 Glu Val Thr Cys Cys Asn Arg Glu Glu Leu Thr Arg Asp Phe Glu Gly 50 55 60 Asn His His Thr Val Ile Arg Ser Arg Ile Ile Gln Tyr Asp Ser Glu 65 70 75 80 Ser Ala Phe Glu Glu Phe Asn Asn Ser Asp Cys Val Val Lys Phe Phe 85 90 95 Leu Glu Thr Gly Ser Val Trp Phe Phe Leu Arg Ser Glu Thr Lys 100
  • DNA molecule includes nucleotides 14667-15284 of SEQ. ID. No. 1 and codes for a third undefined grapevine leafroll virus protein or polypeptide.
  • This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No.
  • the third undefined protein or polypeptide has a deduced amino acid sequence corresponding to SEQ. ID. No. 19 as follows: Met Arg Val Ile Val Ser Pro Tyr Glu Ala Glu Asp Ile Leu Lys Arg 1 5 10 15 Ser Thr Asp Met Leu Arg Asn Ile Asp Ser Gly Val Leu Ser Thr Lys 20 25 30 Glu Cys Ile Lys Ala Phe Ser Thr Ile Thr Arg Asp Leu His Cys Ala 35 40 45 Lys Ala Ser Tyr Gln Trp Gly Val Asp Thr Gly Leu Tyr Gln Arg Asn 50 55 60 Cys Ala Glu Lys Arg Leu Ile Asp Thr Val Glu Ser Asn Ile Arg Leu 65 70 75 80 Ala Gln Pro Leu Val Arg Glu Lys Val Ala Val His Phe Cys Lys Asp 85 90 95 Glu Pro Lys Glu Leu Val Ala Phe Ile Thr Arg Lys Tyr Val Glu Leu 100 105
  • Another DNA molecule of the present invention includes nucleotides 15285-15500 of SEQ. ID. No. 1 and comprises a nucleotide sequence corresponding to SEQ. ID. No. 23 as follows: ACATTGGTTA AGTTTAACGA AAATGATTAG TAAATAATAA ATCGAACGTG GGTGTATCTA 60 CCTGACGTAT CAACTTAAGC TGTTACTGAG TAATTAAACC AACAAGTGTT GGTGTTAATGT 120 GTATGTTGAT GTAGAGAAAA ATCCGTTTGT AGAACGGTGT TTTTCTTC TTTATTTTTA 180 AAAAAAAAAT AAAAAAAAAAAAAAA AAAAAAAAGC GGCCGC 216
  • fragments of the DNA molecules of the present invention are constructed by using appropriate restriction sites, revealed by inspection of the DNA molecule's sequence, to: (i) insert an interposon (Felley et al., “Interposon Mutagenesis of Soil and Water Bacteria: a Family of DNA Fragments Designed for in vitro Insertion Mutagenesis of Gram-negative Bacteria,” Gene, 52: 147-15 (1987), which is hereby incorporated by reference) such that truncated forms of the grapevine leafroll virus coat polypeptide or protein, that lack various amounts of the C-terminus, can be produced or (ii) delete various internal portions of the protein.
  • the sequence can be used to amplify any portion of the coding region, such that it can be cloned into a vector supplying both transcription and translation start signals.
  • Suitable DNA molecules are those that hybridize to a DNA molecule comprising a nucleotide sequence of at least 15 continuous bases of SEQ. ID. No. 1 under stringent conditions characterized by a hybridization buffer comprising 0.9M sodium citrate (“SSC”) buffer at a temperature of 37° C. and remaining bound when subject to washing with SSC buffer at 37° C.; and preferably in a hybridization buffer comprising 20% formamide in 0.9M saline/0.9M SSC buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2 ⁇ SSC buffer at 42° C.
  • SSC sodium citrate
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of nucleotides that have minimal influence on the properties, secondary structure and hydropathic nature of the encoded polypeptide.
  • the nucleotides encoding a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein.
  • the nucleotide sequence may also be altered so that the encoded polypeptide is conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide.
  • the protein or polypeptide of the present invention is preferably produced in purified form (preferably, at least about 80%, more preferably 90%, pure), by conventional techniques.
  • the protein or polypeptide of the present invention is isolated by lysing and sonication. After washing, the lysate pellet is resuspended in buffer containing Tris-HCl. During dialysis, a precipitate forms from this protein solution. The solution is centrifuged, and the pellet is washed and resuspended in the buffer containing Tris-HCl. Proteins are resolved by electrophoresis through an SDS 12% polyacrylamide gel.
  • the DNA molecule encoding the grapevine leafroll virus (type 2) protein or polypeptide of the present invention can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • Recombinant genes may also be introduced into viruses, such as vaccinia virus.
  • Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
  • Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/ ⁇ or KS +/ ⁇ (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see Studier et.
  • viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177
  • host-vector systems may be utilized to express the protein-encoding sequence(s).
  • the vector system must be compatible with the host cell used.
  • Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria or transformed via particle bombardment (i.e. biolistics).
  • the expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
  • mRNA messenger RNA
  • telomere a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis.
  • the DNA sequences of eucaryotic promoters differ from those of procaryotic promoters.
  • eucaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eucaryotic cells.
  • SD Shine-Dalgamo
  • Promoters vary in their “strength” (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E.
  • promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the P R and P L promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced.
  • the addition of specific inducers is necessary for efficient transcription of the inserted DNA.
  • the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside).
  • IPTG isopropylthio-beta-D-galactoside.
  • Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively.
  • the DNA expression vector which contains a promoter, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed.
  • Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
  • Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.
  • the present invention also relates to RNA molecules which encode the various grapevine leafroll virus (type 2) proteins or polypeptides described above.
  • the transcripts can be synthesized using the host cells of the present invention by any of the conventional techniques.
  • the mRNA can be translated either in vitro or in vivo.
  • Cell-free systems typically include wheat-germ or reticulocyte extracts. In vivo translation can be effected, for example, by microinjection into frog oocytes.
  • One aspect of the present invention involves using one or more of the above DNA molecules encoding the various proteins or polypeptides of a grapevine leafroll virus (type 2) to transform grape plants in order to impart grapevine leafroll resistance to the plants.
  • the mechanism by which resistance is imparted is not known.
  • the transformed plant can express a protein or polypeptide of grapevine leafroll virus (type 2), and, when the transformed plant is inoculated by a grapevine leafroll virus, such as GLRaV-1, GLRaV-2, GLRav-3, GLRaV-4, GLRaV-5, or GLRaV-6, or combinations of these, the expressed protein or polypeptide prevents translation of the viral DNA.
  • the subject DNA molecule incorporated in the plant can be constitutively expressed.
  • expression can be regulated by a promoter which is activated by the presence of grapevine leafroll virus.
  • Suitable promoters for these purposes include those from genes expressed in response to grapevine leafroll virus infiltration.
  • the isolated DNA molecules of the present invention can be utilized to impart grapevine leafroll virus resistance for a wide variety of grapevine plants.
  • the DNA molecules are particularly well suited to imparting resistance to Vitis scion or rootstock cultivars.
  • Scion cultivars which can be protected include those commonly referred to as Table or Raisin Grapes, such as Alden, Almeria, Anab-E-Shahi, Autumn Black, Beauty Seedless, Black Corinth, Black Damascus, Black Malvoisie, Black Prince, Blackrose, Bronx Seedless, Burgrave, Calmeria, Campbell Early, Canner, Cardinal, Catawba, Christmas, Concord, Dattier, Delight, Diamond, Dizmar, Duchess, Early Muscat, Emerald Seedless, Emperor, Exotic, Anthony de Lesseps, Fiesta, Flame seedless, Flame Tokay, Gasconade, Gold, Himrod, Hunisa, Hussiene, Isabella, Italia, July Muscat, Khandahar, Katta, Kourgan
  • Rootstock cultivars which can be protected include Couderc 1202, Couderc 1613, Couderc 1616, Couderc 3309, Dog Ridge, Foex 33 EM, Freedom, Ganzin 1 (A ⁇ R #1), Harmony, Kober 5BB, LN33, Millardet & de Grasset 41B, Millardet & de Grasset 420A, Millardet & de Grasset 101-14, Oppenheim 4 (SO 4 ), Paulsen 775, Paulsen 1045, Paulsen 1103, Richter 99, Richter 110, Riparia Gloire, Ruggeri 225, Saint-George, Salt Creek, Teleki 5A, Vitis rupestris Constantia, Vitis california , and Vitis girdiana.
  • GLRaV-2 hsp70-related gene can also be used to produce transgenic plants or cultivars other than grape, such as citrus or sugar beet, which are resistant to closteroviruses other than grapevine leafroll, such as tristeza virus and beet yellows virus.
  • Suitable citrus cultivars include lemon, lime, orange, grapefruit, pineapple, tangerine, and the like, such as Joppa, Maltaise Ovale, Parson (Parson Brown), Pera, Pineapple, Queen, Shamouti, Valencia, Tenerife, Imperial Doblefina, Washington Sanguine, Moro, Sanguinello Moscato, Spanish Sanguinelli, Tarocco, Atwood, Australian, Bahia, Baiana, Cram, Dalmau, Eddy, Fisher, Frost Washington, Gillette, LengNavelina, Washington, Satsuma Mandarin, Dancy, Robinson, Ponkan, Duncan, Marsh, Pink Marsh, Ruby Red, Red Seedless, Smooth Seville, Orlando Tangelo, Eureka, Portugal, Meyer Lemon, Rough Lemon, Sour Orange, Persian Lime, West Indian Lime, Bearss, Sweet Lime, Troyer Citrange, and Citrus Trifoliata. Each of these citrus cultivars is suitable for producing transgenic citrus plants resistant to tristeza virus.
  • Beta vulgaris L. which has four important cultivar types: sugar beet, table beet, fodder beet, and Swiss chard. Each of these beet cultivars is suitable for producing transgenic beet plants resistant to beet yellows virus, as described above.
  • GLRaV-2 has been known to infect tobacco plants (e.g., Nicotiana benthamiana ), it is also desirable to produce transgenic tobacco plants which are resistant to grapevine leafroll viruses, such as GLRaV-2.
  • Plant tissue suitable for transformation include leaf tissue, root tissue, meristems, zygotic and somatic embryos, and anthers. It is particularly preferred to utilize embryos obtained from anther cultures.
  • the expression system of the present invention can be used to transform virtually any plant tissue under suitable conditions.
  • Tissue cells transformed in accordance with the present invention can be grown in vitro in a suitable medium to impart grapevine leafroll virus resistance.
  • Transformed cells can be regenerated into whole plants such that the protein or polypeptide imparts resistance to grapevine leafroll virus in the intact transgenic plants.
  • the plant cells transformed with the recombinant DNA expression system of the present invention are grown and caused to express that DNA molecule to produce one of the above-described grapevine leafroll virus proteins or polypeptides and, thus, to impart grapevine leafroll virus resistance.
  • the DNA construct in a vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA.
  • the genetic material may also be transferred into the plant cell using polyethylene glycol. Krens, et al., Nature, 296: 72-74 (1982), which is hereby incorporated by reference.
  • One technique of transforming plants with the DNA molecules in accordance with the present invention is by contacting the tissue of such plants with an inoculum of a bacteria transformed with a vector comprising a gene in accordance with the present invention which imparts grapevine leafroll resistance.
  • this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28° C.
  • Bacteria from the genus Agrobacterium can be utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacterium tumefaciens (e.g., strains C58, LBA4404, or EHA 105) is particularly useful due to its well-known ability to transform plants.
  • Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the R1 plasmid of A. rhizogenes .
  • the Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science. 237: 1176-83 (1987), which is hereby incorporated by reference.
  • the transformed plant cells After transformation, the transformed plant cells must be regenerated.
  • Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants.
  • the culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
  • the expression cassette After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure so that the DNA construct is present in the resulting plants.
  • transgenic seeds are recovered from the transgenic plants. These seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants.
  • particle bombardment also known as biolistic transformation
  • particle bombardment also known as biolistic transformation
  • this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof.
  • the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA.
  • the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle.
  • Biologically active particles e.g., dried bacterial cells containing the vector and heterologous DNA
  • the transformed tissue is regenerated to form a transgenic plant.
  • regeneration is accomplished by culturing transformed tissue on medium containing the appropriate growth regulators and nutrients to allow for the initiation of shoot meristems. Appropriate antibiotics are added to the regeneration medium to inhibit the growth of Agrobacterium and to select for the development of transformed cells. Following shoot initiation, shoots are allowed to develop tissue culture and are screened for marker gene activity.
  • the DNA molecules of the present invention can be made capable of transcription to a messenger RNA, which, although encoding for a grapevine leafroll virus (type 2) protein or polypeptide, does not translate to the protein. This is known as RNA-mediated resistance.
  • RNA-mediated resistance When a Vitis scion or rootstock cultivar, or a citrus, beet, or tobacco cultivar, is transformed with such a DNA molecule, the DNA molecule can be transcribed under conditions effective to maintain the messenger RNA in the plant cell at low level density readings. Density readings of between 15 and 50 using a Hewlet ScanJet and Image Analysis Program are preferred.
  • a portion of one or more DNA molecules of the present invention as well as other DNA molecules can be used in a transgenic grape plant, citrus plant, beet plant, or tobacco plant in accordance with U.S. patent application Ser. No. 09/025,635, which is hereby incorporated herein by reference.
  • the grapevine leafroll virus (type 2) protein or polypeptide of the present invention can also be used to raise antibodies or binding portions thereof or probes.
  • the antibodies can be monoclonal or polyclonal.
  • Monoclonal antibody production may be effected by techniques which are well-known in the art. Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a mammal (e.g., mouse) which has been previously immunized with the antigen of interest either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies.
  • lymphocytes immune cells
  • mammal e.g., mouse
  • myeloma cells or transformed cells which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line.
  • the resulting fused cells, or hybridomas are cultured, and
  • Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody.
  • a description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, Nature, 256: 495 (1975), which is hereby incorporated by reference.
  • Mammalian lymphocytes are immunized by in vivo immunization of the animal (e.g., a mouse) with the protein or polypeptide of the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Following the last antigen boost, the animals are sacrificed and spleen cells removed.
  • Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol (“PEG”) or other fusing agents.
  • PEG polyethylene glycol
  • This immortal cell line which is preferably murine, but may also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described.
  • Procedures for raising polyclonal antibodies are also well known. Typically, such antibodies can be raised by administering the protein or polypeptide of the present invention subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum.
  • the antigens can be injected at a total volume of 100 ⁇ l per site at six different sites. Each injected material will contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis.
  • the rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost.
  • Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Ultimately, the rabbits are euthenized with pentobarbital 150 mg/Kg IV. This and other procedures for raising polyclonal antibodies are disclosed in Harlow et. al., editors, Antibodies: A Laboratory Manual (1988), which is hereby incorporated by reference.
  • binding portions of such antibodies can be used.
  • binding portions include Fab fragments, F(ab′) 2 fragments, and Fv fragments.
  • These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in Goding, Monoclonal Antibodies: Principles and Practice , New York:Academic Press, pp. 98-118 (1983), which is hereby incorporated by reference.
  • the present invention also relates to probes found either in nature or prepared synthetically by recombinant DNA procedures or other biological procedures.
  • Suitable probes are molecules which bind to grapevine leafroll (type 2) viral antigens identified by the monoclonal antibodies of the present invention.
  • Such probes can be, for example, proteins, peptides, lectins, or nucleic acid probes.
  • the antibodies or binding portions thereof or probes can be administered to grapevine leafroll virus infected scion cultivars or rootstock cultivars. Alternatively, at least the binding portions of these antibodies can be sequenced, and the encoding DNA synthesized.
  • the encoding DNA molecule can be used to transform plants together with a promoter which causes expression of the encoded antibody when the plant is infected by grapevine leafroll virus. In either case, the antibody or binding portion thereof or probe will bind to the virus and help prevent the usual leafroll response.
  • Antibodies raised against the GLRaV-2 proteins or polypeptides of the present invention or binding portions of these antibodies can be utilized in a method for detection of grapevine leafroll virus in a sample of tissue, such as tissue (e.g., scion or rootstock) from a grape plant or tobacco plant.
  • Antibodies or binding portions thereof suitable for use in the detection method include those raised against a helicase, a methyltransferase, a papain-like protease, an RNA-dependent RNA polymerase, a heat shock 70 protein, a heat shock 90 protein, a coat protein, a diverged coat protein, or other proteins or polypeptides in accordance with the present invention.
  • any reaction of the sample with the antibody is detected using an assay system which indicates the presence of grapevine leafroll virus in the sample.
  • assay systems can be employed, such as enzyme-linked immunosorbent assays, radioimmunoassays, gel diffusion precipitin reaction assays, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays, or immunoelectrophoresis assays.
  • grapevine leafroll virus can be detected in such a sample using a nucleotide sequence of the DNA molecule, or a fragment thereof, encoding for a protein or polypeptide of the present invention.
  • the nucleotide sequence is provided as a probe in a nucleic acid hybridization assay or a gene amplification detection procedure (e.g., using a polymerase chain reaction procedure).
  • the nucleic acid probes of the present invention may be used in any nucleic acid hybridization assay system known in the art, including, but not limited to, Southern blots (Southern, E. M., “Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis,” J. Mol.
  • the probes can be used in a gene amplification detection procedure (e.g., a polymerase chain reaction).
  • a gene amplification detection procedure e.g., a polymerase chain reaction.
  • Any reaction with the probe is detected so that the presence of a grapevine leafroll virus in the sample is indicated.
  • detection is facilitated by providing the probe of the present invention with a label.
  • Suitable labels include a radioactive compound, a fluorescent compound, a chemiluminescent compound, an enzymatic compound, or other equivalent nucleic acid labels.
  • probes having nucleotide sequences that correspond with conserved or variable regions of the ORF or UTR For example, to distinguish a grapevine leafroll virus from other related viruses (e.g., other closteroviruses), it is desirable to use probes which contain nucleotide sequences that correspond to sequences more highly conserved among all grapevine leafroll viruses. Also, to distinguish between different grapevine leafroll viruses (i.e., GLRaV-2 from GLRaV-1, GLRaV-3, GLRaV-4, GLRaV-5, and GLRaV-6), it is desirable to utilize probes containing nucleotide sequences that correspond to sequences less highly conserved among the different grapevine leafroll viruses.
  • GLRaV-2 from GLRaV-1, GLRaV-3, GLRaV-4, GLRaV-5, and GLRaV-6
  • Nucleic acid (DNA or RNA) probes of the present invention will hybridize to complementary GLRaV-2 nucleic acid under stringent conditions.
  • stringent conditions are selected to be about 50° C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Nonspecific binding may also be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase. Wash conditions are typically performed at or below stringency. Generally, suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are asas set forth above. More or less stringent conditions may also be selected.
  • Northern hybridization was performed after electrophoresis of the dsRNA of GLRaV-2 in 1% agarose non-denaturing condition gel.
  • the agarose gel was denatured by soaking in 50 mM NaOH containing 0.4 M NaCl for 30 min, and then neutralized with 0.1 M Tris-HCl (PH7.5) containing 0.5 M NaCl for another 30 min.
  • RNA was sandwich blotted overnight onto GenescreenTM plus membrane (Dupont NEN Research Product) in 10 ⁇ SSC buffer and hybridized as described by the manufacturer's instructions (DuPont, NEN).
  • DNA inserts were sequenced in pBluescript SK+ by using T3 and T7 universal primers for the terminal region sequence and additional oligonucleotide primers designed according to the known sequence for the internal region sequence.
  • Purification of plasmid DNA was performed by a modified mini alkaline-lysis/PEG precipitation procedure described by the manufacturer (Applied Biosystems, Inc.).
  • Nucleotide sequencing was performed on both strands of cDNA by using ABI TaqDyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc.). Automatic sequencing was performed on an ABI373 Automated Sequencer (Applied Biosynstems, Inc.) at Cornell University, Geneva, N.Y.
  • nucleotide sequences of GLRaV-2 were assembled and analyzed with the programs of EditSeq and SeqMan, respectively, of DNASTAR package (Madison, Wis.). Amino acid sequences deduced from nucleotide sequences and its encoding open reading frames were conducted using the MapDraw program. Multiple alignments of amino acid sequences, identification of consensus amino acid sequences, and generation of phylogenetic trees were performed using the Clustal method in the MegAlign program. The nucleotide and amino acid sequences of other closteroviruses were obtained with the Entrez Program; and sequence comparisons with nonredundant databases were searched with the Blast Program from the National Center for Biotechnology Information.
  • dsRNA was extracted from phloem tissue of infected grapevines according to the method described by Hu et al., “Characterization of Closterovirus -Like Particles Associated with Grapevine Leafroll Disease,” J. Phytopathology 128: 1-14 (1990), which is hereby incorporated by reference.
  • dsRNA purification of the high molecular weight dsRNA (ca 15 kb) was carried out by electrophoretic separation of the total dsRNA on a 0.7% low melting point agarose gel and extraction by phenol/chloroform following the method described by Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Sping Harbor Laboratory Press, New York (1989), which is hereby incorporated by reference. Concentration of dsRNA was estimated with UV fluorescent density of an ethidium bromide stained dsRNA band in comparison with a known concentration of DNA marker.
  • cDNA synthesis was performed following the method initially described by Jelkmann et al., “Cloning of Four Plant Viruses From Small Quantities of Double-Stranded RNA,” Phytopathology 79: 1250-53 (1989) and modified by Ling et al., “The Coat Protein Gene of Grapevine Leafroll Associated Closterovirus -3: Cloning, Nucleotide Sequencing and Expression in Transgenic Plants,” Arch. Virology 142: 1101-16 (1997), both of which are hereby incorporated by reference.
  • Second strand cDNA was obtained by using RNase H and E. coli DNA polymerase I. Double-stranded cDNA was blunt ended with T4 DNA polymerase and ligated with EcoR I adapters.
  • the cDNA which had EcoR I adapters at the ends, was activated by kinase reaction and ligated into Lambda ZAP II/EcoR I prepared arms following the manufacturer's instruction (Stratagene). The recombinant DNA was then packaged in vitro to Gigapack® II packaging extract (Stratagene). The packaged phage particles were amplified and titered according to the manufacturer's instruction.
  • Two kinds of probes were used to identify GLRaV-2 specific clones from the library.
  • One type was prepared from the synthesized cDNA that was amplified by PCR after ligation to the specific EcoR I Uni-AmpTM adapters (Clontech); and the other type was DNA inserts or PCR products from already sequenced clones.
  • Clones from the cDNA library were selected by colony-lifting hybridization onto the colony/plaque Screen membrane (NEN Research Product) with the probe described above.
  • the probe was prepared by labeling with 32 P [ ⁇ -dATP] using Klenow fragment of E. coli DNA polymerase I. Prehybridization, hybridization, and washing steps were carried out at 65° C. according to the manufacturer's instruction (Dupont, NEN Research Product). Selected plaques were converted to recombinant pBluescript by in vivo excision method according to the manufacturer's instruction (Stratagene).
  • dsRNA was polyadenylated by yeast poly(A) polymerase.
  • cDNA was amplified by RT-PCR with oligo(dT)18 and a specific primer, CP-1/T7R, which is derived from the clone CP-1 and has a nucleotide sequence according to SEQ. ID. No. 20 as follows: TGCTGGAGCT TGAGGTTCTG C 21
  • TA vector Invitrogen
  • a high molecular weight dsRNA of ca. 15 kb was consistently identified from GLRaV-2 infected grapevines, but not from healthy vines.
  • several low molecular weight dsRNAs were also detected from infected tissue.
  • the yield of dsRNA of GLRaV-2 was estimated between 5-10 ng/15 g phloem tissue, which was much lower than that of GLRaV-3 (Hu et al., “Characterization of Closterovirus -Like Particles Associated with Grapevine Leafroll Disease,” J. Phytopathology 128: 1-14 (1990), which is hereby incorporated by reference).
  • Only the high molecular weight dsRNA that was purified from low melting point agarose gel was used for cDNA synthesis, cloning and establishment of the Lambda/ZAP II cDNA library.
  • the initial clones were identified by hybridization with Uni-AmpTM PCR-amplified cDNA as probes.
  • the specificity of these clones e.g., TC-1 ranging from 200 to 1,800 bp in size was confirmed by Northern hybridization to dsRNA of GLRaV-2 as shown in FIG. 1B .
  • TC-1 Uni-AmpTM PCR-amplified cDNA
  • FIG. 1B The specificity of these clones ranging from 200 to 1,800 bp in size was confirmed by Northern hybridization to dsRNA of GLRaV-2 as shown in FIG. 1B .
  • Over 40 different clones ranging form 800 to 7,500 bp in size were identified following hybridization with the probes generated from GLRaV-2 specific cDNA clones or from PCR products. Over 40 clones were then sequenced on the both strands ( FIG. 2 ).
  • ORF6 was the coat protein gene of GLRaV-2
  • the complete ORF6 DNA molecule was subcloned from a PCR product and inserted into the fusion protein expression vector pMAL-C2 (New England Biolabs, Inc.).
  • the specific primers used for the PCR reaction were CP-96F and CP-96R, in which an EcoR I or BamH I site was included to facilitate cloning.
  • CP-96F was designed to include the start codon of the CP and comprises a nucleotide sequence according to SEQ. ID. NO. 21 as follows:
  • CP-96R was 66 nucleotides downstream of the stop codon of the CP and comprises the nucleotide sequence corresponding to SEQ. ID. No.22 as follows: AGCGGATCCA TGGCAGATTC GTGCGTAGCA GTA 33
  • the coat protein was expressed as a fusion protein with maltose binding protein (MBP) of E. coli under the control of a “tac” promoter and suppressed by the “lac” repressor.
  • MBP-CP fusion protein was induced by adding 0.3 mM isopropyl- ⁇ -D-thio-gloactopyranoside (IPTG) and purified by a one step affinity column according to the manufacturer's instruction (New England, Biolabs, Inc).
  • the MBP-CP fusion protein or the coat protein cleaved from the fusion protein was tested to react with specific antiserum of GLRaV-2 (kindly provided by Dr. Charles Greif of INRA, Colmar, France) on Western blot according to the method described by Hu et al., “Characterization of Closterovirus -Like Particles Associated with Grapevine Leafroll Disease,” J. Phytopathology 128: 1-14 (1990), which is hereby incorporated by reference.
  • the non-recombinant plasmids or uninduced cells did not react to the antiserum of GLRaV-2.
  • RNA genome of GLRaV-2 was sequenced and deposited in GenBank (accession number AF039204). About 85% of the total RNA genome was revealed from at least two different clones. The sequence in the coat protein gene region was determined and confirmed from several different overlapping clones.
  • GenBank accession number AF0392064
  • the genome organization of GLRaV-2, shown in FIG. 2 includes nine open reading frames (e.g., ORF1a, 1b-8).
  • ORF1a and ORF1b Analysis of the amino acid sequence of the N-terminal portion of GLRaV-2 ORF 1 a encoded product revealed two putative papain-like protease domains, which showed significant similarity to the papain-like leader protease of BYV (Agranovsky et al., “Beet Yellows Closterovirus : Complete Genome Structure and Identification of a Papain-like Thiol Protease,” Virology 198: 311-24 (1994), which is hereby incorporated by reference). Thus, it allowed prediction of the catalytic cysteine and histidine residues for the putative GLRaV-2 protease.
  • the C-terminal portion of the ORF1a was identified as a helicase domain, the sequence of which showed a high similarity (57.1% identity in a 315-residues alignment) to the helicase domain of BYV and contained the seven conserved motifs characteristic of the Superfamily I helicase of positive-strand RNA viruses ( FIG. 3C ) (Hodgman, “A New Superfamily of Replicative Proteins,” Nature 333: 22-23 (1988); Koonin and Dolja, “Evolution and Taxonomy of Positive-strand RNA Viruses: Implications of Comparative Analysis of Amino Acid Sequences,” Crit. Rev. in Biochem. and Mol. Biol. 28: 375-430 (1993), both of which are hereby incorporated by reference).
  • ORF1b encoded a 460 amino acid polypeptide with a molecular mass of 52, 486 Da, counting from the frameshifting site.
  • Database searching with the RdRP showed a significant similarity to the RDRP domains of positive strand RNA viruses.
  • Comparison of the RdRP domains of GLRaV-2 and BYV showed the presence of the eight conserved motifs of RdRP ( FIG. 3D ).
  • a tentative phylogenetic tree of the RdRP of GLRaV-2 with respect to other closteroviruses shows that it is closely related to the monopartite closteroviruses BYV, BYSV, and CTV.
  • ORF2 encodes a small protein consisting of 171 bp (57 amino acid) with a molecular mass of 6,297 Da. As predicted, the deduced amino acid sequence includes a stretch of nonpolar amino acids, which is presumed to form a transmembrane helix.
  • a small hydrophobic analogous protein is also present in BYV, BYSV, CTV, LIYV, and LChV (Agranovsky et al. “Nucleotide Sequence of the 3′-Terminal Half of Beet Yellows Closterovirus RNA Genome Unique Arrangement of Eight Virus Genes,” J.
  • ORF3 encodes a 600 amino acid polypeptide with a molecular mass of 65,111 Da, which is homologous to the HSP70 cellular heat shock protein.
  • HSP70 is highly conserved among closteroviruses and is probably involved in ATPase activity and the protein to protein interaction for chaperone activity (Agranovsky et al. “The Beet Yellows Closterovirus p65 Homologue of HSP70 Chaperones has ATPase Activity Associated with its conserveed N-terminal Domain but Interact with Unfolded Protein Chains,” J.
  • ORF4 encodes a 551 amino acid protein with a molecular mass of 63,349 Da.
  • Database searching with the ORF4 protein product did not identify similar proteins except those of its counterparts in closteroviruses, BYV (P64), BYSV (P61), CTV (P61), LIYV (P59), and LChV (P61).
  • This protein is believed to be a putative heat shock 90 protein.
  • FIG. 9 two conserved motifs which were present in BYV (Agranovsky et al. “Nucleotide Sequence of the 3′-Terminal Half of Beet Yellows Closterovirus RNA Genome Unique Arrangement of Eight Virus Genes,” J.
  • ORF5 and ORF6 encode polypeptides with molecular mass of 24,803 Da and 21,661 Da, respectively.
  • the start codon for both ORFs is in a favorable context for translation.
  • ORF6 was identified as the coat protein gene of GLRaV-2 based on the sequence comparison with other closteroviruses.
  • the calculated molecular mass of the protein product of ORF6 (21,662 Da) is in good agreement with the previously estimated 22 ⁇ 26 kDa based on SDS-PAGE (Zimmermann et al., “Characterization and Serological Detection of Four Closterovirus -like Particles Associated with Leafroll Disease on Grapevine,” J.
  • ORF6 as the coat protein gene was further confirmed by Western blot following expression of a fusion protein, consisting of a 22 kDa of ORF6 CP and a 42 kDa of maltose binding protein, produced by transformed E. coli as described in Example 5 supra. As shown in FIG.
  • GLRaV-2 is more closely related to aphid transmissible closteroviruses (BYV, BYSV, and CTV) (Candresse, “Closteroviruses and Clostero-like Elongated Plant Viruses,” in Encyclopedia of Virology , pp.
  • ORF7 and ORF8 encode polypeptides of 162 amino acid with a molecular mass of 18,800 Da and of 206 amino acid with a molecular mass of 23,659 Da, respectively.
  • Database searching with the ORF7 and ORF8 showed no significant similarity with any other proteins. Nevertheless, these genes were of similar in size and location as those observed in the sequence of other closteroviruses, BYV (P20, P21), BYSV (P18, P22), and LChV (P21, P27) ( FIG. 7 ). However, conserved regions were not observed between the ORF7 or ORF8 and its counterparts in BYV, BYSV, and LChV.
  • the 3′ terminal untranslated region (3′-UTR) consists of 216 nucleotides. Nucleotide sequence analysis revealed a long oligo(A) tract close to the end of the GLRaV-2 genome which is similar to that observed in the genome of BYV and BYSV (Agranovsky et al. “Nucleotide Sequence of the 3′-Terminal Half of Beet Yellows Closterovirus RNA Genome Unique Arrangement of Eight Virus Genes,” J.
  • BYV ends in CCC, BYSV, and CTV ends in CC with an additional G or A in the double-stranded replicative form of BYSV (Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221: 199-207 (1996), which is hereby incorporated by reference) and CTV (Karasev et al., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,” Virology 208: 511-20 (1995), which is hereby incorporated by reference), respectively.
  • GLRaV-2 had CGC at the 3′ terminus of the genome.
  • the closteroviruses studied so far have apparent similarities in genome organization, which include replication associated genes that consist of MT, HEL, and RdRP conserved domains and a five-gene array unique for closteroviruses (Dolja et al. “Molecular Biology and Evolution of Closteroviruses: Sophisticated Build-up of Large RNA Genomes,” Annual Rev. Photopathology 32: 261-85 (1994); Agranovsky “Principles of Molecular Organization, Expression, and Evolution of Closteroviruses: Over the Barriers,” Adv. in Virus Res.
  • GLRaV-2 is a closterovirus .
  • two putative papain-like proteases were identified and an autoproteolytic cleavage process was predicted.
  • the replication associated proteins consisting of MT, HEL, and RdRP conserved motifs were also identified, which were phylogenetically closely related to the replication associated proteins of other closteroviruses.
  • ORF1b Like all other closteroviruses, the expression of ORF1b is suspected to occur via a +1 ribosomal frameshift and the 3′ proximal ORFs are probably expressed via formation of a nested set of subgenomic RNAs.
  • GLRaV-2 is more closely related to monopartite closteroviruses BYV, BYSV, and CTV than to GLRaV-3 ( FIG. 7 ) (Ling et al., “Nucleotide Sequence of the 3′ Terminal Two-Thirds of the Grapevine Leafroll Associated Virus-3 Genome Reveals a Typical Monopartite Closterovirus,” J.
  • GLRaV-2 is the only closterovirus that matches with the genome organization of BYV, the type member of the genus Closterovirus .
  • genomic RNA of GLRaV-2 is about the same size as that of BYV; however, the transmission vector of GLRaV-2 is unknown.
  • the genome organization of GLRaV-2 is more closely related to the aphid transmissible closteroviruses (BYV and CTV) than to whitefly (LIYV) or mealybug transmissible closteroviruses (LChV and GLRaV-3).
  • BYV and CTV aphid transmissible closteroviruses
  • LIYV whitefly
  • LChV and GLRaV-3 mealybug transmissible closteroviruses
  • GLRaV-2 and GLRaV-3 (Ling et al., “Nucleotide Sequence of the 3′ Terminal Two-Thirds of the Grapevine Leafroll Associated Virus-3 Genome Reveals a Typical Monopartite Closterovirus,” J. General Virology 79(5): 1289-1301 (1998), which is hereby incorporated by reference) are the first grapevine leafroll associated closteroviruses that have been almost completely sequenced.
  • the above data clearly justify the inclusion of GLRaV-2 into the genus Closterovirus .
  • the information regarding the genome of GLRaV-2 would provide a better understanding of this and related GLRaVs, and add fundamental knowledge to the group of closteroviruses.
  • GLRaV-2 infected Vitis vinifera cv Pinot Noir grapevines originated from a vineyard in central New York was used as the virus isolate, from which the cp gene of GLRaV-2 was identified.
  • the sense primer CP-96F (SEQ. ID. No. 21) starts from the ATG initiation codon of the coat protein gene and the complementary primer CP-96R (SEQ. ID. No. 22) starts from 56 nucleotides downstream of the stop codon of the CP gene.
  • a Nco I restriction site (11 bp in SEQ. ID. No. 21 and 13 bp in SEQ. ID. No.
  • the coat protein gene of GLRaV-2 was amplified from dsRNA extracted from GLRaV-2 infected grapevine using reverse transcriptase polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcriptase polymerase chain reaction
  • the PCR-amplified CP product was purified from low melting temperature agarose gel, digested with Nco I and cloned into the same enzyme digested plant expression vector pEPT8 (shown at FIG. 11 ). After screening, the orientation of recombinant construct was checked by using the internal restriction site of the CP gene and directly sequencing the CP gene.
  • the plant expression cassette which consisted of a double cauliflower mosaic virus (CaMV) 35S-enhancer, a CaMV 35S-promoter, an alfalfa mosaic virus (ALMV) RNA4 5′ leader sequence, a coat protein gene of GLRaV-2 (CP-GLRaV-2), and a CaMV 35S 3′ untranslated region as a terminator, was cut using the EcoR I restriction enzyme, isolated from low melting point temperature agarose gel, and cloned into the same restriction enzyme treated binary vector pGA482GG or pGA482G (a derivative of pGA482 (An et al., “Binary Vectors,” in Plant Molecular Biology Manual, pp.
  • the resulting recombinants constructs are pGA482GG/EPT8CP-GLRaV2 (shown at FIG. 11A ), which contain both neomycin phosphotransferase (npt II) and P-glucuronidase (GUS) at the internal region of the T-DNA, and pGA482G/EPT8CP-GLRaV2 (shown at FIG. 11B ) without GUS.
  • NPT II-ELISA Double-antibody sandwich enzyme linked immuosorbent assay (DAS-ELISA) was used to detect the npt II enzyme with an NPT II-ELISA kit (5′ prime to 3′ prime, Inc., Boulder, Co.).
  • DAS-ELISA Double-antibody sandwich enzyme linked immuosorbent assay
  • Indirect ELISA Polyclonal antibodies to GLRaV-2, which were prepared from the coat protein expressed in E. coli , were used. Plates were coated with homogenized samples in extraction buffer (1:10, w/v) (phosphate buffered saline containing 0.05% Tween 20 and 2% polyvinyl pyrrolidone) and incubated overnight at 4° C. After washing with phosphate buffered saline containing 0.05% Tween 20 (PBST), the plates were blocked with blocking buffer (phosphate buffered saline containing 2% BSA) and incubated at room temperature for 1 hr. The anti-GLRaV-2 IgG was added at 2 ⁇ g/ml after washing with PBST.
  • Genomic DNA was extracted from leaves of putative transgenic and non-transgenic plants according to the method described by Cheung et al., “A Simple and Rapid DNA Microextraction Method for Plants, Animal, and Insect Suitable for RAPD and other PCR analysis,” PCR Methods and Applications 3: 69 (1996), which is incorporated herein by reference.
  • the extracted total DNA served as the template for PCR reaction.
  • the primers CP-96F and CP-96R SEQ. ID. Nos. 21 and 22, respectively
  • npt II 5′- and 3′-primers were used for PCR analysis.
  • PCR reaction was performed at the 94° C. ⁇ 3 min for one cycle, followed by 30 cycles of 94° C. ⁇ 1 min, 50° C. ⁇ 1 min, and 72° C. ⁇ 2:30 min with an additional extension at 72° C. for 10 min.
  • the PCR product was analyzed on agarose gel.
  • kanamycin resistant Nicotiana benthamiana lines (R 0 ) were obtained, of which the leaf samples were tested by NPT II enzyme activity. Among them, 37 lines were NPT II positive by ELISA, which took about 88.0% of total transformants. However, some of NPT II negative plants were obtained among these selected kanamycin resistant plants. All of the transgenic plants were self-pollinated in a greenhouse, and the seeds from these transgenic lines were germinated for further analysis.
  • GLRaV-2 CP in transgenic plants was detected by indirect ELISA prior to inoculation, and the results showed that GLRaV-2 CP gene expression was not detectable in all transgenic plants tested. This result was further confirmed with Western blot. Using the antibody to GLRaV-2, the production of the CP was not detected in the transgenic and nontransgenic control plants. However, a protein of expected size ( ⁇ 22 kDa) was detected in GLRaV-2 infected positive control plants. This result was consistent with the ELISA result. The presence of the CP gene of GLRaV-2 in transgenic plants was detected from total genomic DNA extracted from plants tissue by PCR analysis ( FIG. 12 ).
  • the DNA product of expected size (653 bp) was amplified from twenty tested transgenic lines, but not in non-transgenic plants. The result indicated that the CP gene of GLRaV-2 was present at these transgenic lines, which was also confirmed by Northern blot analysis.
  • R 1 and R2 transgenic Nicotiana benthamiana Plants Are Resistant to GLRaV-2
  • GLRaV-2 isolate 94/970 which was originally identified and transmitted from grapevine to Nicotiana benthamiana in South Africa (Goszczynski et al., “Detection of Two Strains of Grapevine Leafroll-Associated Virus 2,” Vitis 35: 133-35 (1996), which is incorporated herein by reference), was used as inoculum.
  • the CP gene of isolate 94/970 was sequenced; and it is identical to the CP gene used in construction. Nicotiana benthamiana is an experimental host of GLRaV-2. The infection on it produces chlorotic and occasional necrotic lesions followed by systemic vein clearing. The vein clearing results in vein necrosis. Eventually the infected plants died, starting from the top to the bottom.
  • Inoculum was prepared by grinding 1.0 g GLRaV-2 infected Nicotiana benthamiana leaf tissue in 5 ml of phosphate buffer (0.01 MK2HPO 4 , PH7.0). The tested plants were dusted with carborundum and rubbed with the prepared inoculum. Non-transformed plants were simultaneously inoculated as above. The plants were observed for symptom development every other day for 60 days after inoculation. Resistant R1 transgenic plants were carried on to R2 generation for further evaluation.
  • Transgenic progenies from 20 R 0 lines were initially screened for the resistance to GLRaV-2 followed by inoculation with GLRaV-2 isolate 94/970.
  • the seedlings of the transgenic plants (NPT II positive), and nontransformed control plants were inoculated with GLRaV-2.
  • the reaction of tested plants were divided into three types: highly susceptible (i.e. typical symptoms were observed two to four weeks postinoculation); tolerant (i.e. no symptom was developed in the early stage and typical symptoms was shown four to eight weeks postinoculation); and resistant (i.e. the plants remained asymptomatic eight weeks postinoculation). Based on the plant reaction, the resistant plants were obtained from fourteen different lines (listed in Table 1 below).
  • Table 2 below shows the symptom development in transgenic plants relative to non-transgenic control plants in the five selected lines in separate experiments.
  • Non-transgenic control plants were all infected two to four weeks after inoculation, which showed typical GLRaV-2 symptoms on Nicotiana benthamiana , including chlorotic and local lesions followed by systemic vein clearingand vein necrosis on the leaves.
  • Three of the tested lines (1, 4, and 19) showed some resistance that was manifested by either an absence or a delay in symptom development.
  • Two other lines, 12 and 13 developed symptoms at nearly the same time as the non-transformed control plants. From top to bottom, the leaves of infected plants gradually became yellow, wilted, and dried, and, eventually, the whole plants died. No matter when infection occurred, the eventual result was the same.
  • Line include transgenic lines and nontransformed control; No.: Number of transgenic and nontransformed plants tested; HS: highly susceptible; typical symptoms were observed two to four weeks after inoculation; T: tolerant, the symptoms were observed five to eight weeks postinoculation; and HR: plants remain without asymptoms after eight weeks inoculation.
  • ELISA was performed at 6 weeks postinoculation to test the GLRaV-2 replication in the plants. Presumably, the increased level of CP reflected virus replication. The result showed that the absorbance value in symptomatic plants reached (OD) 0.7 to 3.2, compared to (OD) 0.10-0.13 prior to inoculation. In contrast, GLRaV-2 was not detected in asymptomatic plants, of which the absorbance value was the same or nearly the same as that of healthy nontransformed control plants. The data confirmed that virus replicated in symptomatic plants, but not in asymptomatic plants. The replication of GLRaV-2 was suppressed in asymptomatic plants. This result implicated that another mechanism other than the CP-mediated resistance was probably involved.
  • R2 progenies derived from transgenic resistant plants of lines 1, 4, and 19 were generated and utilized to examine the stable transmission and whether resistance was maintained in R2 generation. These results are shown in Table 3 below. NPT II analysis revealed that R2 progeny were still segregating. The CP expression in R2 progeny was still undetectable. After inoculation, all the nontransgenic plants were infected and showed GLRaV-2 symptoms on the leaves after 24 days postinoculation. In contrast, the inoculated transgenic R 2 progeny showed different levels of resistance from those highly susceptible to highly resistant. The tolerant and resistant plants were manifested by a delay in symptom development and absence of symptoms, respectively.
  • RNA extracted from susceptible plants from R1 progeny of lines 5, 12, and 13 by Northern hybridization. There was little or no signal detected in the transgenic plants from R1 progeny of line 1, 4, and 19. This RNA is not present in nontransformed control plants.
  • the size of the hybridization signal was estimated to an approximately 0.9 kb nucleic acid, which was about the same as estimated ( FIG. 14 ). In lines of 1, 4, and 19, the steady state level of RNA expression was also low in R2 progeny. This data showed that susceptible plants from lines 12 and 13 had high mRNA level and all transgenic plants from lines 1, 4, and 19 had low mRNA level.
  • Plant materials The rootstock cultivars Couderc 3309 (3309C) ( V. riparia ⁇ V rupestris ), Vitis riparia ‘Gloire de adjoin’ (Gloire), Teleki 5C (5C) ( V. berlandieri ⁇ V. riparia ), Millardet et De Grasset 101-14 (101-14 MGT) ( V. riparia ⁇ V. rupestris ), and Richter 110 (10R) ( V. rupestris ⁇ V. berlandieri ) were utilized.
  • Initial embryogenic calli of Gloire were provided by Mozsar and Süle (Plant Protection Institute, Hungarian Academy of Science, Budapest). All other plant materials came from a vineyard at the New York State Agricultural Experiment Station, Geneva, N.Y.
  • Buds were removed from the clusters and surface sterilized in 70% ethanol for 1-2 min.
  • the buds (from the greenhouse and the field) were transferred to 1% sodium hypochlorite for 15 min, then rinsed three times in sterile, double-distilled water.
  • Anthers were excised aseptically from flower buds with the aid of a stereo microscope.
  • the pollen was crushed on a microscope slide under a coverslip with a drop of acetocarmine to observe the cytological stage. This was done to determine which stage was most favorable for callus induction.
  • Somatic embryogenesis and regeneration Anthers were plated under aseptic conditions at a density of 40 to 50 per 9 cm diameter Petri dish containing MSE. Plates were cultured at 28° C. in the dark. Callus was initiated, and, after 60 days, embryos were induced and were transferred to hormone-free HMG medium for differentiation. Torpedo stage embryos were then transferred from HMG to MGC medium to promote embryo germination. Cultures were maintained in the dark at 26-28° C. and transferred to fresh medium at 3-4 week intervals. Elongated embryos were transferred to rooting medium in baby food jars (5-8 embryos per jar). The embryos were grown in a tissue culture room at 25° C. with a daily 16 h photoperiod (76:mol. s) to induce shoot and root formation. After plants developed roots, they were transplanted to soil in the greenhouse.
  • Embryogenic calli were co-cultivated with the bacteria for 48 h in the dark at 28° C. Then, the plant material was washed in MS liquid plus cefotaxime (300 mg/ml) and carbenicillin (200 mg/ml) 2-3 times. To select transgenic embryos, the material was transferred to HMG medium containing either 20 or 40 mg/L kanamycin, 300 mg/L cefotaxime, and 200 mg/L carbenicillin. Alternatively, after co-cultivation, embryogenic calli were transferred to initiation MSE medium containing 25 mg/l kanamycin plus the same antibiotics listed above. All plant materials were incubated in continuous dark at 28° C.

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Abstract

The present invention relates to isolated proteins or polypeptides of grapevine leafroll virus (type 2). The encoding DNA molecules either alone in isolated form or in an expression system, a host cell, or a transgenic grape plant are also disclosed. Other aspects of the present invention relates to a method of imparting grapevine leafroll resistance to grape and tobacco plants by transforming them with the DNA molecules of the present invention, a method of imparting beet yellows virus resistance to a beet plant, a method of imparting tristeza virus resistance to a citrus plant, and a method of detecting the presence of a grapevine leafroll virus, such as GRLaV-2, in a sample.

Description

  • This application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/047,194, filed May 20, 1997.
  • This work was supported by the U.S. Department of Agriculture Cooperative Grant No. 58-2349-9-01. The U.S. Government may have certain rights in the invention.
    • FIELD OF THE INVENTION
  • The present invention relates to grapevine leafroll virus (type 2) proteins, DNA molecules encoding these proteins, and their uses.
    • BACKGROUND OF THE INVENTION
  • The world's most widely grown fruit crop, the grape (Vitis sp.), is cultivated on all continents except Antarctica. However, major grape production centers are in European countries (including Italy, Spain, and France), which constitute about 70% of the world grape production (Mullins et al., Biology of the Grapevine, Cambridge, U.K.:University Press (1992)). The United States, with 300,000 hectares of grapevines, is the eighth largest grape grower in the world. Although grapes have many uses, a major portion of grape production (˜80%) is used for wine production. Unlike cereal crops, most of the world's vineyards are planted with traditional grapevine cultivars, which have been perpetuated for centuries by vegetative propagation. Several important grapevine virus and virus-like diseases, such as grapevine leafroll, corky bark, and Rupestris stem pitting, are transmitted and spread through the use of infected vegetatively propagated materials. Thus, propagation of certified, virus-free materials is one of the most important disease control measures. Traditional breeding for disease resistance is difficult due to the highly heterozygous nature and outcrossing behavior of grapevines, and due to polygenic patterns of inheritance. Moreover, introduction of a new cultivar may be prohibited by custom or law. Recent biotechnology developments have made possible the introduction of special traits, such as disease resistance, into an established cultivar without altering its horticultural characteristics.
  • Many plant pathogens, such as fungi, bacteria, phytoplasmas, viruses, and nematodes can infect grapes, and the resultant diseases can cause substantial losses in production (Pearson et al., Compendium of Grape Diseases, American Phytopathological Society Press (1988)). Among these, viral diseases constitute a major hindrance to profitable growing of grapevines. About 34 viruses have been isolated and characterized from grapevines. The major virus diseases are grouped into: (1) the grapevine degeneration caused by the fanleaf nepovirus, other European nepoviruses, and American nepoviruses, (2) the leafroll complex, and (3) the rugose wood complex (Martelli, ed., Graft Transmissible Diseases of Grapevines, Handbook for Detection and Diagnosis, FAO, UN, Rome, Italy (1993)).
  • Of the major virus diseases, the grapevine leafroll complex is the most widely distributed throughout the world. According to Goheen (“Grape Leafroll,” in Frazier et al., eds., Virus Diseases of Small Fruits and Grapevines (A Handbook), University of California, Division of Agricultural Sciences, Berkeley, Calif., USA, pp. 209-212 (1970) (“Goheen (1970)”), grapevine leafroll-like disease was described as early as the 1850s in German and French literature. However, the viral nature of the disease was first demonstrated by Scheu (Scheu, “Die Rollkrankheit des Rebstockes (Leafroll of grapevine),” D. D. Weinbau 14: 222-358 (1935) (“Scheu (1935)”)). In 1946, Harmon and Snyder (Harmon et al., “Investigations on the Occurrence, Transmission, Spread and Effect of ‘White’ Fruit Colour in the Emperor Grape,” Proc. Am. Soc. Hort. Sci. 74: 190-194 (1946)) determined the viral nature of White Emperor disease in California. It was later proven by Goheen et al. (Goheen et al., “Leafroll (White Emperor Disease) of Grapes in California, Phytopathology, 48: 51-54 (1958) (“Goheen (1958)”)) that both leafroll and “White Emperor” diseases were the same, and only the name “leafroll” was retained.
  • Leafroll is a serious viral disease of grapes and occurs wherever grapes are grown. This wide distribution of the disease has come about through the propagation of diseased vines. It affects almost all cultivated and rootstock varieties of Vitis. Although the disease is not lethal, it causes yield losses and reduction of sugar content. Scheu estimated in 1936 that 80 percent of all grapevines planted in Germany were infected (Scheu, Mein Winzerbuch, Berlin:Reichsnahrstand-Verlags (1936)). In many California wine grape vineyards, the incidence of leafroll (based on a survey of field symptoms conducted in 1959) agrees with Scheu's initial observation in German vineyards (Goheen et al., “Studies of Grape Leafroll in California,” Amer. J. Enol. Vitic., 10: 78-84 (1959)). The current situation on leafroll disease does not seem to be any better (Goheen, “Diseases Caused by Viruses and Viruslike Agents,” The American Phytopathological Society, St. Paul, Minn.:APS Press, 1: 47-54 (1988) (“Goheen (1988)”). Goheen also estimated that the disease causes an annual loss of about 5-20 percent of the total grape production (Goheen (1970) and Goheen (1988)). The amount of sugar in individual berries of infected vines is only about ½ to ⅔ that of berries from noninfected vines (Goheen (1958)).
  • Symptoms of leafroll disease vary considerably depending upon the cultivar, environment, and time of the year. On red or dark-colored fruit varieties, the typical downward rolling and interveinal reddening of basal, mature leaves is the most prevalent in autumn; but not in spring or early summer. On light-colored fruit varieties however, symptoms are less conspicuous, usually with downward rolling accompanied by interveinal chlorosis. Moreover, many infected rootstock cultivars do not develop symptoms. In these cases, the disease is usually diagnosed with a woody indicator indexing assay using Vitis vivifera cv. Carbernet Franc (Goheen (1988)).
  • Ever since Scheu demonstrated that leafroll was graft transmissible, a virus etiology has been suspected (Scheu (1935)). Several virus particle types have been isolated from leafroll diseased vines. These include potyvirus-like (Tanne et al., “Purification and Characterization of a Virus Associated with the Grapevine Leafroll Disease,” Phytopathology 67: 442-447 (1977)), isometric virus-like (Castellano et al., “Virus-like Particles and Ultrastructural Modifications in the Phloem of Leafroll-affected Grapevines,” Vitis, 22: 23-39 (1983) (“Castellano (1983)”) and Namba et al., “A Small Spherical Virus Associated with the Ajinashika Disease of Koshu Grapevine, Ann. Phytopathol. Soc. Japan, 45: 70-73 (1979)), and closterovirus-like (Namba, “Grapevine Leafroll Virus, a Possible Member of Closteroviruses, Ann. Phytopathol. Soc. Japan, 45: 497-502 (1979)) particles. In recent years, however, long flexuous closteroviruses ranging from 1,400 to 2,200 nm have been most consistently associated with leafroll disease (FIG. 1) (Castellano (1983), Faoro et al., “Association of a Possible Closterovirus with Grapevine Leafroll in Northern Italy,” Riv. Patol. Veg., Ser IV, 17: 183-189 (1981), Gugerli et al., “L'enroulement de la vigne: mise en évidence de particules virales et développement d'une méthode immuno-enzymatique pour le diagnostic rapide (Grapevine Leafroll: Presence of Virus Particles and Development of an Immuno-enzyme method for Diagnosis and Detection),” Rev. Suisse Viticult. Arboricult, Hort., 16: 299-304 (1984) (“Gugerli (1984)”), Hu et al., “Characterization of Closterovirus-like Particles Associated with Grapevine Leafroll Disease,” J. Phptopathol., 128: 1-14 (1990) (“Hu (1990)”), Milne et al., “Closterovirus-like Particles of Two Types Associated with Diseased Grapevines,” Phytopathol. Z., 110: 360-368 (1984), Zee et al., “Cytopathology of Leafroll-diseased Grapevines and the Purification and Serology of Associated Closterovirus like Particles,” Phytopathology 77: 1427-1434 (1987) (“Zee (1987)”), and Zimmermann et al., “Characterization and Serological Detection of Four Closterovirus-like Particles Associated with Leafroll Disease on Grapevine,” J. Phytopathol., 130: 205-218 (1990) (“Zimmermann (1990)”)). These closteroviruses are referred to as grapevine leafroll associated viruses (“GLRaV”). At least six serologically distinct types of GLRaV's (GLRaV-1 to −6) have been detected from leafroll diseased vines (Table 1) (Boscia et al., “Nomenclature of Grapevine Leafroll-associated Putative Closteroviruses, Vitis, 34: 171-175 (1995) (“Boscia (1995)”) and (Martelli, “Leafroll,” pp. 37-44 in Martelli, ed., Graft Transmissible Diseases of Graipevines. Handbook for Detection and Diagnosis, FAO, Rome Italy, (1993) (“Martelli I”)). The first five of these were confirmed in the 10th. Meeting of the International Council for the Study of Virus and Virus Diseases of the Grapevine (“ICVG”) (Volos, Greece, 1990).
    TABLE 1
    Particle Coat
    length protein Mr
    Type (nm) (X103) Reference
    GLRaV-1 1,400-2,200 39 Gugerli (1984)
    GLRaV-2 1,400-1,800 26 Gugerli (1984)
    Zimmermann (1990)
    GLRaV-3 1,400-2,200 43 Zee (1987)
    GLRaV-4 1,400-2,200 36 Hu (1990)
    GLRaV-5 1,400-2,200 36 Zimmermann (1990)
    GLRaV-6 1,400-2,200 36 Gugerli (1993)
  • Through the use of monoclonal antibodies, however, the original GLRaV II described in Gugerli (1984) has been shown to be an apparent mixture of at least two components, IIa and IIb (Gugerli et al., “Grapevine Leafroll Associated Virus II Analyzed by Monoclonal Antibodies,” 11th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine, Montreux, Switzerland, pp. 23-24 (1993) (“Gugerli (1993)”)). Recent investigation with comparative serological assays (Boscia (1995)) demonstrated that the IIb component of cv. Chasselas 8/22 is the same as the GLRaV-2 isolate from France (Zimmermann (1990)) which also include the isolates of grapevine corky bark associated closteroviruses from Italy (GCBaV-BA) (Boscia (1995)) and from the United States (GCBaV-NY) (Namba et al., “Purification and Properties of Closterovirus-like Particles Associated with Grapevine Corky Bark Disease,” Phytopathology, 81: 964-970 (1991) (“Namba (1991)”)). The Ia component of cv. Chasselas 8/22 was given the provisional name of grapevine leafroll associated virus 6 (GLRaV-6). Furthermore, the antiserum to the CA-5 isolate of GLRaV-2 produced by Boscia et al. (Boscia et al., “Characterization of Grape Leafroll Associated Closterovirus (GLRaV) Serotype II and Comparison with GLRaV Serotype III,” Phytopathology, 80: 117 (1990)) was shown to contain antibodies to both GLRaV-2 and GLRaV-1, with a prevalence of the latter (Boscia (1995)).
  • Virions of GLRaV-2 are flexuous, filamentous particles about 1,400-1,800 nm in length (Gugerli et al., “L'enroulement de la Vigne: Mise en Evidence de Particles Virales et Development d'une Methode Immuno-enzymatique Pour le Diagnostic Rapide (Grapevine Leafroll: Presence of Virus Particles and Development of an Immuno-enzyme Method for Diagnosis and Detection),” Rev. Suisse Viticult. Arboricult. Horticult. 16: 299-304 (1984)). A double-stranded RNA (dsRNA) of about 15 kb was consistently isolated from GLRaV-2 infected tissues (Goszczynski et al., “Detection of Two Strains of Grapevine Leafroll-Associated Virus 2,” Vitis 35: 133-35 (1996)). The coat protein of GLRaV-2 is ca 22˜26 kDa (Zimmermann et al., “Characterization and Serological Detection of Four Closterovirus-like Particles Associated with Leafroll Disease on Grapevine,” J. Phytopathology 130: 205-18 (1990); Gugerli and Ramel, Extended abstracts: “Grapevine Leafroll Associated Virus II Analyzed by Monoclonal Antibodies,” 11th ICVG at Montreux, Switzerland, Gugerli, ed., Federal Agricultural Research Station of Changins, CH-1260 Nyon, Switzerland, p. 23-24 (1993); Boscia et al., “Nomenclature of Grapevine Leafroll-Associated Putative Closteroviruses,” Vitis 34: 171-75 (1995)), which is considerably smaller than other GLRaVs (35-43 kDa) (Zee et al., “Cytopathology of Leafroll-Diseased Grapevines and the Purification and Serology of Associated Closterovirus Like Particles,” Phytopathology 77: 1427-34 (1987); Hu et al., “Characterization of Closterovirus-Like Particles Associated with Grapevine Leafroll Disease,” J. of Phytopathology 128: 1-14 (1990); Ling et al., “The Coat Protein Gene of Grapevine Leafroll Associated Closterovirus-3: Cloning, Nucleotide Sequencing and Expression in Transgenic Plants,” Arch. of Virologv 142: 1101-16 (1997)). Although GLRaV-2 has been classififed as a member of the genus Closterovirus based on particle morphology and cytopathology (Martelli, Circular of ICTV-Plant Virus Subcommittee Study Group on Closterolike Viruses” (1996)), its molecular and biochernical properties are not well characterized.
  • In the closterovirus group, several viruses have recently been sequenced. The partial or complete genome sequences of beet yellows virus (BYV) (Agranovsky et al. “Nucleotide Sequence of the 3′-Terminal Half of Beet Yellows Closterovirus RNA Genome Unique Arrangement of Eight Virus Genes,” J. General Virology 72: 15-24 (1991); Agranovsky et al., “Beet Yellows Closterovirus: Complete Genome Structure and Identification of a Papain-like Tiol Protease,” Virology 198: 311-24 (1994)), beet yellow stunt virus (BYSV) (Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221: 199-207 (1996)), citrus tristeza virus (CTV) (Pappu et al., “Nucleotide Sequence and Organization of Eight 3′ Open Reading Frames of the Citrus Tristeza Closterovirus Genome,” Virology 199: 35-46 (1994); Karasev et al., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,” Virology 208: 511-20 (1995)), lettuce infectious yellows virus (LIYV) (Klaassen et al., “Partial Characterization of the Lettuce Infectious Yellows Virus Genomic RNAs, Identification of the Coat Protein Gene and Comparison of its Amino Acid Sequence With Those of Other Filamentous RNA Plant Viruses,” J. General Virology 75: 1525-33 (1994); Klaassen et al., “Genome Structure and Phylogenetic Analysis of Lettuce Infectious Yellows Virus, a Whitefly-Transmitted, Bipartite Closterovirus,” Virology 208: 99-110 (1995)), little cherry virus (LChV) (Keim and Jelkmann, “Genome Analysis of the 3′-Terminal Part of the Little Cherry Disease Associated dsRNA Reveals a Monopartite Clostero-Like Virus,” Arch. Virology 141: 1437-51 (1996); Jelkmann et al., “Complete Genome Structure and Phylogenetic Analysis of Little Cherry Virus, a Mealybug-Transmissible Closterovirus,” J. General Virology 78: 2067-71 (1997)), and GLRaV-3 (Ling et al., “Nucleotide Sequence of the 3′ Terminal Two-Thirds of the Grapevine Leafroll Associated Virus-3 Genome Reveals a Typical Monopartite Closterovirus,” J. Gen. Virology 79(5): 1289-1301 (1998)) revealed several common features of the closteroviruses, including the presence of HSP70 chaperone heat shock protein and a duplicate of the coat protein gene (Agranovsky “Principles of Molecular Organization, Expression, and Evolution of Closteroviruses: Over the Barriers,” Adv. in Virus Res. 47: 119-218 (1996); Dolja et al. “Molecular Biology and Evolution of Closteroviruses: Sophisticated Build-up of Large RNA Genomes,” Annual Rev. Photopathology 32: 261-85 (1994); Boyko et al.,. “Coat Protein Gene Duplication in a Filamentous RNA Virus of Plants,” Proc. Nat. Acad. Sci. USA 89: 9156-60 (1992)). Characterization of the genome organization of GLRaVs would provide molecular information on the serologically distinct closteroviruses that cause similar leafroll symptoms in grapevine.
  • Several shorter closteroviruses (particle length 800 nm long) have also been isolated from grapevines. One of these, called grapevine virus A (“GVA”) has also been found associated, though inconsistently, with the leafroll disease (Agran et al., “Occurrence of Grapevine Virus A (GVA) and Other Closteroviruses in Tunisian Grapevines Affected by Leafroll Disease,” Vitis, 29: 43-48 (1990), Conti, et al., “Closterovirus Associated with Leafroll and Stem Pitting in Grapevine,” Phytopathol. Mediterr., 24: 110-113 (1985), and Conti et al., “A Closterovirus from a Stem-pitting-diseased Grapevine,” Phytopathology, 70: 394-399 (1980)). The etiology of GVA is not really known; however, it appears to be more consistently associated with rugose wood sensu lato (Rosciglione at al., “Maladies de l'enroulement et du bois strie de la vigne: analyse microscopique et serologique (Leafroll and Stem Pitting of Grapevine: Microscopical and Serological Analysis),” Rev. Suisse Vitic Arboric. Hortic., 18: 207-211 (1986) (“Rosciglione (1986)”), and Zimmermann (1990)). Moreover, another short closterovirus (800 nm long) named grapevine virus B (“GVB”) has been isolated and characterized from corky bark-affected vines (Boscia et al., “Properties of a Filamentous Virus Isolated from Grapevines Affected by Corky Bark,” Arch. Virol., 130: 109-120 (1993) and Namba (1991)).
  • As suggested by Martelli I, leafroll symptoms may be induced by more than one virus or they may be simply a general plant physiological response to invasion by an array of phloem-inhabiting viruses. Evidence accumulated in the last 15 years strongly favors the idea that grapevine leafroll is induced by one (or a complex) of long closteroviruses (particle length 1,400 to 2,200 nm).
  • Grapevine leafroll is transmitted primarily by contaminated scions and rootstocks. However, under field conditions, several species of mealybugs have been shown to be the vector of leafroll (Engelbrecht et al., “Transmission of Grapevine Leafroll Disease and Associated Closteroviruses by the Vine Mealybug Planococcus-ficus,” Phytophylactica, 22: 341-346 (1990), Rosciglione, et al., “Transmission of Grapevine Leafroll Disease and an Associated Closterovirus to Healthy Grapevine by the Mealybug Planococcus ficus,” (Abstract), Phytoparasitica, 17: 63-63 (1989), and Tanne, “Evidence for the Transmission by Mealybugs to Healthy Grapevines of a Closter-like Particle Associated with Grapevine Leafroll Disease,” Phytoparasitica, 16: 288 (1988)). Natural spread of leafroll by insect vectors is rapid in various parts of the world. In New Zealand, observations of three vineyards showed that the number of infected vines nearly doubled in a single year (Jordan et al., “Spread of Grapevine Leafroll and its Associated Virus in New Zealand Vineyards,” 11th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine, Montreux, Switzerland, pp. 113-114 (1993)). One vineyard became 90% infected 5 years after GLRaV-3 was first observed. Prevalence of leafroll worldwide may increase as chemical control of mealybugs becomes more difficult due to the unavailability of effective insecticides.
  • In view of the serious risk grapevine leafroll virus poses to vineyards and the absence of an effective treatment of it, the need to prevent this affliction continues to exist. The present invention is directed to overcoming this deficiency in the art.
    • SUMMARY OF INVENTION
  • The present invention relates to an isolated protein or polypeptide corresponding to a protein or polypeptide of a grapevine leafroll virus (type 2). The encoding RNA and DNA molecules, in either isolated form or incorporated in an expression system, a host cell, a transgenic Vitis or citrus scion or rootstock cultivar, or a transgenic Nicotiana plant or beet plant are also disclosed.
  • Another aspect of the present invention relates to a method of imparting grapevine leafroll virus (type 2) resistance to Vitis scion or rootstock cultivars or Nicotiana plants by transforming them with a DNA molecule encoding the protein or polypeptide corresponding to a protein or polypeptide of a grapevine leafroll virus (type 2). Other aspects of the present invention relate to a method of imparting beet yellows virus resistance to beet plants and a method of imparting tristeza virus resistance to citrus scion or rootstock cultivars, both by transforming the plants or cultivars with a DNA molecule encoding the protein or polypeptide corresponding to a protein or polypeptide of a grapevine leafroll virus (type 2).
  • The present invention also relates to an antibody or binding portion thereof or probe which recognizes the protein or polypeptide.
  • Grapevine leafroll virus resistant transgenic variants of the current commercial grape cultivars and rootstocks allows for more complete control of the virus, while retaining the varietal characteristics of specific cultivars. Furthermore, these variants permit control of GLRaV-2 transmitted either by contaminated scions or rootstocks or by a presently uncharacterized insect vector. With respect to the latter mode of transmission, the present invention circumvents increased restriction of pesticide use which has made chemical control of insect infestation increasingly difficult. In this manner, the interests of the environment and the economics of grape cultivation and wine making are all furthered by the present invention.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGS. 1A and 1B are a comparison of a double-stranded RNA (dsRNA) profile (FIG. 1A) of GLRaV-2 and its Northern hybridization analysis (FIG. 1B). In FIG. 1A: lane M, lambda Hind III DNA marker; and lane 1, dsRNA pattern in 1% agarose gel stained with ethidium bromide. FIG. 1B is a northern hybridization of isolated high molecular weight dsRNA of GLRaV-2 with a probe prepared with 32P [α-dATP] labeled cDNA insert from GLRaV-2 specific cDNA clone TC-1. Lane 1, high molecular weight dsRNA of GLRaV-2. Lane 2, total RNA extracted from healthy grapevine.
  • FIG. 2 displays the genome organization of GLRaV-2 and its sequencing strategy. Boxes represent ORFs encoded by deduced amino acid sequences of GLRaV-2, numbered lines represent nucleotide coordinates, beginning from 5′-terminal of RNA in kilobases (kb). The lines below GLRaV-2 RNA genome represent the cDNA clones used to determine the nucleotide sequences.
  • FIG. 3A-3D are comparisons between ORF1a/ORF1b of GLRaV-2 and BYV. FIG. 3A-3D show the conserved domains of two papain-like proteases (P-PRO), methyltransferase (MT/MTR), helicase (HEL), and RNA-dependent RNA polymerase (RdRP), respectively. Exclamation marks indicate the predicted catalytic residues of the leader papain-like protease; slashes indicate the predicted cleavage sites. The conserved motifs of the MT, HEL, and RdRP domains are highlighted with overlines marked with respective letters. The alignment is constructed using the MegAlign program in DNASTAR.
  • FIGS. 4A and 4B are alignments of the nucleotide (FIG. 4A) and deduced amino acid (FIG. 4B) sequences of ORF1a/ORF1b overlapping region of GLRaV-2, BYV, BYSV, and CTV. Identical nucleotides and amino acids are shown in consensus. GLRaV-2 putative +1 frameshift site (TAGC) and its corresponding sites of BYV (TAGC) and BYSV (TAGC) and CTV (CGGC) at nucleotide and amino acid sequences are highlighted with underlines.
  • FIG. 5 is an alignment of the amino acid sequence of HSP70 protein of GLRaV-2 and BYV. The conserved motifs (A to H) are indicated with overlines and marked with respective letters. The alignment was conducted with the MegAlign program of DNASTAR.
  • FIG. 6A is a comparison of the coat protein (CP) and coat protein duplicate (CPd) of GLRaV-2 with other closteroviruses. The amino acid sequence of the GLRaV-2 CP and CPd are aligned with the CP and CPd of BYV, BYSV, and CTV. The conserved amino acid residues are in bold and the consensus sequences are indicated. Sequence alignment and phylogenetic tree were constructed by Clustal Method in the MegAlign Program of DNASTAR. FIG. 6B is a tentative phylogenetic tree of the CP and CPd of GLRaV-2 with BYV, BYSV, CTV, LIYV, LChV, and GLRaV-3. To facilitate the alignment, only the C-terminal 250 amino acids of CP and CPd of LIYV, LChV, and GLRaV-3 were used. The scale beneath the phylogenetic tree represents the distance between sequences. Units indicate the number of substitution events.
  • FIG. 7 is a comparison of the genome organization of GLRaV-2, BYV, BYSV, CTV, LIYV, LChV, and GLRaV-3. P—PRO, papain-like protease; MT/MTR, methyltransferase; HEL, helicase; RdRP, RNA-dependent RNA polymerase; HSP70, heat shock protein 70; CP, coat protein; CPd, coat protein duplicate.
  • FIG. 8 is a tentative phylogenetic tree showing the relationship of RdRP of GLRaV-2 with respect to BYV, BYSV, CTV, and LIYV. The phylogenetic tree was constructed using the Clustal method with the MegAlign program in DNASTAR.
  • FIG. 9 is an alignment of the amino acid sequence of HSP90 protein of GLRaV-2 with respect to other closteroviruses, BYS, BYSV, and CTV. The most conserved motifs (I to II) are indicated with the highlighted lines and marked with respective letters.
  • FIG. 10 is an alignment of the nucleotide sequence of 3′-terminal untranslated region of GLRaV-2 with respect to the closteroviruses BYV (Agranovsky et al., “Beet Yellows Closterovirus: Complete Genome Structure and Identification of a Papain-like Thiol Protease,” Virology 198: 311-24 (1994), which is hereby incorporated by reference), BYSV (Karasev et al., Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221: 199-207 (1996), which is hereby incorporated by reference), and CTV (Karasev et al., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,” Virology 208: 511-20 (1995), which is hereby incorporated by reference). The consensus sequences are shown, and the distance to the 3′-end is indicated. A complementary region capable of forming a “hair-pin” structure is underlined.
  • FIGS. 11A and 11B are genetic maps of the transformation vectors pGA482GG/EPT8CP-GLRaV-2 and pGA482G/EPT8CP-GLRaV-2, respectively. As shown in FIGS. 11A and 11B, the plant expression cassette (EPT8CP-GLRaV-2), which consists of a double cauliflower mosaic virus (CaMV) 35S-enhancer, a CaMV 35S-promoter, an alfalfa mosaic virus (ALMV) RNA4 5′ leader sequence, a coat protein gene of GLRaV-2 (CP-GLRaV-2), and a CaMV 35S 3′ untranslated region as a terminator, was cloned into the transformation vector by EcoR I restriction site. The CP of GLRaV-2 was cloned into the plant expression vector by Nco I restriction site.
  • FIG. 12 is a PCR analysis of DNA molecules extracted from the leaves of putative transgenic plants using both the CP gene of GLRaV-2 and NPT II gene specific primers. An ethidium bromide-stained gel shows a 720 bp amplified DNA fragment for NPT II gene, and a 653 bp DNA fragment for the entire coding sequence of the CP gene. Lane 1, φ 174/Hae III DNA Marker; lanes 2-6, transgenic plants from different lines; lane 7, the cp gene of GLRaV-2 of positive control; and lane 8, NPT II gene of positive control.
  • FIG. 13 is a comparison of resistant (right side 3 plants) and susceptible (left side 3 plants) transgenic Nicotiana benthamiana plants. Plants are shown 48 days after inoculation with GLRaV-2.
  • FIG. 14 is a northern blot analysis of transgenic Nicotiana benthamiana plants. An aliquot of 10 g of total RNA extracted from putative transgenic plants was denatured and loaded onto 1% agarose gel containing formaldehyde. The separated RNAs were transferred to Gene Screen Plus membrane and hybridized with a 32P-labeled DNA probe containing the 3′ one third CP gene sequence. Lanes 1, 3, and 4 represent nontransformed control plants without RNA expression. The remaining lanes represent transgenic plants from different lines: lanes 2, 14-17, and 22-27 represent plants with high RNA expression level which are susceptible to GLRaV-2; all other lanes represent plants with undetectable or low RNA expression level which are resistant to GLRaV-2.
    • DETAILED DESCRIPTION OF TH INVENTION
  • The present invention relates to isolated DNA molecules encoding for the proteins or polypeptides of a grapevine leafroll virus (type 2). A substantial portion of the grapevine leafroll virus (type-2) (“GLRaV-2”) genome has been sequenced. Within the genome are a plurality of open reading frames (“ORFs”) and a 3′ untranscribed region (“UTR”), each containing DNA molecules in accordance with the present invention. The DNA molecule which constitutes a substantial portion of the GLRaV-2 genome comprises the nucleotide sequence corresponding to SEQ. ID. No. 1 as follows:
    TAAACATTGC GAGAGAACCC CATTAGCGTC TCCGGGGTGA ACTTGGGAAG GTCTGCCGCC    60
    GCTCAGGTTA TTTATTTCGG CAGTTTCACG CAGCCCTTCG CGTTGTATCC GCGCCAAGAG   120
    AGCGCGATCG TAAAAACGCA ACTTCCACCG GTCAGTGTAG TGAAGGTGGA GTGCGTAGCT   180
    GCGGAGGTAG CTCCCGACAG GGGCGTGGTC GACAAGAAAC CTACGTCTGT TGGCGTTCCC   240
    CCGCAGCGCG GTGTGCTTTC TTTTCCGACG GTGGTTCGGA ACCGCGGCGA CGTGATAATC   300
    ACAGGGGTGG TGCATGAAGC CCTGAAGAAA ATTAAAGACG GGCTCTTACG CTTCCGCGTA   360
    GGCGGTGACA TGCGTTTTTC GAGATTTTTC TCATCGAACT ACGGCTGCAG ATTCGTCGCG   420
    AGCGTGCGTA CGAACACTAC AGTTTGGCTA AATTGCACGA AAGCGAGTGG TGAGAAATTC   480
    TCACTCGCCG CCGCGTGCAC GGCGGATTAC GTGGCGATGC TGCGTTATGT GTGTGGCGGG   540
    AAATTTCCAC TCGTCCTCAT GAGTAGAGTT ATTTACCCGG ATGGGCGCTG TTACTTGGCC   600
    CATATGAGGT ATTTGTGCGC CTTTTACTGT CGCCCGTTTA GAGAGTCGGA TTATGCCCTC   660
    GGAATGTGGC CTACGGTGGC GCGTCTCAGG GCATGCGTTG AGAAGAACTT CGGTGTCGAA   720
    GCTTGTGGCA TAGCTCTTCG TGGCTATTAC ACCTCTCGCA ATGTTTATCA CTGTGATTAT   780
    GACTCTGCTT ATGTAAAATA TTTTAGAAAC CTTTCCGGCC GCATTGGCGG TGGTTCGTTG   840
    GATCCGACAT CTTTAACCTC CGTAATAACG GTGAAGATTA GCGGTCTTCC AGGTGGTCTT   900
    CCTAAAAATA TAGCGTTTGG TGCCTTCCTG TGCGATATAC GTTACGTCGA ACCGGTAGAC   960
    TCGGGCGGCA TTCAATCGAG CGTTAAGACG AAACGTGAAG ATGCGCACCG AACCGTAGAG  1020
    GAACGGGCGG CCGGCGGATC CGTCGAGCAA CCGCGACAAA AGAGGATAGA TGAGAAAGGT  1080
    TGCGGCAGAG TTCCTAGTGG AGGTTTTTCG CATCTCCTGG TCGGCAACCT TAACGAAGTT  1140
    AGGAGGAAGG TAGCTGCCGG ACTTCTACGC TTTCGCGTTG GCGGTGATAT GGATTTTCAT  1200
    CGCTCGTTCT CCACCCAAGC GGGCCACCGC TTGCTGGTGT GGCGCCGCTC GAGCCGGAGC  1260
    GTGTGCCTTG AACTTTACTC ACCATCTAAA AACTTTTTGC GTTACGATGT CTTGGCCTGT  1320
    TCTGGAGACT ATGCAGCGAT GTTTTCTTTC GCGGCGGGCG GCCGTTTCCC TTTAGTTTTG  1380
    ATGACTAGAA TTAGATACCC GAACGGGTTT TGTTACTTGG CTCACTGCCG GTACGCGTGC  1440
    GCGTTTCTCT TAAGGGGTTT TGATCCGAAG CGTTTCGACA TCGGTGCTTT CCCCACCGCG  1500
    GCCAAGCTCA GAAACCGTAT GGTTTCGGAG CTTGGTGAAA GAAGTTTAGG TTTGAACTTG  1560
    TACGGCGCAT ATACGTCACG CGGCGTCTTT CACTGCGATT ATGACGCTAA GTTTATAAAG  1620
    GATTTGCGTC TTATGTCAGC AGTTATAGCT GGAAAGGACG GGGTGGAAGA GGTGGTACCT  1680
    TCTGACATAA CTCCTGCCAT GAAGCAGAAA ACGATCGAAG CCGTGTATGA TAGATTATAT  1740
    GGCGGCACTG ACTCGTTGCT GAAACTGAGC ATCGAGAAAG ACTTAATCGA TTTCAAAAAT  1800
    GACGTGCAGA GTTTGAAGAA AGATCGGCCG ATTGTCAAAG TGCCCTTTTA CATGTCGGAA  1860
    GCAACACAGA ATTCGGTGAC GCGTTTCTAC CCTCAGTTCG AACTTAAGTT TTCGCACTCC  1920
    TCGCATTCAG ATCATCCCGC CGCCGCCGCT TCTAGACTGC TGGAAAATGA AACGTTAGTG  1980
    CGCTTATGTG GTAATAGCGT TTCAGATATT GGAGGTTGTC CTCTTTTCCA TTTGCATTCC  2040
    AAGACGCAAA GACGGGTTCA CGTATGTAGG CCTGTGTTGG ATGGCAAGGA TGCGCAGCGT  2100
    CGCGTGGTGC GTGATTTGCA GTATTCCAAC GTGCGTTTGG GAGACGATGA TAAAATTTTG  2160
    GAAGGGCCAC GCAATATCGA CATTTGCCAC TATCCTCTGG GCGCGTGTGA CCACGAAAGT  2220
    AGTGCTATGA TGATGGTGCA GGTGTATGAC GCGTCCCTTT ATGAGATATG TGGCGCCATG  2280
    ATCAAGAAGA AAAGCCGCAT AACGTACTTA ACCATGGTCA CGCCCGGCGA GTTTCTTGAC  2340
    GGACGCGAAT GCGTCTACAT GGAGTCGTTA GACTGTGAGA TTGAAGTTGA TGTGCACGCG  2400
    GACGTCGTAA TGTACAAATT CGGTAGTTCT TGCTATTCGC ACAAGCTTTC AATCATCAAG  2460
    GACATCATGA CCACTCGGTA CTTGACACTA GGTGGTTTTC TATTCAGCGT GGAGATGTAT  2520
    GAGGTGCGTA TGGGCGTGAA TTACTTCAAG ATTACGAAGT CCGAAGTATC GCCTAGCATT  2580
    AGCTGCACCA AGCTCCTGAG ATACCGAAGA GCTAATAGTG ACGTGGTTAA AGTTAAACTT  2640
    CCACGTTTCG ATAAGAAACG TCGCATGTGT CTGCCTGGGT ATGACACCAT ATACCTAGAT  2700
    TCGAAGTTTG TGAGTCGCGT TTTCGATTAT GTCGTGTGTA ATTGCTCTGC CGTGAACTCA  2760
    AAAACTTTCG AGTGGGTGTG GAGTTTCATT AAGTCTAGTA AGTCGAGGGT GATTATTAGC  2820
    GGTAAAATAA TTCACAAGGA TGTGAATTTG GACCTCAAGT ACGTCGAGAG TTTCGCCGCG  2880
    GTTATGTTGG CCTCTGGCGT GCGCAGTAGA CTAGCGTCCG AGTACCTTGC TAAGAACCTT  2940
    AGTCATTTTT CGGGAGATTG CTCCTTTATT GAAGCCACGT CTTTCGTGTT GCGTGAGAAA  3000
    ATCAGAAACA TGACTCTGAA TTTTAACGAA AGACTTTTAC AGTTAGTGAA GCGCGTTGCC  3060
    TTTGCGACCT TGGACGTGAG TTTTCTAGAT TTAGATTCAA CTCTTGAATC AATAACTGAT  3120
    TTTGCCGAGT GTAAGGTAGC GATTGAACTC GACGAGTTGG GTTGCTTGAG AGCGGAGGCC  3180
    GAGAATGAAA AAATCAGGAA TCTGGCGGGA GATTCGATTG CGGCTAAACT CGCGAGCGAG  3240
    ATAGTGGTCG ATATTGACTC TAAGCCTTCA CCGAAGCAGG TGGGTAATTC GTCATCCGAA  3300
    AACGCCGATA AGCGGGAAGT TCAGAGGCCC GGTTTGCGTG GTGGTTCTAG AAACGGGGTT  3360
    GTTGGGGAGT TCCTTCACTT CGTCGTGGAT TCTGCCTTGC GTCTTTTCAA ATACGCGACG  3420
    GATCAACAAC GGATCAAGTC TTACGTGCGT TTCTTGGACT CGGCGGTCTC ATTCTTGGAT  3480
    TACAACTACG ATAATCTATC GTTTATACTG CGAGTGCTTT CGGAAGGTTA TTCGTGTATG  3540
    TTCGCGTTTT TGGCGAATCG CGGCGACTTA TCTAGTCGTG TCCGTAGCGC GGTGTGTGCT  3600
    GTGAAAGAAG TTGCTACCTC ATGCGCGAAC GCGAGCGTTT CTAAAGCCAA GGTTATGATT  3660
    ACCTTCGCAG CGGCCGTGTG TGCTATGATG TTTAATAGCT GCGGTTTTTC AGGCGACGGT  3720
    CGGGAGTATA AATCGTATAT AGATCGTTAC ACGCAAGTAT TGTTTGACAC TATCTTTTTT  3780
    GAGGACAGCA GTTACCTACC CATAGAAGTT CTGAGTTCGG CGATATGCGG TGCTATCGTC  3840
    ACACTTTTCT CCTCGGGCTC GTCCATAAGT TTAAACGCCT TCTTACTTCA AATTACCAAA  3900
    GGATTCTCCC TAGAGGTTGT CGTCCGGAAT GTTGTGCGAG TCACGCATGG TTTGAGCACC  3960
    ACAGCGACCG ACGGCGTCAT ACGTGGGGTT TTCTCCCAAA TTGTGTCTCA CTTACTTGTT  4020
    GGAAATACGG GTAATGTGGC TTACCAGTCA GCTTTCATTG CCGGGGTGGT GCCTCTTTTA  4080
    GTTAAAAAGT GTGTGAGCTT AATCTTCATC TTGCGTGAAG ATACTTATTC CGGTTTTATT  4140
    AAGCACGGAA TCAGTGAATT CTCTTTCCTT AGTAGTATTC TGAAGTTCTT GAAGGGTAAG  4200
    CTTGTGGACG AGTTGAAATC GATTATTCAA GGGGTTTTTG ATTCCAACAA GCACGTGTTT  4260
    AAAGAAGCTA CTCAGGAAGC GATTCGTACG ACGGTCATGC AAGTGCCTGT CGCTGTAGTG  4320
    GATGCCCTTA AGAGCGCCGC GGGAAAAATT TATAACAATT TTACTAGTCG ACGTACCTTT  4380
    GGTAAGGATG AAGGCTCCTC TAGCGACGGC GCATGTGAAG AGTATTTCTC ATGCGACGAA  4440
    GGTGAAGGTC CGGGTCTGAA AGGGGGTTCC AGCTATGGCT TCTCAATTTT AGCGTTCTTT  4500
    TCACGCATTA TGTGGGGAGC TCGTCGGCTT ATTGTTAAGG TGAAGCATGA GTGTTTTGGG  4560
    AAACTTTTTG AATTTCTATC GCTCAAGCTT CACGAATTCA GGACTCGCGT TTTTGGGAAG  4620
    AATAGAACGG ACGTGGGAGT TTACGATTTT TTGCCCACGG GCATCGTGGA AACGCTCTCA  4680
    TCGATAGAAG AGTGCGACCA AATTGAAGAA CTTCTCGGCG ACGACCTGAA AGGTGACAAG  4740
    GATGCTTCGT TGACCGATAT GAATTACTTT GAGTTCTCAG AAGACTTCTT AGCCTCTATC  4800
    GAGGAGCCGC CTTTCGCTGG ATTGCGAGGA GGTAGCAAGA ACATCGCGAT TTTGGCGATT  4860
    TTGGAATACG CGCATAATTT GTTTCGCATT GTCGCAAGCA AGTGTTCGAA ACGACCTTTA  4920
    TTTCTTGCTT TCGCCGAACT CTCAAGCGCC CTTATCGAGA AATTTAAGGA GGTTTTCCCT  4980
    CGTAAGAGCC AGCTCGTCGC TATCGTGCGC GAGTATACTC AGAGATTCCT CCGAAGTCGC  5040
    ATGCGTGCGT TGGGTTTGAA TAACGAGTTC GTGGTAAAAT CTTTCGCCGA TTTGCTACCC  5100
    GCATTAATGA AGCGGAAGGT TTCAGGTTCG TTCTTAGCTA GTGTTTATCG CCCACTTAGA  5160
    GGTTTCTCAT ATATGTGTGT TTCAGCGGAG CGACGTGAAA AGTTTTTTGC TCTCGTGTGT  5220
    TTAATCGGGT TAAGTCTCCC TTTCTTCGTG CGCATCGTAG GAGCGAAAGC GTGCGAAGAA  5280
    CTCGTGTCCT CAGCGCGTCG CTTTTATGAG CGTATTAAAA TTTTTCTAAG GCAGAAGTAT  5340
    GTCTCTCTTT CTAATTTCTT TTGTCACTTG TTTAGCTCTG ACGTTGATGA CAGTTCCGCA  5400
    TCTGCAGGGT TGAAAGGTGG TGCGTCGCGA ATGACGCTCT TCCACCTTCT GGTTCGCCTT  5460
    GCTAGTGCCC TCCTATCGTT AGGGTGGGAA GGGTTAAAGC TACTCTTATC GCACCACAAC  5520
    TTGTTATTTT TGTGTTTTGC ATTGGTTGAC GATGTGAACG TCCTTATCAA AGTTCTTGGG  5580
    GGTCTTTCTT TCTTTGTGCA ACCAATCTTT TCCTTGTTTG CGGCGATGCT TCTACAACCG  5640
    GACAGGTTTG TGGAGTATTC CGAGAAACTT GTTACAGCGT TTGAATTTTT CTTAAAATGT  5700
    TCGCCTCGCG CGCCTGCACT ACTCAAAGGG TTTTTTGAGT GCGTGGCGAA CAGCACTGTG  5760
    TCAAAAACCG TTCGAAGACT TCTTCGCTGT TTCGTGAAGA TGCTCAAACT TCGAAAAGGG  5820
    CGAGGGTTGC GTGCGGATGG TAGGGGTCTC CATCGGCAGA AAGCCGTACC CGTCATACCT  5880
    TCTAATCGGG TCGTGACCGA CGGGGTTGAA AGACTTTCGG TAAAGATGCA AGGAGTTGAA  5940
    GCGTTGCGTA CCGAATTGAG AATCTTAGAA GATTTAGATT CTGCCGTGAT CGAAAAACTC  6000
    AATAGACGCA GAAATCGTGA CACTAATGAC GACGAATTTA CGCGCCCTGC TCATGAGCAG  6060
    ATGCAAGAAG TCACCACTTT CTGTTCGAAA GCCAACTCTG CTGGTTTGGC CCTGGAAAGG  6120
    GCAGTGCTTG TGGAAGACGC TATAAAGTCG GAGAAACTTT CTAAGACGGT TAATGAGATG  6180
    GTGAGGAAAG GGAGTACCAC CAGCGAAGAA GTGGCCGTCG CTTTGTCGGA CGATGAAGCC  6240
    GTGGAAGAAA TCTCTGTTGC TGACGAGCGA GACGATTCGC CTAAGACAGT CAGGATAAGC  6300
    GAATACCTAA ATAGGTTAAA CTCAAGCTTC GAATTCCCGA AGCCTATTGT TGTGGACGAC  6360
    AACAAGGATA CCGGGGGTCT AACGAACGCC GTGAGGGAGT TTTATTATAT GCAAGAACTT  6420
    GCTCTTTTCG AAATCCACAG CAAACTGTGC ACCTACTACG ATCAACTGCG CATAGTCAAC  6480
    TTCGATCGTT CCGTAGCACC ATGCAGCGAA GATGCTCAGC TGTACGTACG GAAGAACGGC  6540
    TCAACGATAG TGCAGGGTAA AGAGGTACGT TTGCACATTA AGGATTTCCA CGATCACGAT  6600
    TTCCTGTTTG ACGGAAAAAT TTCTATTAAC AAGCGGCGGC GAGGCGGAAA TGTTTTATAT  6660
    CACGACAACC TCGCGTTCTT GGCGAGTAAT TTGTTCTTAG CCGGCTACCC CTTTTCAAGG  6720
    AGCTTCGTCT TCACGAATTC GTCGGTCGAT ATTCTCCTCT ACGAAGCTCC ACCCGGAGGT  6780
    GGTAAGACGA CGACGCTGAT TGACTCGTTC TTGAAGGTCT TCAAGAAAGG TGAGGTTTCC  6840
    ACCATGATCT TAACCGCCAA CAAAAGTTCG CAGGTTGAGA TCCTAAAGAA AGTGGAGAAG  6900
    GAAGTGTCTA ACATTGAATG CCAGAAACGT AAAGACAAAA GATCTCCGAA AAAGAGCATT  6960
    TACACCATCG ACGCTTATTT AATGCATCAC CGTGGTTGTG ATGCAGACGT TCTTTTCATC  7020
    GATGAGTGTT TCATGGTTCA TGCGGGTAGC GTACTAGCTT GCATTGAGTT CACGAGGTGT  7080
    CATAAAGTAA TGATCTTCGG GGATAGCCGG CAGATTCACT ACATTGAAAG GAACGAATTG  7140
    GACAAGTGTT TGTATGGGGA TCTCGACAGG TTCGTGGACC TGCAGTGTCG GGTTTATGGT  7200
    AATATTTCGT ACCGTTGTCC ATGGGATGTG TGCGCTTGGT TAAGCACAGT GTATGGCAAC  7260
    CTAATCGCCA CCGTGAAGGG TGAAAGCGAA GGTAAGAGCA GCATGCGCAT TAACGAAATT  7320
    AATTCAGTCG ACGATTTAGT CCCCGACGTG GGTTCCACGT TTCTGTGTAT GCTTCAGTCG  7380
    GAGAAGTTGG AAATCAGCAA GCACTTTATT CGCAAGGGTT TGACTAAACT TAACGTTCTA  7440
    ACGGTGCATG AGGCGCAAGG TGAGACGTAT GCGCGTGTGA ACCTTGTGCG ACTTAAGTTT  7500
    CAGGAGGATG AACCCTTTAA ATCTATCAGG CACATAACCG TCGCTCTTTC TCGTCACACC  7560
    GACAGCTTAA CTTATAACGT CTTAGCTGCT CGTCGAGGTG ACGCCACTTG CGATGCCATC  7620
    CAGAAGGCTG CGGAATTGGT GAACAAGTTT CGCGTTTTTC CTACATCTTT TGGTGGTAGT  7680
    GTTATCAATC TCAACGTGAA GAAGGACGTG GAAGATAACA GTAGGTGCAA GGCTTCGTCG  7740
    GCACCATTGA GCGTAATCAA CGACTTTTTG AACGAAGTTA ATCCCGGTAC TGCGGTGATT  7800
    GATTTTGGTG ATTTGTCCGC GGACTTCAGT ACTGGGCCTT TTGAGTGCGG TGCCAGCGGT  7860
    ATTGTGGTGC GGGACAACAT CTCCTCCAGC AACATCACTG ATCACGATAA GCAGCGTGTT  7920
    TAGCGTAGTT CGGTCGCAGG CGATTCCGCG TAGAAAACCT TCTCTACAAG AAAATTTGTA  7980
    TTCGTTTGAA GCGCGGAATT ATAACTTCTC GACTTGCGAC CGTAACACAT CTGCTTCAAT  8040
    GTTCGGAGAG GCTATGGCGA TGAACTGTCT TCGTCGTTGC TTCGACCTAG ATGCCTTTTC  8100
    GTCCCTGCGT GATGATGTGA TTAGTATCAC ACGTTCAGGC ATCGAACAAT GGCTGGAGAA  8160
    ACGTACTCCT AGTCAGATTA AAGCATTAAT GAAGGATGTT GAATCGCCTT TGGAAATTGA  8220
    CGATGAAATT TGTCGTTTTA AGTTGATGGT GAAGCGTGAC GCTAAGGTGA AGTTAGACTC  8280
    TTCTTGTTTA ACTAAACACA GCGCCGCTCA AAATATCATG TTTCATCGCA AGAGCATTAA  8340
    TGCTATCTTC TCTCCTATCT TTAATGAGGT GAAAAACCGA ATAATGTGCT GTCTTAAGCC  8400
    TAACATAAAG TTTTTTACGG AGATGACTAA CAGGGATTTT GCTTCTGTTG TCAGCAACAT  8460
    GCTTGGTGAC GACGATGTGT ACCATATAGG TGAAGTTGAT TTCTCAAAGT ACGACAAGTC  8520
    TCAAGATGCT TTCGTGAAGG CTTTTGAAGA AGTAATGTAT AAGGAACTCG GTGTTGATGA  8580
    AGAGTTGCTG GCTATCTGGA TGTGCGGCGA GCGGTTATCG ATAGCTAACA CTCTCGATGG  8640
    TCAGTTGTCC TTCACGATCG AGAATCAAAG GAAGTCGGGA GCTTCGAACA CTTGGATTGG  8700
    TAACTCTCTC GTCACTTTGG GTATTTTAAG TCTTTACTAC GACGTTAGAA ATTTCGAGGC  8760
    GTTGTACATC TCGGGCGATG ATTCTTTAAT TTTTTCTCGC AGCGAGATTT CGAATTATGC  8820
    CGACGACATA TGCACTGACA TGGGTTTTGA GACAAAATTT ATGTCCCCAA GTGTCCCGTA  8880
    CTTTTGTTCT AAATTTGTTG TTATGTGTGG TCATAAGACG TTTTTTGTTC CCGACCCGTA  8940
    CAAGCTTTTT GTCAAGTTGG GAGCAGTCAA AGAGGATGTT TCAATGGATT TCCTTTTCGA  9000
    GACTTTTACC TCCTTTAAAG ACTTAACCTC CGATTTTAAC GACGAGCGCT TAATTCAAAA  9060
    GCTCGCTGAA CTTGTGGCTT TAAAATATGA GGTTCAAACC GGCAACACCA CCTTGGCGTT  9120
    AAGTGTGATA CATTGTTTGC GTTCGAATTT CCTCTCGTTT AGCAAGTTAT ATCCTCGCGT  9180
    GAAGGGATGG CAGGTTTTTT ACACGTCGGT TAAGAAAGCG CTTCTCAAGA GTGGGTGTTC  9240
    TCTCTTCGAC AGTTTCATGA CCCCTTTTGG TCAGGCTGTC ATGGTTTGGG ATGATGAGTA  9300
    GCGCTAACTT GTGCGCAGTT TCTTTGTTCG TGACATACAC CTTGTGTGTC ACCGTGCGTT  9360
    TATAATGAAT CAGGTTTTGC AGTTTGAATG TTTGTTTCTG CTGAATCTCG CGGTTTTTGC  9420
    TGTGACTTTC ATTTTCATTC TTCTGGTCTT CCGCGTGATT AAGTCTTTTC GCCAGAAGGG  9480
    TCACGAAGCA CCTGTTCCCG TTGTTCGTGG CGGGGGTTTT TCAACCGTAG TGTAGTCAAA  9540
    AGACGCGCAT ATGGTAGTTT TCGGTTTGGA CTTTGGCACC ACATTCTCTA CGGTGTGTGT  9600
    GTACAAGGAT GGACGAGTTT TTTCATTCAA GCAGAATAAT TCGGCGTACA TCCCCACTTA  9660
    CCTCTATCTC TTCTCCGATT CTAACCACAT GACTTTTGGT TACGAGGCCG AATCACTGAT  9720
    GAGTAATCTG AAAGTTAAAG GTTCGTTTTA TAGAGATTTA AAACGTTGGG TGGGTTGCGA  9780
    TTCGAGTAAC CTCGACGCGT ACCTTGACCG TTTAAAACCT CATTACTCGG TCCGCTTGGT  9840
    TAAGATCGGC TCTGGCTTGA ACGAAACTGT TTCAATTGGA AACTTCGGGG GCACTGTTAA  9900
    GTCTGAGGCT CATCTGCCAG GGTTGATAGC TCTCTTTATT AAGGCTGTCA TTAGTTGCGC  9960
    GGAGGGCGCG TTTGCGTGCA CTTGCACCGG GGTTATTTGT TCAGTACCTG CCAATTATGA 10020
    TAGCGTTCAA AGGAATTTCA CTGATCAGTG TGTTTCACTC AGCGGTTATC AGTGCGTATA 10080
    TATGATCAAT GAACCTTCAG CGGCTGCGCT ATCTGCGTGT AATTCGATTG GAAAGAAGTC 10140
    CGCAAATTTG GCTGTTTACG ATTTCGGTGG TGGGACCTTC GACGTGTCTA TCATTTCATA 10200
    CCGCAACAAT ACTTTTGTTG TGCGAGCTTC TGGAGGCGAT CTAAATCTCG GTGGAAGGGA 10260
    TGTTGATCGT GCGTTTCTCA CGCACCTCTT CTCTTTAACA TCGCTGGAAC CTGACCTCAC 10320
    TTTGGATATC TCGAATCTGA AAGAATCTTT ATCAAAAACG GACGCAGAGA TAGTTTACAC 10380
    TTTGAGAGGT GTCGATGGAA GAAAAGAAGA CGTTAGAGTA AACAAAAACA TTCTTACGTC 10440
    GGTGATGCTC CCCTACGTGA ACAGAACGCT TAAGATATTA GAGTCAACCT TAAAATCGTA 10500
    TGCTAAGAGT ATGAATGAGA GTGCGCGAGT TAAGTGCGAT TTAGTGCTGA TAGGAGGATC 10560
    TTCATATCTT CCTGGCCTGG CAGACGTACT AACGAAGCAT CAGAGCGTTG ATCGTATCTT 10620
    AAGAGTTTCG GATCCTCGGG CTGCCGTGGC CGTCGGTTGC GCATTATATT CTTCATGCCT 10680
    CTCAGGATCT GGGGGGTTGC TACTGATCGA CTGTGCAGCT CACACTGTCG CTATAGCGGA 10740
    CAGAAGTTGT CATCAAATCA TTTGCGCTCC AGCGGGGGCA CCGATCCCCT TTTCAGGAAG 10800
    CATGCCTTTG TACTTAGCCA GGGTCAACAA GAACTCGCAG CGTGAAGTCG CCGTGTTTGA 10860
    AGGGGAGTAC GTTAAGTGCC CTAAGAACAG AAAGATCTGT GGAGCAAATA TAAGATTTTT 10920
    TGATATAGGA GTGACGGGTG ATTCGTACGC AGCCGTTACC TTCTATATGG ATTTCTCCAT 10980
    TTCAAGCGTA GGAGCCGTTT CATTCGTGGT GAGAGGTCCT GAGGGTAAGC AAGTGTCACT 11040
    CACTGGAACT CCAGCGTATA ACTTTTCGTC TGTGGCTCTC GGATCACGCA GTGTCCGAGA 11100
    ATTGCATATT AGTTTAAATA ATAAAGTTTT TCTCGGTTTG CTTCTACATA GAAAGGCGGA 11160
    TCGACGAATA CTTTTCACTA AGGATGAAGC GATTCGATAC GCCGATTCAA TTGATATCGC 11220
    GGATGTGCTA AAGGAATATA AAAGTTACGC GGCCAGTGCC TTACCACCAG ACGAGGATGT 11280
    CGAATTACTC CTGGGAAAGT CTGTTCAAAA AGTTTTACGG GGAAGCAGAC TGGAAGAAAT 11340
    ACCTCTCTAG GAGCATAGCA GCACACTCAA GTGAAATTAA AACTCTACCA GACATTCGAT 11400
    TGTACGGCGG TAGGGTTGTA AAGAAGTCCG AATTCGAATC AGCACTTCCT AATTCTTTTG 11460
    AACAGGAATT AGGACTGTTC ATACTGAGCG AAGGGGAAGT GGGATGGAGC AAATTATGCG 11520
    GAATAACGGT GGAAGAAGCA GCATACGATC TTACGAATCC CAAGGCTTAT AAATTCACTG 11580
    CCGAGAGATG TAGCCGGGAT GTAAAAGGTG AAGGACAAAA ATACTCTATG GAAGACGTGA 11640
    TGAATTTCAT GCGTTTATCA AATCTGGATG TTAACGACAA GATGCTGACG GAACAGTGTT 11700
    GGTCGCTGTC CAATTCATGC GGTGAATTGA TCAACCCAGA CGACAAAGGG CGATTCGTGG 11760
    CTCTCACCTT TAAGGACAGA GACACAGCTG ATGACACGGG TGCCGCCAAC GTGGAATGTC 11820
    GCGTGGGCGA CTATCTAGTT TACGCTATGT CCCTGTTTGA GCAGAGGACC CAAAAATCGC 11880
    AGTCTGGCAA CATCTCTCTG TACGAAAAGT ACTGTGAATA CATCAGGACC TACTTAGGGA 11940
    GTACAGACCT GTTCTTCACA GCGCCGGACA GGATTCCGTT ACTTACGGGC ATCCTATACG 12000
    ATTTTTGTAA GGAATACAAC GTTTTCTACT CGTCATATAA GAGAAACGTC GATAATTTCA 12060
    GATTCTTCTT GGCGAATTAT ATGCCTTTGA TATCTGACGT CTTTGTCTTC CAGTGGGTAA 12120
    AACCCGCGCC GGATGTTCGG CTGCTTTTTG AGTTAAGTGC AGCGGAACTA ACGCTGGAGG 12180
    TTCCCACACT GAGTTTGATA GATTCTCAAG TTGTGGTAGG TCATATCTTA AGATACGTAG 12240
    AATCCTACAC ATCAGATCCA GCCATCGACG CGTTAGAAGA CAAACTGGAA GCGATACTGA 12300
    AAAGTAGCAA TCCCCGTCTA TCGACAGCGC AACTATGGGT TGGTTTCTTT TGTTACTATG 12360
    GTGAGTTTCG TACGGCTCAA AGTAGAGTAG TGCAAAGACC AGGCGTATAC AAAACACCTG 12420
    ACTCAGTGGG TGGATTTGAA ATAAACATGA AAGATGTTGA GAAATTCTTC GATAAACTTC 12480
    AGAGAGAATT GCCTAATGTA TCTTTGCGGC GTCAGTTTAA CGGAGCTAGA GCGCATGAGG 12540
    CTTTCAAAAT ATTTAAAAAC GGAAATATAA GTTTCAGACC TATATCGCGT TTAAACGTGC 12600
    CTAGAGAGTT CTGGTATCTG AACATAGACT ACTTCAGGCA CGCGAATAGG TCCGGGTTAA 12660
    CCGAAGAAGA AATACTCATC CTAAACAACA TAAGCGTTGA TGTTAGGAAG TTATGCGCTG 12720
    AGAGAGCGTG CAATACCCTA CCTAGCGCGA AGCGCTTTAG TAAAAATCAT AAGAGTAATA 12780
    TACAATGATC ACGCGAAGAG CGGAGGATTA AAGACCCATT GGTAGTCCTG AAAGACACTT 12840
    TATATGAGTT CCAACACAAG CGTGCCGGTT GGGGGTCTCG AAGCACTCGA GACCTCGGGA 12900
    GTCGTGCTGA CCACGCGAAA GGAAGCGGTT GATAAGTTTT TTAATGAACT AAAAAACGAA 12960
    AATTACTCAT CAGTTGACAG CAGCCGATTA AGCGATTCGG AAGTAAAAGA AGTGTTAGAG 13020
    AAAAGTAAAG AAAGTTTCAA AAGCGAACTG GCCTCCACTG ACGAGCACTT CGTCTACCAC 13080
    ATTATATTTT TCTTAATCCG ATGTGCTAAG ATATCGACAA GTGAAAAGGT GAAGTACGTT 13140
    GGTAGTCATA CGTACGTGGT CGACGGAAAA ACGTACACCG TTCTTGACGC TTGGGTATTC 13200
    AACATGATGA AAAGTCTCAC GAAGAAGTAC AAACGAGTGA ATGGTCTGCG TGCGTTCTGT 13260
    TGCGCGTGCG AAGATCTATA TCTAACCGTC GCACCAATAA TGTCAGAACG CTTTAAGACT 13320
    AAAGCCGTAG GGATGAAAGG TTTGCCTGTT GGAAAGGAAT ACTTAGGCGC CGACTTTCTT 13380
    TCGGGAACTA GCAAACTGAT GAGCGATCAC GACAGGGCGG TCTCCATCGT TGCAGCGAAA 13440
    AACGCTGTCG ATCGTAGCGC TTTCACGGGT GGGGAGAGAA AGATAGTTAG TTTGTATGAT 13500
    CTAGGGAGGT ACTAAGCACG GTGTGCTATA GTGCGTGCTA TAATAATAAA CACTAGTGCT 13560
    TAAGTCGGGC AGAAGAAAAC GCTATGGAGT TGATGTCCGA CAGCAACCTT AGCAACCTGG 13620
    TGATAACCGA CGCCTCTAGT CTAAATGGTG TCGACAAGAA GCTTTTATCT GCTGAAGTTG 13680
    AAAAAATGTT GGTGCAGAAA GGGGCTCCTA ACGAGGGTAT AGAAGTGGTG TTCGGTCTAC 13740
    TCCTTTACGC ACTCGCGGCA AGAACCACGT CTCCTAAGGT TCAGCGCGCA GATTCAGACG 13800
    TTATATTTTC AAATAGTTTC GGAGAGAGGA ATGTGGTAGT AACAGAGGGT GACCTTAAGA 13860
    AGGTACTCGA CGGGTGTGCG CCTCTCACTA GGTTCACTAA TAAACTTAGA ACGTTCGGTC 13920
    GTACTTTCAC TGAGGCTTAC GTTGACTTTT GTATCGCGTA TAAGCACAAA TTACCCCAAC 13980
    TCAACGCCGC GGCGGAATTG GGGATTCCAG CTGAAGATTC GTACTTAGCT GCAGATTTTC 14040
    TGGGTACTTG CCCGAAGCTC TCTGAATTAC AGCAAAGTAG GAAGATGTTC GCGAGTATGT 14100
    ACGCTCTAAA AACTGAAGGT GGAGTGGTAA ATACACCAGT GAGCAATCTG CGTCAGCTAG 14160
    GTAGAAGGGA AGTTATGTAA TGGAAGATTA CGAAGAAAAA TCCGAATCGC TCATACTGCT 14220
    ACGCACGAAT CTGAACACTA TGCTTTTAGT GGTCAAGTCC GATGCTAGTG TAGAGCTGCC 14280
    TAAACTACTA ATTTGCGGTT ACTTACGAGT GTCAGGACGT GGGGAGGTGA CGTGTTGCAA 14340
    CCGTGAGGAA TTAACAAGAG ATTTTGAGGG CAATCATCAT ACGGTGATCC GTTCTAGAAT 14400
    CATACAATAT GACAGCGAGT CTGCTTTTGA GGAATTCAAC AACTCTGATT GCGTAGTGAA 14460
    GTTTTTCCTA GAGACTGGTA GTGTCTTTTG GTTTTTCCTT CGAAGTGAAA CCAAAGGTAG 14520
    AGCGGTGCGA CATTTGCGCA CCTTCTTCGA AGCTAACAAT TTCTTCTTTG GATCGCATTG 14580
    CGGTACCATG GAGTATTGTT TGAAGCAGGT ACTAACTGAA ACTGAATCTA TAATCGATTC 14640
    TTTTTGCGAA GAAAGAAATC GTTAAGATGA GGGTTATAGT GTCTCCTTAT GAAGCTGAAG 14700
    ACATTCTGAA AAGATCGACT GACATGTTAC GAAACATAGA CAGTGGGGTC TTGAGCACTA 14760
    AAGAATGTAT CAAGGCATTC TCGACGATAA CGCGAGACCT ACATTGTGCG AAGGCTTCCT 14820
    ACCAGTGGGG TGTTGACACT GGGTTATATC AGCGTAATTG CGCTGAAAAA CGTTTAATTG 14880
    ACACGGTGGA GTCAAACATA CGGTTGGCTC AACCTCTCGT GCGTGAAAAA GTGGCGGTTC 14940
    ATTTTTGTAA GGATGAACCA AAAGAGCTAG TAGCATTCAT CACGCGAAAG TACGTGGAAC 15000
    TCACGGGCGT GGGAGTGAGA GAAGCGGTGA AGAGGGAAAT GCGCTCTCTT ACCAAAACAG 15060
    TTTTAAATAA AATGTCTTTG GAAATGGCGT TTTACATGTC ACCACGAGCG TGGAAAAACG 15120
    CTGAATGGTT AGAACTAAAA TTTTCACCTG TGAAAATCTT TAGAGATCTG CTATTAGACG 15180
    TGGAAACGCT CAACGAATTG TGCGCCGAAG ATGATGTTCA CGTCGACAAA GTAAATGAGA 15240
    ATGGGGACGA AAATCACGAC CTCGAACTCC AAGACGAATG TTAAACATTG GTTAAGTTTA 15300
    ACGAAAATGA TTAGTAAATA ATAAATCGAA CGTGGGTGTA TCTACCTGAC GTATCAACTT 15360
    AAGCTGTTAC TGAGTAATTA AACCAACAAG TGTTGGTGTA ATGTGTATGT TGATGTAGAG 15420
    AAAAATCCGT TTGTAGAACG GTGTTTTTCT CTTCTTTATT TTTAAAAAAA AAATAAAAAA 15480
    AAAAAAAAAA AAGCGGCCGC 15500
  • Another DNA molecule of the present invention (GLRaV-2 ORF1a) includes nucleotides 4-7923 of SEQ. ID. No. 1 and is believed to code for a large, grapevine leafroll virus polyprotein containing the conserved domains characteristic of two papain-like proteases, a methyltransferase, and a helicase. This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 2 as follows:
    ACATTGCGAG AGAACCCCAT TAGCGTCTCC GGGGTGAACT TGGGAAGGTC TGCCGCCGCT   60
    CAGGTTATTT ATTTCGGCAG TTTCACGCAG CCCTTCGCGT TGTATCCGCG CCAAGAGAGC  120
    GCGATCGTAA AAACGCAACT TCCACCGGTC AGTGTAGTGA AGGTGGAGTG CGTAGCTGCG  180
    GAGGTAGCTC CCGACAGGGG CGTGGTCGAC AAGAAACCTA CGTCTGTTGG CGTTCCCCCG  240
    CAGCGCGGTG TGCTTTCTTT TCCGACGGTG GTTCGGAACC GCGGCGACGT GATAATCACA  300
    GGGGTGGTGC ATGAAGCCCT GAAGAAAATT AAAGACGGGC TCTTACGCTT CCGCGTAGGC  360
    GGTGACATGC GTTTTTCGAG ATTTTTCTCA TCGAACTACG GCTGCAGATT CGTCGCGAGC  420
    GTGCGTACGA ACACTACAGT TTGGCTAAAT TGCACGAAAG CGAGTGGTGA GAAATTCTCA  480
    CTCGCCGCCG CGTGCACGGC GGATTACGTG GCGATGCTGC GTTATGTGTG TGGCGGGAAA  540
    TTTCCACTCG TCCTCATGAG TAGAGTTATT TACCCGGATG GGCGCTGTTA CTTGGCCCAT  600
    ATGAGGTATT TGTGCGCCTT TTACTGTCGC CCGTTTAGAG AGTCGGATTA TGCCCTCGGA  660
    ATGTGGCCTA CGGTGGCGCG TCTCAGGGCA TGCGTTGAGA AGAACTTCGG TGTCGAAGCT  720
    TGTGGCATAG CTCTTCGTGG CTATTACACC TCTCGCAATG TTTATCACTG TGATTATGAC  780
    TCTGCTTATG TAAAATATTT TAGAAACCTT TCCGGCCGCA TTGGCGGTGG TTCGTTCGAT  840
    CCGACATCTT TAACCTCCGT AATAACGGTG AAGATTAGCG GTCTTCCAGG TGGTCTTCCT  900
    AAAAATATAG CGTTTGGTGC CTTCCTGTGC GATATACGTT ACGTCGAACC GGTAGACTCG  960
    GGCGGCATTC AATCGAGCGT TAAGACGAAA CGTGAAGATG CGCACCGAAC CGTAGAGGAA 1020
    CGGGCGGCCG GCGGATCCGT CGAGCAACCG CGACAAAAGA GGATAGATGA GAAAGGTTGC 1080
    GGCAGAGTTC CTAGTGGAGG TTTTTCGCAT CTCCTGGTCG GCAACCTTAA CGAAGTTAGG 1140
    AGGAAGGTAG CTGCCGGACT TCTACGCTTT CGCGTTGGCG GTGATATGGA TTTTCATCGC 1200
    TCGTTCTCCA CCCAAGCGGG CCACCGCTTG CTGGTGTGGC GCCGCTCGAG CCGGAGCGTG 1260
    TGCCTTGAAC TTTACTCACC ATCTAAAAAC TTTTTGCGTT ACGATGTCTT GCCCTGTTCT 1320
    GGAGACTATG CAGCGATGTT TTCTTTCGCG GCGGGCGGCC GTTTCCCTTT AGTTTTGATG 1380
    ACTAGAATTA GATACCCGAA CGGGTTTTGT TACTTGGCTC ACTGCCGGTA CGCGTGCGCG 1440
    TTTCTCTTAA GGGGTTTTGA TCCGAAGCGT TTCGACATCG GTGCTTTCCC CACCGCGGCC 1500
    AAGCTCAGAA ACCGTATGGT TTCGGAGCTT GGTGAAAGAA GTTTAGGTTT GAACTTGTAC 1560
    GGCGCATATA CGTCACGCGG CGTCTTTCAC TGCGATTATG ACGCTAAGTT TATAAAGGAT 1620
    TTGCGTCTTA TGTCAGCAGT TATAGCTGGA AAGGACGGGG TGGAAGAGGT GGTACCTTCT 1680
    GACATAACTC CTGCCATGAA GCAGAAAACG ATCGAAGCCG TGTATGATAG ATTATATGGC 1740
    GGCACTGACT CGTTGCTGAA ACTGAGCATC GAGAAAGACT TAATCGATTT CAAAAATGAC 1800
    GTGCAGAGTT TGAAGAAAGA TCGGCCGATT GTCAAAGTGC CCTTTTACAT GTCGGAAGCA 1860
    ACACAGAATT CGCTGACGCG TTTCTACCCT CAGTTCGAAC TTAAGTTTTC GCACTCCTCG 1920
    CATTCAGATC ATCCCGCCGC CGCCGCTTCT AGACTGCTGG AAAATGAAAC GTTAGTGCGC 1980
    TTATGTGGTA ATAGCGTTTC AGATATTGGA GGTTGTCCTC TTTTCCATTT GCATTCCAAG 2040
    ACGCAAAGAC GGGTTCACGT ATGTAGGCCT GTGTTGGATG GCAAGGATGC GCAGCGTCGC 2100
    GTGGTGCGTG ATTTGCAGTA TTCCAACGTG CGTTTGGGAG ACGATGATAA AATTTTGGAA 2160
    GGGCCACGCA ATATCGACAT TTGCCACTAT CCTCTGGGCG CGTGTGACCA CGAAAGTAGT 2220
    GCTATGATGA TGGTGCAGGT GTATGACGCG TCCCTTTATG AGATATGTGG CGCCATGATC 2280
    AAGAAGAAAA GCCGCATAAC GTACTTAACC ATGGTCACGC CCGGCGAGTT TCTTGACGGA 2340
    CGCGAATGCG TCTACATGGA GTCGTTAGAC TGTGAGATTG AAGTTGATGT GCACGCGGAC 2400
    GTCGTAATGT ACAAATTCGG TAGTTCTTGC TATTCGCACA AGCTTTCAAT CATCAAGGAC 2460
    ATCATGACCA CTCCGTACTT GACACTAGGT GGTTTTCTAT TCAGCGTGGA GATGTATGAG 2520
    GTGCGTATGG GCGTGAATTA CTTCAAGATT ACGAAGTCCG AAGTATCGCC TAGCATTAGC 2580
    TGCACCAAGC TCCTGAGATA CCGAAGAGCT AATAGTGACG TGGTTAAAGT TAAACTTCCA 2640
    CGTTTCGATA AGAAACGTCG CATGTGTCTG CCTGGGTATG ACACCATATA CCTAGATTCG 2700
    AAGTTTGTGA GTCGCGTTTT CGATTATGTC GTGTGTAATT GCTCTGCCGT GAACTCAAAA 2760
    ACTTTCGAGT GGGTGTGGAG TTTCATTAAG TCTAGTAAGT CGAGGGTGAT TATTAGCGGT 2820
    AAAATAATTC ACAAGGATGT GAATTTGGAC CTCAAGTACG TCGAGAGTTT CGCCGCGGTT 2880
    ATGTTGGCCT CTGGCGTGCG CAGTAGACTA GCGTCCGAGT ACCTTGCTAA GAACCTTAGT 2940
    CATTTTTCGG GAGATTGCTC CTTTATTGAA GCCACGTCTT TCGTGTTGCG TGAGAAAATC 3000
    AGAAACATGA CTCTGAATTT TAACGAAAGA CTTTTACAGT TAGTGAAGCG CGTTGCCTTT 3060
    GCGACCTTGG ACGTGAGTTT TCTAGATTTA GATTCAACTC TTGAATCAAT AACTGATTTT 3120
    GCCGAGTGTA AGGTAGCGAT TGAACTCGAC GAGTTGGGTT GCTTGAGAGC GGAGGCCGAG 3180
    AATGAAAAAA TCAGGAATCT GGCGGGAGAT TCGATTGCGG CTAAACTCGC GAGCGAGATA 3240
    GTGGTCGATA TTGACTCTAA GCCTTCACCG AAGCAGGTGG GTAATTCGTC ATCCGAAAAC 3300
    GCCGATAAGC GGGAAGTTCA GAGGCCCGGT TTGCGTGGTG GTTCTAGAAA CGGGGTTGTT 3360
    GGGGAGTTCC TTCACTTCGT CGTGGATTCT GCCTTGCGTC TTTTCAAATA CGCGACGGAT 3420
    CAACAACGGA TCAAGTCTTA CGTGCGTTTC TTGGACTCGG CGGTCTCATT CTTGGATTAC 3480
    AACTACGATA ATCTATCGTT TATACTGCGA GTGCTTTCGG AAGGTTATTC GTGTATGTTC 3540
    GCGTTTTTGG CGAATCGCGG CGACTTATCT AGTCGTGTCC GTAGCGCGGT GTGTGCTGTG 3600
    AAAGAAGTTG CTACCTCATG CGCGAACGCG AGCGTTTCTA AAGCCAAGGT TATGATTACC 3660
    TTCGCAGCGG CCGTGTGTGC TATGATGTTT AATAGCTGCG GTTTTTCAGG CGACGGTCGG 3720
    GAGTATAAAT CGTATATACA TCGTTACACG CAAGTATTGT TTGACACTAT CTTTTTTGAG 3780
    GACAGCAGTT ACCTACCCAT AGAAGTTCTG AGTTCGGCGA TATGCGGTGC TATCGTCACA 3840
    CTTTTCTCCT CGGGCTCGTC CATAAGTTTA AACGCCTTCT TACTTCAAAT TACCAAAGGA 3900
    TTCTCCCTAG AGGTTGTCGT CCGGAATGTT GTGCGAGTCA CGCATGGTTT GAGCACCACA 3960
    GCGACCGACG GCGTCATACG TGGGGTTTTC TCCCAAATTG TGTCTCACTT ACTTGTTGGA 4020
    AATACGGGTA ATGTGGCTTA CCAGTCAGCT TTCATTGCCG GGGTGGTGCC TCTTTTAGTT 4080
    AAAAAGTGTG TGAGCTTAAT CTTCATCTTG CGTGAAGATA CTTATTCCGG TTTTATTAAG 4140
    CACGGAATCA GTGAATTCTC TTTCCTTAGT AGTATTCTGA AGTTCTTGAA GGGTAAGCTT 4200
    GTGGACGAGT TGAAATCGAT TATTCAAGGG GTTTTTGATT CCAACAAGCA CGTGTTTAAA 4260
    GAAGCTACTC AGGAAGCGAT TCGTACGACG GTCATGCAAG TGCCTGTCGC TGTAGTGGAT 4320
    GCCCTTAAGA GCGCCGGGGG AAAAATTTAT AACAATTTTA CTAGTCGACG TACCTTTGGT 4380
    AAGGATGAAG GCTCGTCTAG CGACGGCGCA TGTGAAGAGT ATTTCTCATG CGACGAAGGT 4440
    GAAGGTCCGG GTCTGAAAGG GGGTTCCAGC TATGGCTTCT CAATTTTAGC GTTCTTTTCA 4500
    CGCATTATGT GGGGAGCTCG TCGGCTTATT GTTAAGGTGA AGCATGAGTG TTTTGGGAAA 4560
    CTTTTTGAAT TTCTATCGCT GAAGCTTCAC GAATTCAGGA CTCGCGTTTT TGGGAAGAAT 4620
    AGAACGGACG TGGGAGTTTA CGATTTTTTG CCCACGGGCA TCGTGGAAAC GCTCTCATCG 4680
    ATAGAAGAGT GCGACCAAAT TGAAGAACTT CTCGGCGACG ACCTGAAAGG TGACAAGGAT 4740
    GCTTCGTTGA CCGATATGAA TTACTTTGAG TTCTCAGAAG ACTTCTTAGC CTCTATCGAG 4800
    GAGCCGCCTT TCGCTGGATT GCGAGGAGGT AGCAAGAACA TCGCGATTTT GGCGATTTTG 4860
    GAATACGCGG ATAATTTGTT TCGCATTGTC GCAAGCAAGT GTTCGAAACG ACCTTTATTT 4920
    CTTGCTTTCG CCGAACTCTC AAGCGCCCTT ATCGAGAAAT TTAAGGAGGT TTTCCCTCGT 4980
    AAGAGCCAGC TCGTCGCTAT CGTGCGCGAG TATACTCAGA GATTCCTCCG AAGTCGCATG 5040
    CGTGCGTTGG GTTTGAATAA CGAGTTCGTG GTAAAATCTT TCGCCGATTT GCTACCCGCA 5100
    TTAATGAAGC GGAAGGTTTC AGGTTCGTTC TTAGCTAGTG TTTATCGCCC ACTTAGAGGT 5160
    TTCTCATATA TGTGTGTTTC AGCGGAGCGA CGTGAAAAGT TTTTTGCTCT CGTGTGTTTA 5220
    ATCGGGTTAA GTCTCCCTTT CTTCGTGCGC ATCGTAGGAG CGAAAGCGTG CGAAGAACTC 5280
    GTGTCCTCAG CGCGTCGCTT TTATGAGCGT ATTAAAATTT TTCTAAGGCA GAAGTATGTC 5340
    TCTCTTTCTA ATTTCTTTTG TCACTTGTTT AGCTCTGACG TTGATGACAG TTCCGCATCT 5400
    GCAGGGTTGA AAGGTGGTGC GTCGCGAATG ACGCTCTTCC ACCTTCTGGT TCGCCTTGCT 5460
    AGTGCCCTCC TATCGTTAGG GTGGGAAGGG TTAAAGCTAC TCTTATCGCA CCACAACTTG 5520
    TTATTTTTGT GTTTTGCATT GGTTGACGAT GTGAACGTCC TTATCAAAGT TCTTGGGGGT 5580
    CTTTCTTTCT TTGTGCAACC AATCTTTTCC TTGTTTGCGG CGATGCTTCT ACAACCGGAC 5640
    AGGTTTGTGG AGTATTCCGA GAAACTTGTT ACAGCGTTTG AATTTTTCTT AAAATGTTCG 5700
    CCTCGCGCGC CTGCACTACT CAAAGGGTTT TTTGAGTGCG TGGCGAACAG CACTGTGTCA 5760
    AAAACCGTTC GAAGACTTCT TCGCTGTTTC GTGAAGATGC TCAAACTTCG AAAAGGGCGA 5820
    GGGTTGCGTG CGGATGGTAG GGGTCTCCAT CGGCAGAAAG CCGTACCCGT CATACCTTCT 5880
    AATCGGGTCG TGACCGACGG GGTTGAAAGA CTTTCGGTAA AGATGCAAGG AGTTGAAGCG 5940
    TTGCGTACCG AATTGAGAAT CTTAGAAGAT TTAGATTCTG CCGTGATCGA AAAACTCAAT 6000
    AGACGCAGAA ATCGTGACAC TAATGACGAC GAATTTACGC GCCCTGCTCA TGAGCAGATG 6060
    CAAGAAGTCA CCACTTTCTG TTCGAAAGCC AACTCTGCTG GTTTGGCCCT GGAAAGGGCA 6120
    GTGCTTGTGG AAGACGCTAT AAAGTCGGAG AAACTTTCTA AGACGGTTAA TGAGATGGTG 6180
    AGGAAAGGGA GTACCACCAG CGAAGAAGTG GCCGTCGCTT TGTCGGACGA TGAAGCCGTG 6240
    GAAGAAATCT CTGTTGCTGA CGAGCGAGAC GATTCGCCTA AGACAGTCAG GATAAGCGAA 6300
    TACCTAAATA GGTTAAACTC AAGCTTCGAA TTCCCGAAGC CTATTGTTGT GGACGACAAC 6360
    AAGGATACCG GGGGTCTAAC GAACGCCGTG AGGGAGTTTT ATTATATGCA AGAACTTGCT 6420
    CTTTTCGAAA TCCACAGCAA ACTGTGCACC TACTACGATC AACTGCGCAT AGTCAACTTC 6480
    GATCGTTCCG TAGCACCATG CAGCGAAGAT GCTCAGCTGT ACGTACGGAA GAACGGCTCA 6540
    ACGATAGTGC AGGGTAAAGA GGTACGTTTG CACATTAAGG ATTTCCACGA TCACGATTTC 6600
    CTGTTTGACG GAAAAATTTC TATTAACAAG CGGCGGCGAG GCGGAAATGT TTTATATCAC 6660
    GACAACCTCG CGTTCTTGGC GAGTAATTTG TTCTTAGCCG GCTACCCCTT TTCAAGGAGC 6720
    TTCGTCTTCA CGAATTCGTC GGTCGATATT CTCCTCTACG AAGCTCCACC CGGAGGTGGT 6780
    AAGACGACGA CGCTGATTGA CTCGTTCTTG AAGGTCTTCA AGAAAGGTGA GGTTTCCACC 6840
    ATGATCTTAA CCGCCAACAA AAGTTCGCAG GTTGAGATCC TAAAGAAAGT GGAGAAGGAA 6900
    GTGTCTAACA TTGAATGCCA GAAACGTAAA GACAAAAGAT CTCCGAAAAA GAGCATTTAC 6960
    ACCATCGACG CTTATTTAAT GCATCACCGT GGTTGTGATG CAGACGTTCT TTTCATCGAT 7020
    GAGTGTTTCA TGGTTCATGC GGGTAGCGTA CTAGCTTGCA TTGAGTTCAC GAGGTGTCAT 7080
    AAAGTAATGA TCTTCGGGGA TAGCCGGCAG ATTCACTACA TTGAAAGGAA CGAATTGGAC 7140
    AAGTGTTTGT ATGGGGATCT CGACAGGTTC GTGGACCTGC AGTGTCGGGT TTATGGTAAT 7200
    ATTTCGTACC GTTGTCCATG GGATGTGTGC GCTTGGTTAA GCACAGTGTA TGGCAACCTA 7260
    ATCGCCACCG TGAAGGGTGA AAGCGAAGGT AAGAGCAGCA TGCGCATTAA CGAAATTAAT 7320
    TCAGTCGACG ATTTAGTCCC CGACGTGGGT TCCACGTTTC TGTGTATGCT TCAGTCGGAG 7380
    AAGTTGGAAA TCAGCAAGCA CTTTATTCGC AAGGGTTTGA CTAAACTTAA CGTTCTAACG 7440
    GTGCATGAGG CGCAAGGTGA GACGTATGCG CGTGTGAACC TTGTGCGACT TAAGTTTCAG 7500
    GAGGATGAAC CCTTTAAATC TATCAGGCAC ATAACCGTCG CTCTTTCTCG TCACACCGAC 7560
    AGCTTAACTT ATAACGTCTT AGCTGCTCGT CGAGGTGACG CCACTTGCGA TGCCATCCAG 7620
    AAGGCTGCGG AATTGGTGAA CAAGTTTCGC GTTTTTCCTA CATCTTTTGG TGGTAGTGTT 7680
    ATCAATCTCA ACGTGAAGAA GGACGTGGAA GATAACAGTA GGTGCAAGGC TTCGTCGGCA 7740
    CCATTGAGCG TAATCAACGA CTTTTTGAAC GAAGTTAATC CCGGTACTGC GGTGATTGAT 7800
    TTTGGTGATT TGTCCGCGGA CTTCAGTACT GGGCCTTTTG AGTGCGGTGC CAGCGGTATT 7860
    GTGGTGCGGG ACAACATCTC CTCCAGCAAC ATCACTGATC ACGATAAGCA GCGTGTTTAG 7920
  • The large polyprotein (papain-like proteases, methyltransferase, and helicase) has an amino acid sequence corresponding to SEQ. ID. No. 3 as follows:
    Thr Leu Arg Glu Asn Pro Ile Ser Val Ser Gly Val Asn Leu Gly Arg
    1               5                   10                  15
    Ser Ala Ala Ala Gln Val Ile Tyr Phe Gly Ser Phe Thr Gln Pro Phe
                20                  25                  30
    Ala Leu Tyr Pro Arg Gln Glu Ser Ala Ile Val Lys Thr Gln Leu Pro
            35                  40                  45
    Pro Val Ser Val Val Lys Val Glu Cys Val Ala Ala Glu Val Ala Pro
        50                  55                  60
    Asp Arg Gly Val Val Asp Lys Lys Pro Thr Ser Val Gly Val Pro Pro
    65                  70                  75                  80
    Gln Arg Gly Val Leu Ser Phe Pro Thr Val Val Arg Asn Arg Gly Asp
                    85                  90                  95
    Val Ile Ile Thr Gly Val Val His Glu Ala Leu Lys Lys Ile Lys Asp
                100                 105                 110
    Gly Leu Leu Arg Phe Arg Val Gly Gly Asp Met Arg Phe Ser Arg Phe
            115                 120                 125
    Phe Ser Ser Asn Tyr Gly Cys Arg Phe Val Ala Ser Val Arg Thr Asn
        130                 135                 140
    Thr Thr Val Trp Leu Asn Cys Thr Lys Ala Ser Gly Glu Lys Phe Ser
    145                 150                 155                 160
    Leu Ala Ala Ala Cys Thr Ala Asp Tyr Val Ala Met Leu Arg Tyr Val
                    165                 170                 175
    Cys Gly Gly Lys Phe Pro Leu Val Leu Met Ser Arg Val Ile Tyr Pro
                180                 185                 190
    Asp Gly Arg Cys Tyr Leu Ala His Met Arg Tyr Leu Cys Ala Phe Tyr
            195                 200                 205
    Cys Arg Pro Phe Arg Glu Ser Asp Tyr Ala Leu Gly Met Trp Pro Thr
        210                 215                 220
    Val Ala Arg Leu Arg Ala Cys Val Glu Lys Asn Phe Gly Val Glu Ala
    225                 230                 235                 240
    Cys Gly Ile Ala Leu Arg Gly Tyr Tyr Thr Ser Arg Asn Val Tyr His
                    245                 250                 255
    Cys Asp Tyr Asp Ser Ala Tyr Val Lys Tyr Phe Arg Asn Leu Ser Gly
                260                 265                 270
    Arg Ile Gly Gly Gly Ser Phe Asp Pro Thr Ser Leu Thr Ser Val Ile
            275                 280                 285
    Thr Val Lys Ile Ser Gly Leu Pro Gly Gly Leu Pro Lys Asn Ile Ala
        290                 295                 300
    Phe Gly Ala Phe Leu Cys Asp Ile Arg Tyr Val Glu Pro Val Asp Ser
    305                 310                 315                 320
    Gly Gly Ile Gln Ser Ser Val Lys Thr Lys Arg Glu Asp Ala His Arg
                    325                 330                 335
    Thr Val Glu Glu Arg Ala Ala Gly Gly Ser Val Glu Gln Pro Arg Gln
                340                 345                 350
    Lys Arg Ile Asp Glu Lys Gly Cys Gly Arg Val Pro Ser Gly Gly Phe
            355                 360                 365
    Ser His Leu Leu Val Gly Asn Leu Asn Glu Val Arg Arg Lys Val Ala
        370                 375                 380
    Ala Gly Leu Leu Arg Phe Arg Val Gly Gly Asp Met Asp Phe His Arg
    385                 390                 395                 400
    Ser Phe Ser Thr Gln Ala Gly His Arg Leu Leu Val Trp Arg Arg Ser
                    405                 410                 415
    Ser Arg Ser Val Cys Leu Glu Leu Tyr Ser Pro Ser Lys Asn Phe Leu
                420                 425                 430
    Arg Tyr Asp Val Leu Pro Cys Ser Gly Asp Tyr Ala Ala Met Phe Ser
            435                 440                 445
    Phe Ala Ala Gly Gly Arg Phe Pro Leu Val Leu Met Thr Arg Ile Arg
        450                 455                 460
    Tyr Pro Asn Gly Phe Cys Tyr Leu Ala His Cys Arg Tyr Ala Cys Ala
    465                 470                 475                 480
    Phe Leu Leu Arg Gly Phe Asp Pro Lys Arg Phe Asp Ile Gly Ala Phe
                    485                 490                 495
    Pro Thr Ala Ala Lys Leu Arg Asn Arg Met Val Ser Glu Leu Gly Glu
                500                 505                 510
    Arg Ser Leu Gly Leu Asn Leu Tyr Gly Ala Tyr Thr Ser Arg Gly Val
            515                 520                 525
    Phe His Cys Asp Tyr Asp Ala Lys Phe Ile Lys Asp Leu Arg Leu Met
        530                 535                 540
    Ser Ala Val Ile Ala Gly Lys Asp Gly Val Glu Glu Val Val Pro Ser
    545                 550                 555                 560
    Asp Ile Thr Pro Ala Met Lys Gln Lys Thr Ile Glu Ala Val Tyr Asp
                    565                 570                 575
    Arg Leu Tyr Gly Gly Thr Asp Ser Leu Leu Lys Leu Ser Ile Glu Lys
                580                 585                 590
    Asp Leu Ile Asp Phe Lys Asn Asp Val Gln Ser Leu Lys Lys Asp Arg
            595                 600                 605
    Pro Ile Val Lys Val Pro Phe Tyr Met Ser Glu Ala Thr Gln Asn Ser
        610                 615                 620
    Leu Thr Arg Phe Tyr Pro Gln Phe Glu Leu Lys Phe Ser His Ser Ser
    625                 630                 635                 640
    His Ser Asp His Pro Ala Ala Ala Ala Ser Arg Leu Leu Glu Asn Glu
                    645                 650                 655
    Thr Leu Val Arg Leu Cys Gly Asn Ser Val Ser Asp Ile Gly Gly Cys
                660                 665                 670
    Pro Leu Phe His Leu His Ser Lys Thr Gln Arg Arg Val His Val Cys
            675                 680                 685
    Arg Pro Val Leu Asp Gly Lys Asp Ala Gln Arg Arg Val Val Arg Asp
        690                 695                 700
    Leu Gln Tyr Ser Asn Val Arg Leu Gly Asp Asp Asp Lys Ile Leu Glu
    705                 710                 715                 720
    Gly Pro Arg Asn Ile Asp Ile Cys His Tyr Pro Leu Gly Ala Cys Asp
                    725                 730                 735
    His Glu Ser Ser Ala Met Met Met Val Gln Val Tyr Asp Ala Ser Leu
                740                 745                 750
    Tyr Glu Ile Cys Gly Ala Met Ile Lys Lys Lys Ser Arg Ile Thr Tyr
            755                 760                 765
    Leu Thr Met Val Thr Pro Gly Glu Phe Leu Asp Gly Arg Glu Cys Val
        770                 775                 780
    Tyr Met Glu Ser Leu Asp Cys Glu Ile Glu Val Asp Val His Ala Asp
    785                 790                 795                 800
    Val Val Met Tyr Lys Phe Gly Ser Ser Cys Tyr Ser His Lys Leu Ser
                    805                 810                 815
    Ile Ile Lys Asp Ile Met Thr Thr Pro Tyr Leu Thr Leu Gly Gly Phe
                820                 825                 830
    Leu Phe Ser Val Glu Met Tyr Glu Val Arg Met Gly Val Asn Tyr Phe
            835                 840                 845
    Lys Ile Thr Lys Ser Glu Val Ser Pro Ser Ile Ser Cys Thr Lys Leu
        850                 855                 860
    Leu Arg Tyr Arg Arg Ala Asn Ser Asp Val Val Lys Val Lys Leu Pro
    865                 870                 875                 880
    Arg Phe Asp Lys Lys Arg Arg Met Cys Leu Pro Gly Tyr Asp Thr Ile
                    885                 890                 895
    Tyr Leu Asp Ser Lys Phe Val Ser Arg Val Phe Asp Tyr Val Val Cys
                900                 905                 910
    Asn Cys Ser Ala Val Asn Ser Lys Thr Phe Glu Trp Val Trp Ser Phe
            915                 920                 925
    Ile Lys Ser Ser Lys Ser Arg Val Ile Ile Ser Gly Lys Ile Ile His
        930                 935                 940
    Lys Asp Val Asn Leu Asp Leu Lys Tyr Val Glu Ser Phe Ala Ala Val
    945                 950                 955                 960
    Met Leu Ala Ser Gly Val Arg Ser Arg Leu Ala Ser Glu Tyr Leu Ala
                    965                 970                 975
    Lys Asn Leu Ser His Phe Ser Gly Asp Cys Ser Phe Ile Glu Ala Thr
                980                 985                 990
    Ser Phe Val Leu Arg Glu Lys Ile Arg Asn Met Thr Leu Asn Phe Asn
            995                 1000                1005
    Glu Arg Leu Leu Gln Leu Val Lys Arg Val Ala Phe Ala Thr Leu Asp
        1010                1015                1020
    Val Ser Phe Leu Asp Leu Asp Ser Thr Leu Glu Ser Ile Thr Asp Phe
    1025                1030                1035                1040
    Ala Glu Cys Lys Val Ala Ile Glu Leu Asp Glu Leu Gly Cys Leu Arg
                    1045                1050                1055
    Ala Glu Ala Glu Asn Glu Lys Ile Arg Asn Leu Ala Gly Asp Ser Ile
                1060                1065                1070
    Ala Ala Lys Leu Ala Ser Glu Ile Val Val Asp Ile Asp Ser Lys Pro
            1075                1080                1085
    Ser Pro Lys Gln Val Gly Asn Ser Ser Ser Glu Asn Ala Asp Lys Arg
        1090                1095                1100
    Glu Val Gln Arg Pro Gly Leu Arg Gly Gly Ser Arg Asn Gly Val Val
    1105                1110                1115                1120
    Gly Glu Phe Leu His Phe Val Val Asp Ser Ala Leu Arg Leu Phe Lys
                    1125                1130                1135
    Tyr Ala Thr Asp Gln Gln Arg Ile Lys Ser Tyr Val Arg Phe Leu Asp
                1140                1145                1150
    Ser Ala Val Ser Phe Leu Asp Tyr Asn Tyr Asp Asn Leu Ser Phe Ile
            1155                1160                1165
    Leu Arg Val Leu Ser Glu Gly Tyr Ser Cys Met Phe Ala Phe Leu Ala
        1170                1175                1180
    Asn Arg Gly Asp Leu Ser Ser Arg Val Arg Ser Ala Val Cys Ala Val
    1185                1190                1195                1200
    Lys Glu Val Ala Thr Ser Cys Ala Asn Ala Ser Val Ser Lys Ala Lys
                    1205                1210                1215
    Val Met Ile Thr Phe Ala Ala Ala Val Cys Ala Met Met Phe Asn Ser
                1220                1225                1230
    Cys Gly Phe Ser Gly Asp Gly Arg Glu Tyr Lys Ser Tyr Ile His Arg
            1235                1240                1245
    Tyr Thr Gln Val Leu Phe Asp Thr Ile Phe Phe Glu Asp Ser Ser Tyr
        1250                1255                1260
    Leu Pro Ile Glu Val Leu Ser Ser Ala Ile Cys Gly Ala Ile Val Thr
    1265                1270                1275                1280
    Leu Phe Ser Ser Gly Ser Ser Ile Ser Leu Asn Ala Phe Leu Leu Gln
                    1285                1290                1295
    Ile Thr Lys Gly Phe Ser Leu Glu Val Val Val Arg Asn Val Val Arg
                1300                1305                1310
    Val Thr His Gly Leu Ser Thr Thr Ala Thr Asp Gly Val Ile Arg Gly
            1315                1320                1325
    Val Phe Ser Gln Ile Val Ser His Leu Leu Val Gly Asn Thr Gly Asn
        1330                1335                1340
    Val Ala Tyr Gln Ser Ala Phe Ile Ala Gly Val Val Pro Leu Leu Val
    1345                1350                1355                1360
    Lys Lys Cys Val Ser Leu Ile Phe Ile Leu Arg Glu Asp Thr Tyr Ser
                    1365                1370                1375
    Gly Phe Ile Lys His Gly Ile Ser Glu Phe Ser Phe Leu Ser Ser Ile
                1380                1385                1390
    Leu Lys Phe Leu Lys Gly Lys Leu Val Asp Glu Leu Lys Ser Ile Ile
            1395                1400                1405
    Gln Gly Val Phe Asp Ser Asn Lys His Val Phe Lys Glu Ala Thr Gln
        1410                1415                1420
    Glu Ala Ile Arg Thr Thr Val Met Gln Val Pro Val Ala Val Val Asp
    1425                1430                1435                1440
    Ala Leu Lys Ser Ala Ala Gly Lys Ile Tyr Asn Asn Phe Thr Ser Arg
                    1445                1450                1455
    Arg Thr Phe Gly Lys Asp Glu Gly Ser Ser Ser Asp Gly Ala Cys Glu
                1460                1465                1470
    Glu Tyr Phe Ser Cys Asp Glu Gly Glu Gly Pro Gly Leu Lys Gly Gly
            1475                1480                1485
    Ser Ser Tyr Gly Phe Ser Ile Leu Ala Phe Phe Ser Arg Ile Met Trp
        1490                1495                1500
    Gly Ala Arg Arg Leu Ile Val Lys Val Lys His Glu Cys Phe Gly Lys
    1505                1510                1515                1520
    Leu Phe Glu Phe Leu Ser Leu Lys Leu His Glu Phe Arg Thr Arg Val
                    1525                1530                1535
    Phe Gly Lys Asn Arg Thr Asp Val Gly Val Tyr Asp Phe Leu Pro Thr
                1540                1545                1550
    Gly Ile Val Glu Thr Leu Ser Ser Ile Glu Glu Cys Asp Gln Ile Glu
            1555                1560                1565
    Glu Leu Leu Gly Asp Asp Leu Lys Gly Asp Lys Asp Ala Ser Leu Thr
        1570                1575                1580
    Asp Met Asn Tyr Phe Glu Phe Ser Glu Asp Phe Leu Ala Ser Ile Glu
    1585                1590                1595                1600
    Glu Pro Pro Phe Ala Gly Leu Arg Gly Gly Ser Lys Asn Ile Ala Ile
                    1605                1610                1615
    Leu Ala Ile Leu Glu Tyr Ala His Asn Leu Phe Arg Ile Val Ala Ser
                1620                1625                1630
    Lys Cys Ser Lys Arg Pro Leu Phe Leu Ala Phe Ala Glu Leu Ser Ser
            1635                1640                1645
    Ala Leu Ile Glu Lys Phe Lys Glu Val Phe Pro Arg Lys Ser Gln Leu
        1650                1655                1660
    Val Ala Ile Val Arg Glu Tyr Thr Gln Arg Phe Leu Arg Ser Arg Met
    1665                1670                1675                1680
    Arg Ala Leu Gly Leu Asn Asn Glu Phe Val Val Lys Ser Phe Ala Asp
                    1685                1690                1695
    Leu Leu Pro Ala Leu Met Lys Arg Lys Val Ser Gly Ser Phe Leu Ala
                1700                1705                1710
    Ser Val Tyr Arg Pro Leu Arg Gly Phe Ser Tyr Met Cys Val Ser Ala
            1715                1720                1725
    Glu Arg Arg Glu Lys Phe Phe Ala Leu Val Cys Leu Ile Gly Leu Ser
        1730                1735                1740
    Leu Pro Phe Phe Val Arg Ile Val Gly Ala Lys Ala Cys Glu Glu Leu
    1745                1750                1755                1760
    Val Ser Ser Ala Arg Arg Phe Tyr Glu Arg Ile Lys Ile Phe Leu Arg
                    1765                1770                1775
    Gln Lys Tyr Val Ser Leu Ser Asn Phe Phe Cys His Leu Phe Ser Ser
                1780                1785                1790
    Asp Val Asp Asp Ser Ser Ala Ser Ala Gly Leu Lys Gly Gly Ala Ser
                    1795                1800                1805
    Arg Met Thr Leu Phe His Leu Leu Val Arg Leu Ala Ser Ala Leu Leu
        1810                1815                1820
    Ser Leu Gly Trp Glu Gly Leu Lys Leu Leu Leu Ser His His Asn Leu
    1825                1830                1835                1840
    Leu Phe Leu Cys Phe Ala Leu Val Asp Asp Val Asn Val Leu Ile Lys
                    1845                1850                1855
    Val Leu Gly Gly Leu Ser Phe Phe Val Gln Pro Ile Phe Ser Leu Phe
                1860                1865                1870
    Ala Ala Met Leu Leu Gln Pro Asp Arg Phe Val Glu Tyr Ser Glu Lys
            1875                1880                1885
    Leu Val Thr Ala Phe Glu Phe Phe Leu Lys Cys Ser Pro Arg Ala Pro
        1890                1895                1900
    Ala Leu Leu Lys Gly Phe Phe Glu Cys Val Ala Asn Ser Thr Val Ser
    1905                1910                1915                1920
    Lys Thr Val Arg Arg Leu Leu Arg Cys Phe Val Lys Met Leu Lys Leu
                    1925                1930                1935
    Arg Lys Gly Arg Gly Leu Arg Ala Asp Gly Arg Gly Leu His Arg Gln
                1940                1945                1950
    Lys Ala Val Pro Val Ile Pro Ser Asn Arg Val Val Thr Asp Gly Val
            1955                1960                1965
    Glu Arg Leu Ser Val Lys Met Gln Gly Val Glu Ala Leu Arg Thr Glu
        1970                1975                1980
    Leu Arg Ile Leu Glu Asp Leu Asp Ser Ala Val Ile Glu Lys Leu Asn
    1985                1990                1995                2000
    Arg Arg Arg Asn Arg Asp Thr Asn Asp Asp Glu Phe Thr Arg Pro Ala
                    2005                2010                2015
    His Glu Gln Met Gln Glu Val Thr Thr Phe Cys Ser Lys Ala Asn Ser
                2020                2025                2030
    Ala Gly Leu Ala Leu Glu Arg Ala Val Leu Val Glu Asp Ala Ile Lys
            2035                2040                2045
    Ser Glu Lys Leu Ser Lys Thr Val Asn Glu Met Val Arg Lys Gly Ser
        2050                2055                2060
    Thr Thr Ser Glu Glu Val Ala Val Ala Leu Ser Asp Asp Glu Ala Val
    2065                2070                2075                2080
    Glu Glu Ile Ser Val Ala Asp Glu Arg Asp Asp Ser Pro Lys Thr Val
                    2085                2090                2095
    Arg Ile Ser Glu Tyr Leu Asn Arg Leu Asn Ser Ser Phe Glu Phe Pro
                2100                2105                2110
    Lys Pro Ile Val Val Asp Asp Asn Lys Asp Thr Gly Gly Leu Thr Asn
            2115                2120                2125
    Ala Val Arg Glu Phe Tyr Tyr Met Gln Glu Leu Ala Leu Phe Glu Ile
        2130                2135                2140
    His Ser Lys Leu Cys Thr Tyr Tyr Asp Gln Leu Arg Ile Val Asn Phe
    2145                2150                2155                2160
    Asp Arg Ser Val Ala Pro Cys Ser Glu Asp Ala Gln Leu Tyr Val Arg
                    2165                2170                2175
    Lys Asn Gly Ser Thr Ile Val Gln Gly Lys Glu Val Arg Leu His Ile
                2180                2185                2190
    Lys Asp Phe His Asp His Asp Phe Leu Phe Asp Gly Lys Ile Ser Ile
            2195                2200                2205
    Asn Lys Arg Arg Arg Gly Gly Asn Val Leu Tyr His Asp Asn Leu Ala
        2210                2215                2220
    Phe Leu Ala Ser Asn Leu Phe Leu Ala Gly Tyr Pro Phe Ser Arg Ser
    2225                2230                2235                2240
    Phe Val Phe Thr Asn Ser Ser Val Asp Ile Leu Leu Tyr Glu Ala Pro
                    2245                2250                2255
    Pro Gly Gly Gly Lys Thr Thr Thr Leu Ile Asp Ser Phe Leu Lys Val
                2260                2265                2270
    Phe Lys Lys Gly Glu Val Ser Thr Met Ile Leu Thr Ala Asn Lys Ser
            2275                2280                2285
    Ser Gln Val Glu Ile Leu Lys Lys Val Glu Lys Glu Val Ser Asn Ile
        2290                2295                2300
    Glu Cys Gln Lys Arg Lys Asp Lys Arg Ser Pro Lys Lys Ser Ile Tyr
    2305                2310                2315                2320
    Thr Ile Asp Ala Tyr Leu Met His His Arg Gly Cys Asp Ala Asp Val
                    2325                2330                2335
    Leu Phe Ile Asp Glu Cys Phe Met Val His Ala Gly Ser Val Leu Ala
                2340                2345                2350
    Cys Ile Glu Phe Thr Arg Cys His Lys Val Met Ile Phe Gly Asp Ser
            2355                2360                2365
    Arg Gln Ile His Tyr Ile Glu Arg Asn Glu Leu Asp Lys Cys Leu Tyr
        2370                2375                2380
    Gly Asp Leu Asp Arg Phe Val Asp Leu Gln Cys Arg Val Tyr Gly Asn
    2385                2390                2395                2400
    Ile Ser Tyr Arg Cys Pro Trp Asp Val Cys Ala Trp Leu Ser Thr Val
                    2405                2410                2415
    Tyr Gly Asn Leu Ile Ala Thr Val Lys Gly Glu Ser Glu Gly Lys Ser
                2420                2425                2430
    Ser Met Arg Ile Asn Glu Ile Asn Ser Val Asp Asp Leu Val Pro Asp
            2435                2440                2445
    Val Gly Ser Thr Phe Leu Cys Met Leu Gln Ser Glu Lys Leu Glu Ile
        2450                2455                2460
    Ser Lys His Phe Ile Arg Lys Gly Leu Thr Lys Leu Asn Val Leu Thr
    2465                2470                2475                2480
    Val His Glu Ala Gln Gly Glu Thr Tyr Ala Arg Val Asn Leu Val Arg
                    2485                2490                2495
    Leu Lys Phe Gln Glu Asp Glu Pro Phe Lys Ser Ile Arg His Ile Thr
                2500                2505                2510
    Val Ala Leu Ser Arg His Thr Asp Ser Leu Thr Tyr Asn Val Leu Ala
            2515                2520                2525
    Ala Arg Arg Gly Asp Ala Thr Cys Asp Ala Ile Gln Lys Ala Ala Glu
        2530                2535                2540
    Leu Val Asn Lys Phe Arg Val Phe Pro Thr Ser Phe Gly Gly Ser Val
    2545                2550                2555                2560
    Ile Asn Leu Asn Val Lys Lys Asp Val Glu Asp Asn Ser Arg Cys Lys
                    2565                2570                2575
    Ala Ser Ser Ala Pro Leu Ser Val Ile Asn Asp Phe Leu Asn Glu Val
                2580                2585                2590
    Asn Pro Gly Thr Ala Val Ile Asp Phe Gly Asp Leu Ser Ala Asp Phe
            2595                2600                2605
    Ser Thr Gly Pro Phe Glu Cys Gly Ala Ser Gly Ile Val Val Arg Asp
        2610                2615                2620
    Asn Ile Ser Ser Ser Asn Ile Thr Asp His Asp Lys Gln Arg Val
    2625                2630                2635

    and has a molecular weight of about 290 to 300 kDa, preferably 294 kDa.
  • Another such DNA molecule (GLRaV-2 ORFlb) includes nucleotides 7922-9301 of SEQ. ID. No. 1 and codes for a grapevine leafroll virus RNA-dependent RNA polymerase (RdRP). This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 4 as follows:
    AGCGTAGTTC GGTCGCAGGC GATTCCGCGT AGAAAACCTT CTCTACAAGA AAATTTGTAT   60
    TGGTTTGAAG CGCGGAATTA TAACTTCTCG ACTTGCGACC GTAACACATC TGCTTCAATG  120
    TTCGGAGAGG CTATGGCGAT GAACTGTCTT CGTCGTTGCT TCGACCTAGA TGCCTTTTCG  180
    TCCCTGCGTG ATGATGTGAT TAGTATCACA CGTTCAGGCA TCGAACAATG GCTGGAGAAA  240
    CGTACTCCTA GTCAGATTAA AGCATTAATG AAGGATGTTG AATCGCCTTT GGAAATTGAC  300
    GATGAAATTT GTCGTTTTAA GTTGATGGTG AAGCGTGACG CTAAGGTGAA GTTAGACTCT  360
    TCTTGTTTAA CTAAACACAG CGCCGCTCAA AATATCATGT TTCATCGCAA GAGCATTAAT  420
    GCTATCTTCT CTCCTATCTT TAATGAGGTG AAAAACCGAA TAATGTGCTG TCTTAAGCCT  480
    AACATAAAGT TTTTTACGGA GATGACTAAC AGGGATTTTG CTTCTGTTGT CAGCAACATG  540
    CTTGGTGACG ACGATGTGTA CCATATAGGT GAAGTTGATT TCTCAAAGTA CGACAAGTCT  600
    CAAGATGCTT TCGTGAAGGC TTTTGAAGAA GTAATGTATA AGGAACTCGG TGTTGATGAA  660
    GAGTTGCTGG CTATCTGGAT GTGCGGCGAG CGGTTATCGA TAGCTAACAC TCTCGATGGT  720
    CAGTTGTCCT TCACGATCGA GAATCAAAGG AAGTCGGGAG CTTCGAACAC TTGGATTGGT  780
    AACTCTCTCG TCACTTTGGG TATTTTAAGT CTTTACTACG ACGTTAGAAA TTTCGAGGCG  840
    TTGTACATCT CGGGCGATGA TTCTTTAATT TTTTCTCGCA GCGAGATTTC GAATTATGCC  900
    GAGGACATAT GCACTGACAT GGGTTTTGAG ACAAAATTTA TGTCCCCAAG TGTCCCGTAC  960
    TTTTGTTCTA AATTTGTTGT TATGTGTGGT CATAAGACGT TTTTTGTTCC CGACCCGTAC 1020
    AAGCTTTTTG TCAAGTTGGG AGCAGTCAAA GAGGATGTTT CAATGGATTT CCTTTTCGAG 1080
    ACTTTTACCT CCTTTAAAGA CTTAACCTCC GATTTTAACG ACGAGCGCTT AATTCAAAAG 1140
    CTCGCTGAAC TTGTGGCTTT AAAATATGAG GTTCAAACCG GCAACACCAC CTTGGCGTTA 1200
    AGTGTGATAC ATTGTTTGCG TTCGAATTTC CTCTCGTTTA GCAAGTTATA TCCTCGCGTG 1260
    AAGGGATGGC AGGTTTTTTA CACGTCGGTT AAGAAAGCGC TTCTCAAGAG TGGGTGTTCT 1320
    CTCTTCGACA GTTTCATGAC CCCTTTTGGT CAGGCTGTCA TGGTTTGGGA TGATGAGTAG 1380
  • The RNA-dependent RNA polymerase has an amino acid sequence corresponding to SEQ. ID. No. 5 as follows:
    Ser Val Val Arg Ser Gln Ala Ile Pro Arg Arg Lys Pro Ser Leu Gln
    1               5                   10                  15
    Glu Asn Leu Tyr Ser Phe Glu Ala Arg Asn Tyr Asn Phe Ser Thr Cys
                20                  25                  30
    Asp Arg Asn Thr Ser Ala Ser Met Phe Gly Glu Ala Met Ala Met Asn
            35                  40                  45
    Cys Leu Arg Arg Cys Phe Asp Leu Asp Ala Phe Ser Ser Leu Arg Asp
        50                  55                  60
    Asp Val Ile Ser Ile Thr Arg Ser Gly Ile Glu Gln Trp Leu Glu Lys
    65                  70                  75                  80
    Arg Thr Pro Ser Gln Ile Lys Ala Leu Met Lys Asp Val Glu Ser Pro
                    85                  90                  95
    Leu Glu Ile Asp Asp Glu Ile Cys Arg Phe Lys Leu Met Val Lys Arg
                100                 105                 110
    Asp Ala Lys Val Lys Leu Asp Ser Ser Cys Leu Thr Lys His Ser Ala
            115                 120                 125
    Ala Gln Asn Ile Met Phe His Arg Lys Ser Ile Asn Ala Ile Phe Ser
        130                 135                 140
    Pro Ile Phe Asn Glu Val Lys Asn Arg Ile Met Cys Cys Leu Lys Pro
    145                 150                 155                 160
    Asn Ile Lys Phe Phe Thr Glu Met Thr Asn Arg Asp Phe Ala Ser Val
                    165                 170                 175
    Val Ser Asn Met Leu Gly Asp Asp Asp Val Tyr His Ile Gly Glu Val
                180                 185                 190
    Asp Phe Ser Lys Tyr Asp Lys Ser Gln Asp Ala Phe Val Lys Ala Phe
            195                 200                 205
    Glu Glu Val Met Tyr Lys Glu Leu Gly Val Asp Glu Glu Leu Leu Ala
        210                 215                 220
    Ile Trp Met Cys Gly Glu Arg Leu Ser Ile Ala Asn Thr Leu Asp Gly
    225                 230                 235                 240
    Gln Leu Ser Phe Thr Ile Glu Asn Gln Arg Lys Ser Gly Ala Ser Asn
                    245                 250                 255
    Thr Trp Ile Gly Asn Ser Leu Val Thr Leu Gly Ile Leu Ser Leu Tyr
                260                 265                 270
    Tyr Asp Val Arg Asn Phe Glu Ala Leu Tyr Ile Ser Gly Asp Asp Ser
            275                 280                 285
    Leu Ile Phe Ser Arg Ser Glu Ile Ser Asn Tyr Ala Asp Asp Ile Cys
        290                 295                 300
    Thr Asp Met Gly Phe Glu Thr Lys Phe Met Ser Pro Ser Val Pro Tyr
    305                 310                 315                 320
    Phe Cys Ser Lys Phe Val Val Met Cys Gly His Lys Thr Phe Phe Val
                    325                 330                 335
    Pro Asp Pro Tyr Lys Leu Phe Val Lys Leu Gly Ala Val Lys Glu Asp
                340                 345                 350
    Val Ser Met Asp Phe Leu Phe Glu Thr Phe Thr Ser Phe Lys Asp Leu
            355                 360                 365
    Thr Ser Asp Phe Asn Asp Glu Arg Leu Ile Gln Lys Leu Ala Glu Leu
        370                 375                 380
    Val Ala Leu Lys Tyr Glu Val Gln Thr Gly Asn Thr Thr Leu Ala Leu
    385                 390                 395                 400
    Ser Val Ile His Cys Leu Arg Ser Asn Phe Leu Ser Phe Ser Lys Leu
                    405                 410                 415
    Tyr Pro Arg Val Lys Gly Trp Gln Val Phe Tyr Thr Ser Val Lys Lys
                420                 425                 430
    Ala Leu Leu Lys Ser Gly Cys Ser Leu Phe Asp Ser Phe Met Thr Pro
            435                 440                 445
    Phe Gly Gln Ala Val Met Val Trp Asp Asp Glu
        450                 455

    and a molecular weight from about 50 to about 54 kDa, preferably about 52 kDa.
  • Another such DNA molecule (GLRAV-2 ORF2) includes nucleotides 9365-9535 of SEQ. ID. No. 1 and codes for a small, grapevine leafroll virus hydrophobic protein or polypeptide. This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 6 as follows:
    ATGAATCAGG TTTTGCAGTT TGAATGTTTG TTTCTGCTGA ATCTCGCGGT TTTTGCTGTG  60
    ACTTTCATTT TCATTCTTCT GGTCTTCCGC GTGATTAAGT CTTTTCGCCA GAAGGGTCAC 120
    GAAGGACCTG TTCCCGTTGT TCGTGGCGGG GGTTTTTCAA CCGTAGTGTA G 171
  • The small hydrophobic protein or polypeptide has an amino acid sequence corresponding to SEQ. ID. No. 7 as follows:
    Met Asn Gln Val Leu Gln Phe Glu Cys Leu Phe Leu Leu Asn Leu Ala
    1               5                   10                  15
    Val Phe Ala Val Thr Phe Ile Phe Ile Leu Leu Val Phe Arg Val Ile
                20                  25                  30
    Lys Ser Phe Arg Gln Lys Gly His Glu Ala Pro Val Pro Val Val Arg
            35                  40                  45
    Gly Gly Gly Phe Ser Thr Val Val
        50                  55

    and a molecular weight from about 5 to about 7 kDa, preferably about 6 kDa.
  • Another such DNA molecule (GLRaV-2 ORF3) includes nucleotides 9551-11350 of SEQ. ID. No. 1 and encodes for a grapevine leafroll virus heat shock 70 protein. This DNA molecule comprises the nucleotide sequence corresponding to SEQ. ID. No. 8 as follows:
    ATGGTAGTTT TCGGTTTGGA CTTTGGCACC ACATTCTCTA CGGTGTGTGT GTACAAGGAT   60
    GGACGAGTTT TTTCATTCAA GCAGAATAAT TCGGCGTACA TCCCCACTTA CCTCTATCTC  120
    TTCTCCGATT CTAACCACAT GACTTTTGGT TACGAGGCCG AATCACTGAT GAGTAATCTG  180
    AAAGTTAAAG GTTCGTTTTA TAGAGATTTA AAACGTTGGG TGGGTTGCGA TTCGAGTAAC  240
    CTCGACGCGT ACCTTGACCG TTTAAAACCT CATTACTCGG TCCGCTTGGT TAAGATCGGC  300
    TCTGGCTTGA ACGAAACTGT TTCAATTGGA AACTTCGGGG GCACTGTTAA GTCTGAGGCT  360
    CATCTGCCAG GGTTGATAGC TCTCTTTATT AAGGCTGTCA TTAGTTGCGC GGAGGGCGCG  420
    TTTGCGTGCA CTTGCACCGG GGTTATTTGT TCAGTACCTG CCAATTATGA TAGCGTTCAA  480
    AGGAATTTCA CTGATCAGTG TGTTTCACTC AGCGGTTATC AGTGCGTATA TATGATCAAT  540
    GAACCTTCAG CGGCTGCGCT ATCTGCGTGT AATTCGATTG GAAAGAAGTC CGCAAATTTG  600
    GCTGTTTACG ATTTCGGTGG TGGGACCTTC GACGTGTCTA TCATTTCATA CCGCAACAAT  660
    ACTTTTGTTG TGCGAGCTTC TGGAGGCGAT CTAAATCTCG GTGGAAGGGA TGTTGATCGT  720
    GCGTTTCTCA CGCACCTCTT CTCTTTAACA TCGCTGGAAC CTGACCTCAC TTTGGATATC  780
    TCGAATCTGA AAGAATCTTT ATCAAAAACG GACGCAGAGA TAGTTTACAC TTTGAGAGGT  840
    GTCGATGGAA GAAAAGAAGA CGTTAGAGTA AACAAAAACA TTCTTACGTC GGTGATGCTC  900
    CCCTACGTGA ACAGAACGCT TAAGATATTA GAGTCAACCT TAAAATCGTA TGCTAAGAGT  960
    ATGAATGAGA GTGCGCGAGT TAAGTGCGAT TTAGTGCTGA TAGGAGGATC TTCATATCTT 1020
    CCTGGCCTGG CAGACGTACT AACGAAGCAT CAGAGCGTTG ATCGTATCTT AAGAGTTTCG 1080
    GATCCTCGGG CTGCCGTGGC CGTCGGTTGC GCATTATATT CTTCATGCCT CTCAGGATCT 1140
    GGGGGGTTGC TACTGATCGA CTGTGCAGCT CACACTGTCG CTATAGCGGA CAGAAGTTGT 1200
    CATCAAATCA TTTGCGCTCC AGCGGGGGCA CCGATCCCCT TTTCAGGAAG CATGCCTTTG 1260
    TACTTAGCCA GGGTCAACAA GAACTCGCAG CGTGAAGTCG CCGTGTTTGA AGGGGAGTAC 1320
    GTTAAGTGCC CTAAGAACAG AAAGATCTGT GGAGCAAATA TAAGATTTTT TGATATAGGA 1380
    GTGACGGGTG ATTCGTACGC ACCCGTTACC TTCTATATGG ATTTCTCCAT TTCAAGCGTA 1440
    GGAGCCGTTT CATTCGTGGT GAGAGGTCCT GAGGGTAAGC AAGTGTCACT CACTGGAACT 1500
    CCAGCGTATA ACTTTTCGTC TGTGGCTCTC GGATCACGCA GTGTCCGAGA ATTGCATATT 1560
    AGTTTAAATA ATAAAGTTTT TCTCGGTTTG CTTCTACATA GAAAGGCGGA TCGACGAATA 1620
    CTTTTCACTA AGGATGAAGC GATTCGATAC GCCGATTCAA TTGATATCGC GGATGTGCTA 1680
    AAGGAATATA AAAGTTACGC GGCCAGTGCC TTACCACCAG ACGAGGATGT CGAATTACTC 1740
    CTGGGAAAGT CTGTTCAAAA AGTTTTACGG GGAAGCAGAC TGGAAGAAAT ACCTCTCTAG 1800
  • The heat shock 70 protein is believed to function as a chaperone protein and has an amino acid sequence corresponding to SEQ. ID. No. 9 as follows:
    Met Val Val Phe Gly Leu Asp Phe Gly Thr Thr Phe Ser Thr Val Cys
    1               5                   10                  15
    Val Tyr Lys Asp Gly Arg Val Phe Ser Phe Lys Gln Asn Asn Ser Ala
                20                  25                  30
    Tyr Ile Pro Thr Tyr Leu Tyr Leu Phe Ser Asp Ser Asn His Met Thr
            35                  40                  45
    Phe Gly Tyr Glu Ala Glu Ser Leu Met Ser Asn Leu Lys Val Lys Gly
        50                  55                  60
    Ser Phe Tyr Arg Asp Leu Lys Arg Trp Val Gly Cys Asp Ser Ser Asn
    65                  70                  75                  80
    Leu Asp Ala Tyr Leu Asp Arg Leu Lys Pro His Tyr Ser Val Arg Leu
                    85                  90                  95
    Val Lys Ile Gly Ser Gly Leu Asn Glu Thr Val Ser Ile Gly Asn Phe
                100                 105                 110
    Gly Gly Thr Val Lys Ser Glu Ala His Leu Pro Gly Leu Ile Ala Leu
            115                 120                 125
    Phe Ile Lys Ala Val Ile Ser Cys Ala Glu Gly Ala Phe Ala Cys Thr
        130                 135                 140
    Cys Thr Gly Val Ile Cys Ser Val Pro Ala Asn Tyr Asp Ser Val Gln
    145                 150                 155                 160
    Arg Asn Phe Thr Asp Gln Cys Val Ser Leu Ser Gly Tyr Gln Cys Val
                    165                 170                 175
    Tyr Met Ile Asn Glu Pro Ser Ala Ala Ala Leu Ser Ala Cys Asn Ser
                180                 185                 190
    Ile Gly Lys Lys Ser Ala Asn Leu Ala Val Tyr Asp Phe Gly Gly Gly
            195                 200                 205
    Thr Phe Asp Val Ser Ile Ile Ser Tyr Arg Asn Asn Thr Phe Val Val
        210                 215                 220
    Arg Ala Ser Gly Gly Asp Leu Asn Leu Gly Gly Arg Asp Val Asp Arg
    225                 230                 235                 240
    Ala Phe Leu Thr His Leu Phe Ser Leu Thr Ser Leu Glu Pro Asp Leu
                    245                 250                 255
    Thr Leu Asp Ile Ser Asn Leu Lys Glu Ser Leu Ser Lys Thr Asp Ala
                260                 265                 270
    Glu Ile Val Tyr Thr Leu Arg Gly Val Asp Gly Arg Lys Glu Asp Val
            275                 280                 285
    Arg Val Asn Lys Asn Ile Leu Thr Ser Val Met Leu Pro Tyr Val Asn
        290                 295                 300
    Arg Thr Leu Lys Ile Leu Glu Ser Thr Leu Lys Ser Tyr Ala Lys Ser
    305                 310                 315                 320
    Met Asn Glu Ser Ala Arg Val Lys Cys Asp Leu Val Leu Ile Gly Gly
                    325                 330                 335
    Ser Ser Tyr Leu Pro Gly Leu Ala Asp Val Leu Thr Lys His Gln Ser
                340                 345                 350
    Val Asp Arg Ile Leu Arg Val Ser Asp Pro Arg Ala Ala Val Ala Val
            355                 360                 365
    Gly Cys Ala Leu Tyr Ser Ser Cys Leu Ser Gly Ser Gly Gly Leu Leu
        370                 375                 380
    Leu Ile Asp Cys Ala Ala His Thr Val Ala Ile Ala Asp Arg Ser Cys
    385                 390                 395                 400
    His Gln Ile Ile Cys Ala Pro Ala Gly Ala Pro Ile Pro Phe Ser Gly
                    405                 410                 415
    Ser Met Pro Leu Tyr Leu Ala Arg Val Asn Lys Asn Ser Gln Arg Glu
                420                 425                 430
    Val Ala Val Phe Glu Gly Glu Tyr Val Lys Cys Pro Lys Asn Arg Lys
            435                 440                 445
    Ile Cys Gly Ala Asn Ile Arg Phe Phe Asp Ile Gly Val Thr Gly Asp
        450                 455                 460
    Ser Tyr Ala Pro Val Thr Phe Tyr Met Asp Phe Ser Ile Ser Ser Val
    465                 470                 475                 480
    Gly Ala Val Ser Phe Val Val Arg Gly Pro Glu Gly Lys Gln Val Ser
                    485                 490                 495
    Leu Thr Gly Thr Pro Ala Tyr Asn Phe Ser Ser Val Ala Leu Gly Ser
                500                 505                 510
    Arg Ser Val Arg Glu Leu His Ile Ser Leu Asn Asn Lys Val Phe Leu
            515                 520                 525
    Gly Leu Leu Leu His Arg Lys Ala Asp Arg Arg Ile Leu Phe Thr Lys
        530                 535                 540
    Asp Glu Ala Ile Arg Tyr Ala Asp Ser Ile Asp Ile Ala Asp Val Leu
    545                 550                 555                 560
    Lys Glu Tyr Lys Ser Tyr Ala Ala Ser Ala Leu Pro Pro Asp Glu Asp
                    565                 570                 575
    Val Glu Leu Leu Leu Gly Lys Ser Val Gln Lys Val Leu Arg Gly Ser
                580                 585                 590
    Arg Leu Glu Glu Ile Pro Leu
            595

    and a molecular weight from about 63 to about 67 kDa, preferably about 65 kDa.
  • Another such DNA molecule (GLRaV-2 ORF4) includes nucleotides 11277-12932 of SEQ. ID. No. 1 and codes for a putative grapevine leafroll virus heat shock 90 protein. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 10 as follows:
    ATGTCGAATT ACTCCTGGGA AAGTCTGTTC AAAAAGTTTT ACGGGGAAGC AGACTGGAAG   60
    AAATACCTCT CTAGGAGCAT AGCAGCACAC TCAAGTGAAA TTAAAACTCT ACCAGACATT  120
    CGATTGTACG GCGGTAGGGT TGTAAAGAAG TCCGAATTCG AATCAGCACT TCCTAATTCT  180
    TTTGAACAGG AATTAGGACT GTTCATACTG AGCGAACGGG AAGTGGGATG GAGCAAATTA  240
    TGCGGAATAA CGGTGGAAGA AGCAGCATAC GATCTTACGA ATCCCAAGGC TTATAAATTC  300
    ACTGCCGAGA CATGTAGCCC GGATGTAAAA GGTGAAGGAC AAAAATACTC TATGGAAGAC  360
    GTGATGAATT TCATGCGTTT ATCAAATCTG GATGTTAACG ACAAGATGCT GACGGAACAG  420
    TGTTGGTCGC TGTCCAATTC ATGCGGTGAA TTGATCAACC CAGACGACAA AGGGCGATTC  480
    GTGGCTCTCA CCTTTAAGGA CAGAGACACA GCTGATGACA CGGGTGCCGC CAACGTGGAA  540
    TGTCGCGTGG GCGACTATCT AGTTTACGCT ATGTCCCTGT TTGAGCAGAG GACCCAAAAA  600
    TCGCAGTCTG GCAACATCTC TCTGTACGAA AAGTACTGTG AATACATCAG GACCTACTTA  660
    GGGAGTACAG ACCTGTTCTT CACAGCGCCG GACAGGATTC CGTTACTTAC GGGCATCCTA  720
    TACGATTTTT GTAAGGAATA CAACGTTTTC TACTCGTCAT ATAAGAGAAA CGTCGATAAT  780
    TTCAGATTCT TCTTGGCGAA TTATATGCCT TTGATATCTG ACGTCTTTGT CTTCCAGTGG  840
    GTAAAACCCG CGCCGGATGT TCGGCTGCTT TTTGAGTTAA GTGCAGCGGA ACTAACGCTG  900
    GAGGTTCCCA CACTGAGTTT GATAGATTCT CAAGTTGTGG TAGGTCATAT CTTAAGATAC  960
    GTAGAATCCT ACACATCAGA TCCAGCCATC GACGCGTTAG AAGACAAACT GGAAGCGATA 1020
    CTGAAAAGTA GCAATCCCCG TCTATCGACA GCGCAACTAT GGGTTGGTTT CTTTTGTTAC 1080
    TATGGTGAGT TTCGTACGGC TCAAAGTAGA GTAGTGCAAA GACCAGGCGT ATACAAAACA 1140
    CCTGACTCAG TGGGTGGATT TGAAATAAAC ATGAAAGATG TTGAGAAATT CTTCGATAAA 1200
    CTTCAGAGAG AATTGCCTAA TGTATCTTTG CGGCGTCAGT TTAACGGAGC TAGAGCGCAT 1260
    GAGGCTTTCA AAATATTTAA AAACGGAAAT ATAAGTTTCA GACCTATATC GCGTTTAAAC 1320
    GTGCCTAGAG AGTTCTGGTA TCTGAACATA GACTACTTCA GGCACGCGAA TAGGTCCGGG 1380
    TTAACCGAAG AAGAAATACT CATCCTAAAC AACATAAGCG TTGATGTTAG GAAGTTATGC 1440
    GCTGAGAGAG CGTGCAATAC CCTACCTAGC GCGAAGCGCT TTAGTAAAAA TCATAAGAGT 1500
    AATATACAAT CATCACGCCA AGAGCGGAGG ATTAAAGACC CATTGGTAGT CCTGAAAGAC 1560
    ACTTTATATG AGTTCCAACA CAAGCGTGCC GGTTGGGGGT CTCGAAGCAC TCGAGACCTC 1620
    GGGAGTCGTG CTGACCACGC GAAAGGAAGC GGTTGA 1656
  • The heat shock 90 protein has an amino acid sequence corresponding to SEQ. ID. No. 11 as follows:
    Met Ser Asn Tyr Ser Trp Glu Ser Leu Phe Lys Lys Phe Tyr Gly Glu
    1               5                   10                  15
    Ala Asp Trp Lys Lys Tyr Leu Ser Arg Ser Ile Ala Ala His Ser Ser
                20                  25                  30
    Glu Ile Lys Thr Leu Pro Asp Ile Arg Leu Tyr Gly Gly Arg Val Val
            35                  40                  45
    Lys Lys Ser Glu Phe Glu Ser Ala Leu Pro Asn Ser Phe Glu Gln Glu
        50                  55                  60
    Leu Gly Leu Phe Ile Leu Ser Glu Arg Glu Val Gly Trp Ser Lys Leu
    65                  70                  75                  80
    Cys Gly Ile Thr Val Glu Glu Ala Ala Tyr Asp Leu Thr Asn Pro Lys
                    85                  90                  95
    Ala Tyr Lys Phe Thr Ala Glu Thr Cys Ser Pro Asp Val Lys Gly Glu
                100                 105                 110
    Gly Gln Lys Tyr Ser Met Glu Asp Val Met Asn Phe Met Arg Leu Ser
            115                 120                 125
    Asn Leu Asp Val Asn Asp Lys Met Leu Thr Glu Gln Cys Trp Ser Leu
        130                 135                 140
    Ser Asn Ser Cys Gly Glu Leu Ile Asn Pro Asp Asp Lys Gly Arg Phe
    145                 150                 155                 160
    Val Ala Leu Thr Phe Lys Asp Arg Asp Thr Ala Asp Asp Thr Gly Ala
                    165                 170                 175
    Ala Asn Val Glu Cys Arg Val Gly Asp Tyr Leu Val Tyr Ala Met Ser
                180                 185                 190
    Leu Phe Glu Gln Arg Thr Gln Lys Ser Gln Ser Gly Asn Ile Ser Leu
            195                 200                 205
    Tyr Glu Lys Tyr Cys Glu Tyr Ile Arg Thr Tyr Leu Gly Ser Thr Asp
        210                 215                 220
    Leu Phe Phe Thr Ala Pro Asp Arg Ile Pro Leu Leu Thr Gly Ile Leu
    225                 230                 235                 240
    Tyr Asp Phe Cys Lys Glu Tyr Asn Val Phe Tyr Ser Ser Tyr Lys Arg
                    245                 250                 255
    Asn Val Asp Asn Phe Arg Phe Phe Leu Ala Asn Tyr Met Pro Leu Ile
                260                 265                 270
    Ser Asp Val Phe Val Phe Gln Trp Val Lys Pro Ala Pro Asp Val Arg
            275                 280                 285
    Leu Leu Phe Glu Leu Ser Ala Ala Glu Leu Thr Leu Glu Val Pro Thr
        290                 295                 300
    Leu Ser Leu Ile Asp Ser Gln Val Val Val Gly His Ile Leu Arg Tyr
    305                 310                 315                 320
    Val Glu Ser Tyr Thr Ser Asp Pro Ala Ile Asp Ala Leu Glu Asp Lys
                    325                 330                 335
    Leu Glu Ala Ile Leu Lys Ser Ser Asn Pro Arg Leu Ser Thr Ala Gln
                340                 345                 350
    Leu Trp Val Gly Phe Phe Cys Tyr Tyr Gly Glu Phe Arg Thr Ala Gln
            355                 360                 365
    Ser Arg Val Val Gln Arg Pro Gly Val Tyr Lys Thr Pro Asp Ser Val
        370                 375                 380
    Gly Gly Phe Glu Ile Asn Met Lys Asp Val Glu Lys Phe Phe Asp Lys
    385                 390                 395                 400
    Leu Gln Arg Glu Leu Pro Asn Val Ser Leu Arg Arg Gln Phe Asn Gly
                    405                 410                 415
    Ala Arg Ala His Glu Ala Phe Lys Ile Phe Lys Asn Gly Asn Ile Ser
                420                 425                 430
    Phe Arg Pro Ile Ser Arg Leu Asn Val Pro Arg Glu Phe Trp Tyr Leu
            435                 440                 445
    Asn Ile Asp Tyr Phe Arg His Ala Asn Arg Ser Gly Leu Thr Glu Glu
        450                 455                 460
    Glu Ile Leu Ile Leu Asn Asn Ile Ser Val Asp Val Arg Lys Leu Cys
    465                 470                 475                 480
    Ala Glu Arg Ala Cys Asn Thr Leu Pro Ser Ala Lys Arg Phe Ser Lys
                    485                 490                 495
    Asn His Lys Ser Asn Ile Gln Ser Ser Arg Gln Glu Arg Arg Ile Lys
                500                 505                 510
    Asp Pro Leu Val Val Leu Lys Asp Thr Leu Tyr Glu Phe Gln His Lys
            515                 520                 525
    Arg Ala Gly Trp Gly Ser Arg Ser Thr Arg Asp Leu Gly Ser Arg Ala
        530                 535                 540
    Asp His Ala Lys Gly Ser Gly
    545                 550

    and a molecular weight from about 61 to about 65 kDa, preferably about 63 kDa.
  • Yet another DNA molecule of the present invention (GLRaV-2 ORF5) includes nucleotides 12844-13515 of SEQ. ID. No. 1 and codes for a diverged coat protein. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 12 as follows:
    ATGAGTTCCA ACACAAGCGT GCCGGTTGGG GGTCTCGAAG CACTCGAGAC CTCGGGAGTC  60
    GTGCTGACCA CGCGAAAGGA AGCGGTTGAT AAGTTTTTTA ATGAACTAAA AAACGAAAAT 120
    TACTCATCAG TTGACAGCAG CCGATTAAGC GATTCGGAAG TAAAAGAAGT GTTAGAGAAA 180
    AGTAAAGAAA GTTTCAAAAG CGAACTGGCC TCCACTGACG AGCACTTCGT CTACCACATT 240
    ATATTTTTCT TAATCCGATG TGCTAAGATA TCGACAAGTG AAAAGGTGAA GTACGTTGGT 300
    AGTCATACGT ACGTGGTCGA CGGAAAAACG TACACCGTTC TTGACGCTTG GGTATTCAAC 360
    ATGATGAAAA GTCTCACGAA GAAGTACAAA CGAGTGAATG GTCTGCGTGC GTTCTGTTGC 420
    GCGTGCGAAG ATCTATATCT AACCGTCGCA CCAATAATGT CAGAACGCTT TAAGACTAAA 480
    GCCGTAGGGA TGAAAGGTTT GCCTGTTGGA AAGGAATACT TAGGCGCCGA CTTTCTTTCG 540
    GGAACTAGCA AACTGATGAG CGATCACGAC AGGGCGGTCT CCATCGTTGC AGCGAAAAAC 600
    GCTGTCGATC GTAGCGCTTT CACGGGTGGG GAGAGAAAGA TAGTTAGTTT GTATGATCTA 660
    GGGAGGTACT AA 672
  • The diverged coat protein has an amino acid sequence corresponding to SEQ. ID. No. 13 as follows:
    Met Ser Ser Asn Thr Ser Val Pro Val Gly Gly Leu Glu Ala Leu Glu
    1               5                   10                  15
    Thr Ser Gly Val Val Leu Thr Thr Arg Lys Glu Ala Val Asp Lys Phe
                20                  25                  30
    Phe Asn Glu Leu Lys Asn Glu Asn Tyr Ser Ser Val Asp Ser Ser Arg
            35                  40                  45
    Leu Ser Asp Ser Glu Val Lys Glu Val Leu Glu Lys Ser Lys Glu Ser
        50                  55                  60
    Phe Lys Ser Glu Leu Ala Ser Thr Asp Glu His Phe Val Tyr His Ile
    65                  70                  75                  80
    Ile Phe Phe Leu Ile Arg Cys Ala Lys Ile Ser Thr Ser Glu Lys Val
                    85                  90                  95
    Lys Tyr Val Gly Ser His Thr Tyr Val Val Asp Gly Lys Thr Tyr Thr
                100                 105                 110
    Val Leu Asp Ala Trp Val Phe Asn Met Met Lys Ser Leu Thr Lys Lys
            115                 120                 125
    Tyr Lys Arg Val Asn Gly Leu Arg Ala Phe Cys Cys Ala Cys Glu Asp
        130                 135                 140
    Leu Tyr Leu Thr Val Ala Pro Ile Met Ser Glu Arg Phe Lys Thr Lys
    145                 150                 155                 160
    Ala Val Gly Met Lys Gly Leu Pro Val Gly Lys Glu Tyr Leu Gly Ala
                    165                 170                 175
    Asp Phe Leu Ser Gly Thr Ser Lys Leu Met Ser Asp His Asp Arg Ala
                180                 185                 190
    Val Ser Ile Val Ala Ala Lys Asn Ala Val Asp Arg Ser Ala Phe Thr
            195                 200                 205
    Gly Gly Glu Arg Lys Ile Val Ser Leu Tyr Asp Leu Gly Arg Tyr
        210                 215                 220

    and a molecular weight from about 23 to about 27 kDa, preferably about 25 kDa.
  • Another such DNA molecule (GLRaV-2 ORF6) includes nucleotides 13584-14180 of SEQ. ID. No. 1 and codes for a grapevine leafroll virus coat protein. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 14 as follows:
    ATGGAGTTGA TGTCCGACAG CAACCTTAGC AACCTGGTGA TAACCGACGC CTCTAGTCTA  60
    AATGGTGTCG ACAAGAAGCT TTTATCTGCT GAAGTTGAAA AAATGTTGGT GCAGAAAGGG 120
    GCTCCTAACG AGGGTATAGA AGTGGTGTTC GGTCTACTCC TTTACGCACT CGCGGCAAGA 180
    ACCACGTCTC CTAAGGTTCA GCGCGCAGAT TCAGACGTTA TATTTTCAAA TAGTTTCGGA 240
    GAGAGGAATG TGGTAGTAAC AGAGGGTGAC CTTAAGAAGG TACTCGACGG GTGTGCGCCT 300
    CTCACTAGGT TCACTAATAA ACTTAGAACG TTCGGTCGTA CTTTCACTGA GGCTTACGTT 360
    GACTTTTGTA TCGCGTATAA GCACAAATTA CCCCAACTCA ACGCCGCGGC GGAATTGGGG 420
    ATTCCAGCTG AAGATTCGTA CTTAGCTGCA GATTTTCTGG GTACTTGCCC GAAGCTCTCT 480
    GAATTACAGC AAAGTAGGAA GATGTTCGCG AGTATGTACG CTCTAAAAAC TGAAGGTGGA 540
    GTGGTAAATA CACCAGTGAG CAATCTGCGT CAGCTAGGTA GAAGGGAAGT TATGTAA 597
  • The coat protein has an amino acid sequence corresponding to SEQ. ID. No. 15 as follows:
    Met Glu Leu Met Ser Asp Ser Asn Leu Ser Asn Leu Val Ile Thr Asp
    1               5                   10                  15
    Ala Ser Ser Leu Asn Gly Val Asp Lys Lys Leu Leu Ser Ala Glu Val
                20                  25                  30
    Glu Lys Met Leu Val Gln Lys Gly Ala Pro Asn Glu Gly Ile Glu Val
            35                  40                  45
    Val Phe Gly Leu Leu Leu Tyr Ala Leu Ala Ala Arg Thr Thr Ser Pro
        50                  55                  60
    Lys Val Gln Arg Ala Asp Ser Asp Val Ile Phe Ser Asn Ser Phe Gly
    65                  70                  75                  80
    Glu Arg Asn Val Val Val Thr Glu Gly Asp Leu Lys Lys Val Leu Asp
                    85                  90                  95
    Gly Cys Ala Pro Leu Thr Arg Phe Thr Asn Lys Leu Arg Thr Phe Gly
                100                 105                 110
    Arg Thr Phe Thr Glu Ala Tyr Val Asp Phe Cys Ile Ala Tyr Lys His
            115                 120                 125
    Lys Leu Pro Gln Leu Asn Ala Ala Ala Glu Leu Gly Ile Pro Ala Glu
        130                 135                 140
    Asp Ser Tyr Leu Ala Ala Asp Phe Leu Gly Thr Cys Pro Lys Leu Ser
    145                 150                 155                 160
    Glu Leu Gln Gln Ser Arg Lys Met Phe Ala Ser Met Tyr Ala Leu Lys
                    165                 170                 175
    Thr Glu Gly Gly Val Val Asn Thr Pro Val Ser Asn Leu Arg Gln Leu
                180                 185                 190
    Gly Arg Arg Glu Val Met
            195

    and a molecular weight from about 20 to about 24 kDa, preferably about 22 kDa.
  • Another such DNA molecule (GLRaV-2 ORF7) includes nucleotides 14180-14665 of SEQ. ID. No. 1 and codes for a second undefined grapevine leafroll virus protein or polypeptide. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 16 as follows:
    ATGGAAGATT ACGAAGAAAA ATCCGAATCG CTCATACTGC TACGCACGAA TCTGAACACT  60
    ATGCTTTTAG TGGTCAAGTC CGATGCTAGT GTAGAGCTGC CTAAACTACT AATTTGCGGT 120
    TACTTACGAG TGTCAGGACG TGGGGAGGTG ACGTGTTGCA ACCGTGAGGA ATTAACAAGA 180
    GATTTTGAGG GCAATCATCA TACGGTGATC CGTTCTAGAA TCATACAATA TGACAGCGAG 240
    TCTGCTTTTG AGGAATTCAA CAACTCTGAT TGCGTAGTGA AGTTTTTCCT AGAGACTGGT 300
    AGTGTCTTTT GGTTTTTCCT TCGAAGTGAA ACCAAAGGTA GAGCGGTGCG ACATTTGCGC 360
    ACCTTCTTCG AAGCTAACAA TTTCTTCTTT GGATCGCATT GCGGTACCAT GGAGTATTGT 420
    TTGAAGCAGG TACTAACTGA AACTGAATCT ATAATCGATT CTTTTTGCGA AGAAAGAAAT 480
    CGTTAA 486
  • The second undefined grapevine leafroll virus protein or polypeptide has a deduced amino acid sequence corresponding to SEQ. ID. No. 17 as follows:
    Met Glu Asp Tyr Glu Glu Lys Ser Glu Ser Leu Ile Leu Leu Arg Thr
    1               5                   10                  15
    Asn Leu Asn Thr Met Leu Leu Val Val Lys Ser Asp Ala Ser Val Glu
                20                  25                  30
    Leu Pro Lys Leu Leu Ile Cys Gly Tyr Leu Arg Val Ser Gly Arg Gly
            35                  40                  45
    Glu Val Thr Cys Cys Asn Arg Glu Glu Leu Thr Arg Asp Phe Glu Gly
        50                  55                  60
    Asn His His Thr Val Ile Arg Ser Arg Ile Ile Gln Tyr Asp Ser Glu
    65                  70                  75                  80
    Ser Ala Phe Glu Glu Phe Asn Asn Ser Asp Cys Val Val Lys Phe Phe
                    85                  90                  95
    Leu Glu Thr Gly Ser Val Phe Trp Phe Phe Leu Arg Ser Glu Thr Lys
                100                 105                 110
    Gly Arg Ala Val Arg His Leu Arg Thr Phe Phe Glu Ala Asn Asn Phe
            115                 120                 125
    Phe Phe Gly Ser His Cys Gly Thr Met Glu Tyr Cys Leu Lys Gln Val
        130                 135                 140
    Leu Thr Glu Thr Glu Ser Ile Ile Asp Ser Phe Cys Glu Glu Arg Asn
    145                 150                 155                 160
    Arg

    and a molecular weight from about 17 to about 21 kDa, preferably about 19 kDa.
  • Yet another such DNA molecule (GLRaV-2 ORF8) includes nucleotides 14667-15284 of SEQ. ID. No. 1 and codes for a third undefined grapevine leafroll virus protein or polypeptide. This DNA molecule comprises a nucleotide sequence corresponding to SEQ. ID. No. 18 as follows:
    ATGAGGGTTA TAGTGTCTCC TTATGAAGCT GAAGACATTC TGAAAAGATC GACTGACATG  60
    TTACGAAACA TAGACAGTGG GGTCTTGAGC ACTAAAGAAT GTATCAAGGC ATTCTCGACG 120
    ATAACGCGAG ACCTACATTG TGCGAAGGCT TCCTACCAGT GGGGTGTTGA CACTGGGTTA 180
    TATCAGCGTA ATTGCGCTGA AAAACGTTTA ATTGACACGG TGGAGTCAAA CATACGGTTG 240
    GCTCAACCTC TCGTGCGTGA AAAAGTGGCG GTTCATTTTT GTAAGGATGA ACCAAAAGAG 300
    CTAGTAGCAT TCATCACGCG AAAGTACGTG GAACTCACGG GCGTGGGAGT GAGAGAAGCG 360
    GTGAAGAGGG AAATGCGCTG TCTTACCAAA ACAGTTTTAA ATAAAATGTC TTTGGAAATG 420
    GCGTTTTACA TGTCACCACG AGCGTGGAAA AACGCTGAAT GGTTAGAACT AAAATTTTCA 480
    CCTGTGAAAA TCTTTAGAGA TCTGCTATTA GACGTGGAAA CGCTCAACGA ATTGTGCGCC 540
    GAAGATGATG TTCACGTCGA CAAAGTAAAT GAGAATGGGG ACGAAAATCA CGACCTCGAA 600
    CTCCAAGACG AATGTTAA 618
  • The third undefined protein or polypeptide has a deduced amino acid sequence corresponding to SEQ. ID. No. 19 as follows:
    Met Arg Val Ile Val Ser Pro Tyr Glu Ala Glu Asp Ile Leu Lys Arg
    1               5                   10                  15
    Ser Thr Asp Met Leu Arg Asn Ile Asp Ser Gly Val Leu Ser Thr Lys
                20                  25                  30
    Glu Cys Ile Lys Ala Phe Ser Thr Ile Thr Arg Asp Leu His Cys Ala
            35                  40                  45
    Lys Ala Ser Tyr Gln Trp Gly Val Asp Thr Gly Leu Tyr Gln Arg Asn
        50                  55                  60
    Cys Ala Glu Lys Arg Leu Ile Asp Thr Val Glu Ser Asn Ile Arg Leu
    65                  70                  75                  80
    Ala Gln Pro Leu Val Arg Glu Lys Val Ala Val His Phe Cys Lys Asp
                    85                  90                  95
    Glu Pro Lys Glu Leu Val Ala Phe Ile Thr Arg Lys Tyr Val Glu Leu
                100                 105                 110
    Thr Gly Val Gly Val Arg Glu Ala Val Lys Arg Glu Met Arg Ser Leu
            115                 120                 125
    Thr Lys Thr Val Leu Asn Lys Met Ser Leu Glu Met Ala Phe Tyr Met
        130                 135                 140
    Ser Pro Arg Ala Trp Lys Asn Ala Glu Trp Leu Glu Leu Lys Phe Ser
    145                 150                 155                 160
    Pro Val Lys Ile Phe Arg Asp Leu Leu Leu Asp Val Glu Thr Leu Asn
                    165                 170                 175
    Glu Leu Cys Ala Glu Asp Asp Val His Val Asp Lys Val Asn Glu Asn
                180                 185                 190
    Gly Asp Glu Asn His Asp Leu Glu Leu Gln Asp Glu Cys
            195                 200                 205

    and a molecular weight from about 22 to about 26 kDa, preferably about 24 kDa.
  • Another DNA molecule of the present invention (GLRaV-2 3′ UTR) includes nucleotides 15285-15500 of SEQ. ID. No. 1 and comprises a nucleotide sequence corresponding to SEQ. ID. No. 23 as follows:
    ACATTGGTTA AGTTTAACGA AAATGATTAG TAAATAATAA ATCGAACGTG GGTGTATCTA  60
    CCTGACGTAT CAACTTAAGC TGTTACTGAG TAATTAAACC AACAAGTGTT GGTGTTAATGT 120
    GTATGTTGAT GTAGAGAAAA ATCCGTTTGT AGAACGGTGT TTTTCTCTTC TTTATTTTTA 180
    AAAAAAAAAT AAAAAAAAAA AAAAAAAAGC GGCCGC 216
  • Also encompassed by the present invention are fragments of the DNA molecules of the present invention. Suitable fragments capable of imparting grapevine leafroll resistance to grape plants are constructed by using appropriate restriction sites, revealed by inspection of the DNA molecule's sequence, to: (i) insert an interposon (Felley et al., “Interposon Mutagenesis of Soil and Water Bacteria: a Family of DNA Fragments Designed for in vitro Insertion Mutagenesis of Gram-negative Bacteria,” Gene, 52: 147-15 (1987), which is hereby incorporated by reference) such that truncated forms of the grapevine leafroll virus coat polypeptide or protein, that lack various amounts of the C-terminus, can be produced or (ii) delete various internal portions of the protein. Alternatively, the sequence can be used to amplify any portion of the coding region, such that it can be cloned into a vector supplying both transcription and translation start signals.
  • Suitable DNA molecules are those that hybridize to a DNA molecule comprising a nucleotide sequence of at least 15 continuous bases of SEQ. ID. No. 1 under stringent conditions characterized by a hybridization buffer comprising 0.9M sodium citrate (“SSC”) buffer at a temperature of 37° C. and remaining bound when subject to washing with SSC buffer at 37° C.; and preferably in a hybridization buffer comprising 20% formamide in 0.9M saline/0.9M SSC buffer at a temperature of 42° C. and remaining bound when subject to washing at 42° C. with 0.2×SSC buffer at 42° C.
  • Variants may also (or alternatively) be modified by, for example, the deletion or addition of nucleotides that have minimal influence on the properties, secondary structure and hydropathic nature of the encoded polypeptide. For example, the nucleotides encoding a polypeptide may be conjugated to a signal (or leader) sequence at the N-terminal end of the protein which co-translationally or post-translationally directs transfer of the protein. The nucleotide sequence may also be altered so that the encoded polypeptide is conjugated to a linker or other sequence for ease of synthesis, purification, or identification of the polypeptide.
  • The protein or polypeptide of the present invention is preferably produced in purified form (preferably, at least about 80%, more preferably 90%, pure), by conventional techniques. Typically, the protein or polypeptide of the present invention is isolated by lysing and sonication. After washing, the lysate pellet is resuspended in buffer containing Tris-HCl. During dialysis, a precipitate forms from this protein solution. The solution is centrifuged, and the pellet is washed and resuspended in the buffer containing Tris-HCl. Proteins are resolved by electrophoresis through an SDS 12% polyacrylamide gel.
  • The DNA molecule encoding the grapevine leafroll virus (type 2) protein or polypeptide of the present invention can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i.e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.
  • U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture.
  • Recombinant genes may also be introduced into viruses, such as vaccinia virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.
  • Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gt11, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/−or KS +/−(see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see Studier et. al., “Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,” Gene Expression Technology, vol. 185 (1990), which is hereby incorporated by reference), and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1982), which is hereby incorporated by reference.
  • A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); and plant cells infected by bacteria or transformed via particle bombardment (i.e. biolistics). The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.
  • Different genetic signals and processing events control many levels of gene expression (e.g., DNA transcription and messenger RNA (“mRNA”) translation).
  • Transcription of DNA is dependent upon the presence of a promoter which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promoters differ from those of procaryotic promoters. Furthermore, eucaryotic promoters and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promoters are not recognized and do not function in eucaryotic cells.
  • Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes. Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgamo (“SD”) sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymology, 68: 473 (1979), which is hereby incorporated by reference.
  • Promoters vary in their “strength” (i.e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promoters in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promoters may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promoters such as the T7 phage promoter, lac promoter, trp promoter, recA promoter, ribosomal RNA promoter, the PR and PL promoters of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli promoters produced by recombinant DNA or other synthetic DNA techniques may be used to provide for transcription of the inserted gene.
  • Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promoter unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.
  • Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in “strength” as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promoter, may also contain any combination of various “strong” transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno (“SD”) sequence about 7-9 bases 5′ to the initiation codon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.
  • Once the isolated DNA molecules encoding the various grapevine leafroll virus (type 2) proteins or polypeptides, as described above, have been cloned into an expression system, they are ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.
  • The present invention also relates to RNA molecules which encode the various grapevine leafroll virus (type 2) proteins or polypeptides described above. The transcripts can be synthesized using the host cells of the present invention by any of the conventional techniques. The mRNA can be translated either in vitro or in vivo. Cell-free systems typically include wheat-germ or reticulocyte extracts. In vivo translation can be effected, for example, by microinjection into frog oocytes.
  • One aspect of the present invention involves using one or more of the above DNA molecules encoding the various proteins or polypeptides of a grapevine leafroll virus (type 2) to transform grape plants in order to impart grapevine leafroll resistance to the plants. The mechanism by which resistance is imparted is not known. In one hypothetical mechanism, the transformed plant can express a protein or polypeptide of grapevine leafroll virus (type 2), and, when the transformed plant is inoculated by a grapevine leafroll virus, such as GLRaV-1, GLRaV-2, GLRav-3, GLRaV-4, GLRaV-5, or GLRaV-6, or combinations of these, the expressed protein or polypeptide prevents translation of the viral DNA.
  • In this aspect of the present invention the subject DNA molecule incorporated in the plant can be constitutively expressed. Alternatively, expression can be regulated by a promoter which is activated by the presence of grapevine leafroll virus. Suitable promoters for these purposes include those from genes expressed in response to grapevine leafroll virus infiltration.
  • The isolated DNA molecules of the present invention can be utilized to impart grapevine leafroll virus resistance for a wide variety of grapevine plants. The DNA molecules are particularly well suited to imparting resistance to Vitis scion or rootstock cultivars. Scion cultivars which can be protected include those commonly referred to as Table or Raisin Grapes, such as Alden, Almeria, Anab-E-Shahi, Autumn Black, Beauty Seedless, Black Corinth, Black Damascus, Black Malvoisie, Black Prince, Blackrose, Bronx Seedless, Burgrave, Calmeria, Campbell Early, Canner, Cardinal, Catawba, Christmas, Concord, Dattier, Delight, Diamond, Dizmar, Duchess, Early Muscat, Emerald Seedless, Emperor, Exotic, Ferdinand de Lesseps, Fiesta, Flame seedless, Flame Tokay, Gasconade, Gold, Himrod, Hunisa, Hussiene, Isabella, Italia, July Muscat, Khandahar, Katta, Kourgane, Kishmishi, Loose Perlette, Malaga, Monukka, Muscat of Alexandria, Muscat Flame, Muscat Hamburg, New York Muscat, Niabell, Niagara, Olivette blanche, Ontario, Pierce, Queen, Red Malaga, Ribier, Rish Baba, Romulus, Ruby Seedless, Schuyler, Seneca, Suavis (IP 365), Thompson seedless, and Thomuscat. They also include those used in wine production, such as Aleatico, Alicante Bouschet, Aligote, Alvarelhao, Aramon, Baco blanc (22A), Burger, Cabernet franc, Cabernet, Sauvignon, Calzin, Carignane, Charbono, Chardonnay, Chasselas dore, Chenin blanc, Clairette blanche, Early Burgundy, Emerald Riesling, Feher Szagos, Fernao Pires, Flora, French Colombard, Fresia, Furmint, Gamay, Gewurztraminer, Grand noir, Gray Riesling, Green Hungarian, Green Veltliner, Grenache, Grillo, Helena, Inzolia, Lagrein, Lambrusco de Salamino, Malbec, Malvasia bianca, Mataro, Melon, Merlot, Meunier, Mission, Montua de Pilas, Muscadelle du Bordelais, Muscat blanc, Muscat Ottonel, Muscat Saint-Vallier, Nebbiolo, Nebbiolo fino, Nebbiolo Lampia, Orange Muscat, Palomino, Pedro Ximenes, Petit Bouschet, Petite Sirah, Peverella, Pinot noir, Pinot Saint-George, Primitive di Gioa, Red Veltliner, Refosco, Rkatsiteli, Royalty, Rubired, Ruby Cabernet, Saint-Emilion, Saint Macaire, Salvador, Sangiovese, Sauvignon blanc, Sauvignon gris, Sauvignon vert, Scarlet, Seibel 5279, Seibel 9110, Seibel 13053, Semillon, Servant, Shiraz, Souzao, Sultana Crimson, Sylvaner, Tannat, Teroldico, Tinta Madeira, Tinto cao, Touriga, Traminer, Trebbiano Toscano, Trousseau, Valdepenas, Viognier, Walschriesling, White Riesling, and Zinfandel. Rootstock cultivars which can be protected include Couderc 1202, Couderc 1613, Couderc 1616, Couderc 3309, Dog Ridge, Foex 33 EM, Freedom, Ganzin 1 (A×R #1), Harmony, Kober 5BB, LN33, Millardet & de Grasset 41B, Millardet & de Grasset 420A, Millardet & de Grasset 101-14, Oppenheim 4 (SO4), Paulsen 775, Paulsen 1045, Paulsen 1103, Richter 99, Richter 110, Riparia Gloire, Ruggeri 225, Saint-George, Salt Creek, Teleki 5A, Vitis rupestris Constantia, Vitis california, and Vitis girdiana.
  • There exists an extensive similarity in the hsp70-related sequence regions of GLRaV-2 and other closteroviruses, such as tristeza virus and beet yellows virus. Consequently, the GLRaV-2 hsp70-related gene can also be used to produce transgenic plants or cultivars other than grape, such as citrus or sugar beet, which are resistant to closteroviruses other than grapevine leafroll, such as tristeza virus and beet yellows virus.
  • Suitable citrus cultivars include lemon, lime, orange, grapefruit, pineapple, tangerine, and the like, such as Joppa, Maltaise Ovale, Parson (Parson Brown), Pera, Pineapple, Queen, Shamouti, Valencia, Tenerife, Imperial Doblefina, Washington Sanguine, Moro, Sanguinello Moscato, Spanish Sanguinelli, Tarocco, Atwood, Australian, Bahia, Baiana, Cram, Dalmau, Eddy, Fisher, Frost Washington, Gillette, LengNavelina, Washington, Satsuma Mandarin, Dancy, Robinson, Ponkan, Duncan, Marsh, Pink Marsh, Ruby Red, Red Seedless, Smooth Seville, Orlando Tangelo, Eureka, Lisbon, Meyer Lemon, Rough Lemon, Sour Orange, Persian Lime, West Indian Lime, Bearss, Sweet Lime, Troyer Citrange, and Citrus Trifoliata. Each of these citrus cultivars is suitable for producing transgenic citrus plants resistant to tristeza virus.
  • The economically important species of sugar beet is Beta vulgaris L., which has four important cultivar types: sugar beet, table beet, fodder beet, and Swiss chard. Each of these beet cultivars is suitable for producing transgenic beet plants resistant to beet yellows virus, as described above.
  • Because GLRaV-2 has been known to infect tobacco plants (e.g., Nicotiana benthamiana), it is also desirable to produce transgenic tobacco plants which are resistant to grapevine leafroll viruses, such as GLRaV-2.
  • Plant tissue suitable for transformation include leaf tissue, root tissue, meristems, zygotic and somatic embryos, and anthers. It is particularly preferred to utilize embryos obtained from anther cultures.
  • The expression system of the present invention can be used to transform virtually any plant tissue under suitable conditions. Tissue cells transformed in accordance with the present invention can be grown in vitro in a suitable medium to impart grapevine leafroll virus resistance. Transformed cells can be regenerated into whole plants such that the protein or polypeptide imparts resistance to grapevine leafroll virus in the intact transgenic plants. In either case, the plant cells transformed with the recombinant DNA expression system of the present invention are grown and caused to express that DNA molecule to produce one of the above-described grapevine leafroll virus proteins or polypeptides and, thus, to impart grapevine leafroll virus resistance.
  • In producing transgenic plants, the DNA construct in a vector described above can be microinjected directly into plant cells by use of micropipettes to transfer mechanically the recombinant DNA. Crossway, Mol. Gen. Genetics, 202: 179-85 (1985), which is hereby incorporated by reference. The genetic material may also be transferred into the plant cell using polyethylene glycol. Krens, et al., Nature, 296: 72-74 (1982), which is hereby incorporated by reference.
  • One technique of transforming plants with the DNA molecules in accordance with the present invention is by contacting the tissue of such plants with an inoculum of a bacteria transformed with a vector comprising a gene in accordance with the present invention which imparts grapevine leafroll resistance. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28° C.
  • Bacteria from the genus Agrobacterium can be utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacterium tumefaciens (e.g., strains C58, LBA4404, or EHA 105) is particularly useful due to its well-known ability to transform plants.
  • Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the R1 plasmid of A. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science. 237: 1176-83 (1987), which is hereby incorporated by reference.
  • After transformation, the transformed plant cells must be regenerated.
  • Plant regeneration from cultured protoplasts is described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. III (1986), which are hereby incorporated by reference.
  • It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugarcane, sugar beets, cotton, fruit trees, and legumes.
  • Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.
  • After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • Once transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure so that the DNA construct is present in the resulting plants. Alternatively, transgenic seeds are recovered from the transgenic plants. These seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants.
  • Another approach to transforming plant cells with a gene which imparts resistance to pathogens is particle bombardment (also known as biolistic transformation) of the host cell. This can be accomplished in one of several ways. The first involves propelling inert or biologically active particles at cells. This technique is disclosed in U.S. Pat. Nos. 4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., and in Emerschad et al., “Somatic Embryogenesis and Plant Development from Immature Zygotic Embryos of Seedless Grapes (Vitis vinifera),” Plant Cell Reports, 14: 6-12 (1995) (“Emerschad (1995)”), which are hereby incorporated by reference. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells.
  • Once a grape plant tissue, citrus plant tissue, beet plant tissue, or tobacco plant tissue is transformed in accordance with the present invention, the transformed tissue is regenerated to form a transgenic plant. Generally, regeneration is accomplished by culturing transformed tissue on medium containing the appropriate growth regulators and nutrients to allow for the initiation of shoot meristems. Appropriate antibiotics are added to the regeneration medium to inhibit the growth of Agrobacterium and to select for the development of transformed cells. Following shoot initiation, shoots are allowed to develop tissue culture and are screened for marker gene activity.
  • The DNA molecules of the present invention can be made capable of transcription to a messenger RNA, which, although encoding for a grapevine leafroll virus (type 2) protein or polypeptide, does not translate to the protein. This is known as RNA-mediated resistance. When a Vitis scion or rootstock cultivar, or a citrus, beet, or tobacco cultivar, is transformed with such a DNA molecule, the DNA molecule can be transcribed under conditions effective to maintain the messenger RNA in the plant cell at low level density readings. Density readings of between 15 and 50 using a Hewlet ScanJet and Image Analysis Program are preferred.
  • A portion of one or more DNA molecules of the present invention as well as other DNA molecules can be used in a transgenic grape plant, citrus plant, beet plant, or tobacco plant in accordance with U.S. patent application Ser. No. 09/025,635, which is hereby incorporated herein by reference.
  • The grapevine leafroll virus (type 2) protein or polypeptide of the present invention can also be used to raise antibodies or binding portions thereof or probes. The antibodies can be monoclonal or polyclonal.
  • Monoclonal antibody production may be effected by techniques which are well-known in the art. Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a mammal (e.g., mouse) which has been previously immunized with the antigen of interest either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, Nature, 256: 495 (1975), which is hereby incorporated by reference.
  • Mammalian lymphocytes are immunized by in vivo immunization of the animal (e.g., a mouse) with the protein or polypeptide of the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Following the last antigen boost, the animals are sacrificed and spleen cells removed.
  • Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol (“PEG”) or other fusing agents. (See Milstein and Kohler, Eur. J. Immunol., 6: 511 (1976), which is hereby incorporated by reference.) This immortal cell line, which is preferably murine, but may also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described.
  • Procedures for raising polyclonal antibodies are also well known. Typically, such antibodies can be raised by administering the protein or polypeptide of the present invention subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The antigens can be injected at a total volume of 100 μl per site at six different sites. Each injected material will contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Ultimately, the rabbits are euthenized with pentobarbital 150 mg/Kg IV. This and other procedures for raising polyclonal antibodies are disclosed in Harlow et. al., editors, Antibodies: A Laboratory Manual (1988), which is hereby incorporated by reference.
  • In addition to utilizing whole antibodies, binding portions of such antibodies can be used. Such binding portions include Fab fragments, F(ab′)2 fragments, and Fv fragments. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in Goding, Monoclonal Antibodies: Principles and Practice, New York:Academic Press, pp. 98-118 (1983), which is hereby incorporated by reference.
  • The present invention also relates to probes found either in nature or prepared synthetically by recombinant DNA procedures or other biological procedures. Suitable probes are molecules which bind to grapevine leafroll (type 2) viral antigens identified by the monoclonal antibodies of the present invention. Such probes can be, for example, proteins, peptides, lectins, or nucleic acid probes.
  • The antibodies or binding portions thereof or probes can be administered to grapevine leafroll virus infected scion cultivars or rootstock cultivars. Alternatively, at least the binding portions of these antibodies can be sequenced, and the encoding DNA synthesized. The encoding DNA molecule can be used to transform plants together with a promoter which causes expression of the encoded antibody when the plant is infected by grapevine leafroll virus. In either case, the antibody or binding portion thereof or probe will bind to the virus and help prevent the usual leafroll response.
  • Antibodies raised against the GLRaV-2 proteins or polypeptides of the present invention or binding portions of these antibodies can be utilized in a method for detection of grapevine leafroll virus in a sample of tissue, such as tissue (e.g., scion or rootstock) from a grape plant or tobacco plant. Antibodies or binding portions thereof suitable for use in the detection method include those raised against a helicase, a methyltransferase, a papain-like protease, an RNA-dependent RNA polymerase, a heat shock 70 protein, a heat shock 90 protein, a coat protein, a diverged coat protein, or other proteins or polypeptides in accordance with the present invention. Any reaction of the sample with the antibody is detected using an assay system which indicates the presence of grapevine leafroll virus in the sample. A variety of assay systems can be employed, such as enzyme-linked immunosorbent assays, radioimmunoassays, gel diffusion precipitin reaction assays, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays, or immunoelectrophoresis assays.
  • Alternatively, grapevine leafroll virus can be detected in such a sample using a nucleotide sequence of the DNA molecule, or a fragment thereof, encoding for a protein or polypeptide of the present invention. The nucleotide sequence is provided as a probe in a nucleic acid hybridization assay or a gene amplification detection procedure (e.g., using a polymerase chain reaction procedure). The nucleic acid probes of the present invention may be used in any nucleic acid hybridization assay system known in the art, including, but not limited to, Southern blots (Southern, E. M., “Detection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresis,” J. Mol. Biol., 98: 503-17 (1975), which is hereby incorporated by reference), Northern blots (Thomas, P. S., “Hybridization of Denatured RNA and Small DNA Fragments Transferred to Nitrocellulose,” Proc. Nat'l Acad. Sci. USA, 77: 5201-05 (1980), which is hereby incorporated by reference), and Colony blots (Grunstein, M., et al., “Colony Hybridization: A Method for the Isolation of Cloned cDNAs that Contain a Specific Gene,” Proc. Nat'l Acad. Sci. USA, 72: 3961-65 (1975), which is hereby incorporated by reference). Alternatively, the probes can be used in a gene amplification detection procedure (e.g., a polymerase chain reaction). Erlich, H. A., et. al., “Recent Advances in the Polymerase Chain Reaction,” Science 252: 1643-51 (1991), which is hereby incorporated by reference. Any reaction with the probe is detected so that the presence of a grapevine leafroll virus in the sample is indicated. Such detection is facilitated by providing the probe of the present invention with a label. Suitable labels include a radioactive compound, a fluorescent compound, a chemiluminescent compound, an enzymatic compound, or other equivalent nucleic acid labels.
  • Depending upon the desired scope of detection, it is possible to utilize probes having nucleotide sequences that correspond with conserved or variable regions of the ORF or UTR. For example, to distinguish a grapevine leafroll virus from other related viruses (e.g., other closteroviruses), it is desirable to use probes which contain nucleotide sequences that correspond to sequences more highly conserved among all grapevine leafroll viruses. Also, to distinguish between different grapevine leafroll viruses (i.e., GLRaV-2 from GLRaV-1, GLRaV-3, GLRaV-4, GLRaV-5, and GLRaV-6), it is desirable to utilize probes containing nucleotide sequences that correspond to sequences less highly conserved among the different grapevine leafroll viruses.
  • Nucleic acid (DNA or RNA) probes of the present invention will hybridize to complementary GLRaV-2 nucleic acid under stringent conditions. Generally, stringent conditions are selected to be about 50° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. The Tm is dependent upon the solution conditions and the base composition of the probe, and may be calculated using the following equation: T m = ⁢ 79.8 ⁢ °C + ( 18.5 × Log ⁡ [ Na + ] ) + ( 58.4 ⁢ °C × % ⁡ [ G + C ] ) - ⁢ ( 820 / # ⁢ bp ⁢   ⁢ in ⁢   ⁢ duplex ) - ( 0.5 × % ⁢   ⁢ formamide )
    Nonspecific binding may also be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein-containing solutions, addition of heterologous RNA, DNA, and SDS to the hybridization buffer, and treatment with RNase. Wash conditions are typically performed at or below stringency. Generally, suitable stringent conditions for nucleic acid hybridization assays or gene amplification detection procedures are asas set forth above. More or less stringent conditions may also be selected.
    • EXAMPLES
  • The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.
    • Example 1 Northern Hybridization
  • Specificity of the selected clones was confirmed by Northern hybridization. Northern hybridization was performed after electrophoresis of the dsRNA of GLRaV-2 in 1% agarose non-denaturing condition gel. The agarose gel was denatured by soaking in 50 mM NaOH containing 0.4 M NaCl for 30 min, and then neutralized with 0.1 M Tris-HCl (PH7.5) containing 0.5 M NaCl for another 30 min. RNA was sandwich blotted overnight onto Genescreen™ plus membrane (Dupont NEN Research Product) in 10×SSC buffer and hybridized as described by the manufacturer's instructions (DuPont, NEN).
    • Example 2 Sequencing and Computer Assisted Nucleotide and Amino Acid Sequence Analysis
  • DNA inserts were sequenced in pBluescript SK+ by using T3 and T7 universal primers for the terminal region sequence and additional oligonucleotide primers designed according to the known sequence for the internal region sequence. Purification of plasmid DNA was performed by a modified mini alkaline-lysis/PEG precipitation procedure described by the manufacturer (Applied Biosystems, Inc.). Nucleotide sequencing was performed on both strands of cDNA by using ABI TaqDyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc.). Automatic sequencing was performed on an ABI373 Automated Sequencer (Applied Biosynstems, Inc.) at Cornell University, Geneva, N.Y.
  • The nucleotide sequences of GLRaV-2 were assembled and analyzed with the programs of EditSeq and SeqMan, respectively, of DNASTAR package (Madison, Wis.). Amino acid sequences deduced from nucleotide sequences and its encoding open reading frames were conducted using the MapDraw program. Multiple alignments of amino acid sequences, identification of consensus amino acid sequences, and generation of phylogenetic trees were performed using the Clustal method in the MegAlign program. The nucleotide and amino acid sequences of other closteroviruses were obtained with the Entrez Program; and sequence comparisons with nonredundant databases were searched with the Blast Program from the National Center for Biotechnology Information.
    • Example 3 Isolation of dsRNA
  • Several vines of GLRaV-2 infected Vitis vinifera cv Pinot Noir that originated from a central New York vineyard served as the source for dsRNA isolation and cDNA cloning. dsRNA was extracted from phloem tissue of infected grapevines according to the method described by Hu et al., “Characterization of Closterovirus-Like Particles Associated with Grapevine Leafroll Disease,” J. Phytopathology 128: 1-14 (1990), which is hereby incorporated by reference. Purification of the high molecular weight dsRNA (ca 15 kb) was carried out by electrophoretic separation of the total dsRNA on a 0.7% low melting point agarose gel and extraction by phenol/chloroform following the method described by Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Sping Harbor Laboratory Press, New York (1989), which is hereby incorporated by reference. Concentration of dsRNA was estimated with UV fluorescent density of an ethidium bromide stained dsRNA band in comparison with a known concentration of DNA marker.
    • Example 4 cDNA Synthesis and Cloning
  • cDNA synthesis was performed following the method initially described by Jelkmann et al., “Cloning of Four Plant Viruses From Small Quantities of Double-Stranded RNA,” Phytopathology 79: 1250-53 (1989) and modified by Ling et al., “The Coat Protein Gene of Grapevine Leafroll Associated Closterovirus-3: Cloning, Nucleotide Sequencing and Expression in Transgenic Plants,” Arch. Virology 142: 1101-16 (1997), both of which are hereby incorporated by reference. About 100 ng of high molecular weight dsRNA purified from low melting agarose gel was denatured in 20 mM methylmercuric hydroxide and incubated at room temperature for 10 min with 350 ng of random primers. First strand cDNA was synthesized by using avian myeloblastosis virus (AMV) reverse transcriptase. Second strand cDNA was obtained by using RNase H and E. coli DNA polymerase I. Double-stranded cDNA was blunt ended with T4 DNA polymerase and ligated with EcoR I adapters. The cDNA, which had EcoR I adapters at the ends, was activated by kinase reaction and ligated into Lambda ZAP II/EcoR I prepared arms following the manufacturer's instruction (Stratagene). The recombinant DNA was then packaged in vitro to Gigapack® II packaging extract (Stratagene). The packaged phage particles were amplified and titered according to the manufacturer's instruction.
  • Two kinds of probes were used to identify GLRaV-2 specific clones from the library. One type was prepared from the synthesized cDNA that was amplified by PCR after ligation to the specific EcoR I Uni-Amp™ adapters (Clontech); and the other type was DNA inserts or PCR products from already sequenced clones. Clones from the cDNA library were selected by colony-lifting hybridization onto the colony/plaque Screen membrane (NEN Research Product) with the probe described above. The probe was prepared by labeling with 32P [α-dATP] using Klenow fragment of E. coli DNA polymerase I. Prehybridization, hybridization, and washing steps were carried out at 65° C. according to the manufacturer's instruction (Dupont, NEN Research Product). Selected plaques were converted to recombinant pBluescript by in vivo excision method according to the manufacturer's instruction (Stratagene).
  • To obtain clones representing the extreme 3′-terminus of GLRaV-2, dsRNA was polyadenylated by yeast poly(A) polymerase. Using poly(A)-tailed dsRNA as template, cDNA was amplified by RT-PCR with oligo(dT)18 and a specific primer, CP-1/T7R, which is derived from the clone CP-1 and has a nucleotide sequence according to SEQ. ID. No. 20 as follows:
    TGCTGGAGCT TGAGGTTCTG C 21

    The resulting PCR product (3′-PCR) was cloned into a TA vector (Invitrogen) and sequenced.
  • As shown in FIG. 1A, a high molecular weight dsRNA of ca. 15 kb was consistently identified from GLRaV-2 infected grapevines, but not from healthy vines. In addition, several low molecular weight dsRNAs were also detected from infected tissue. The yield of dsRNA of GLRaV-2 was estimated between 5-10 ng/15 g phloem tissue, which was much lower than that of GLRaV-3 (Hu et al., “Characterization of Closterovirus-Like Particles Associated with Grapevine Leafroll Disease,” J. Phytopathology 128: 1-14 (1990), which is hereby incorporated by reference). Only the high molecular weight dsRNA that was purified from low melting point agarose gel was used for cDNA synthesis, cloning and establishment of the Lambda/ZAP II cDNA library.
  • Two kinds of probes were used for screening the cDNA library. The initial clones were identified by hybridization with Uni-Amp™ PCR-amplified cDNA as probes. The specificity of these clones (e.g., TC-1) ranging from 200 to 1,800 bp in size was confirmed by Northern hybridization to dsRNA of GLRaV-2 as shown in FIG. 1B. Additionally, over 40 different clones ranging form 800 to 7,500 bp in size were identified following hybridization with the probes generated from GLRaV-2 specific cDNA clones or from PCR products. Over 40 clones were then sequenced on the both strands (FIG. 2).
    • Example 5 Expression of the Coat Protein in E. coli and Immunoblotting
  • To determine that ORF6 was the coat protein gene of GLRaV-2, the complete ORF6 DNA molecule was subcloned from a PCR product and inserted into the fusion protein expression vector pMAL-C2 (New England Biolabs, Inc.). The specific primers used for the PCR reaction were CP-96F and CP-96R, in which an EcoR I or BamH I site was included to facilitate cloning. CP-96F was designed to include the start codon of the CP and comprises a nucleotide sequence according to SEQ. ID. NO. 21 as follows:
    • CGGAATTCAC CATGGAGTTG ATGTCCGACA G 31
  • CP-96R was 66 nucleotides downstream of the stop codon of the CP and comprises the nucleotide sequence corresponding to SEQ. ID. No.22 as follows:
    AGCGGATCCA TGGCAGATTC GTGCGTAGCA GTA 33

    The coat protein was expressed as a fusion protein with maltose binding protein (MBP) of E. coli under the control of a “tac” promoter and suppressed by the “lac” repressor. The MBP-CP fusion protein was induced by adding 0.3 mM isopropyl-β-D-thio-gloactopyranoside (IPTG) and purified by a one step affinity column according to the manufacturer's instruction (New England, Biolabs, Inc). The MBP-CP fusion protein or the coat protein cleaved from the fusion protein was tested to react with specific antiserum of GLRaV-2 (kindly provided by Dr. Charles Greif of INRA, Colmar, France) on Western blot according to the method described by Hu et al., “Characterization of Closterovirus-Like Particles Associated with Grapevine Leafroll Disease,” J. Phytopathology 128: 1-14 (1990), which is hereby incorporated by reference. In contrast, the non-recombinant plasmids or uninduced cells did not react to the antiserum of GLRaV-2.
    • Example 6 Sequence Analysis and Genome Organization of GLRaV-2
  • A total of 15,500 bp of the RNA genome of GLRaV-2 was sequenced and deposited in GenBank (accession number AF039204). About 85% of the total RNA genome was revealed from at least two different clones. The sequence in the coat protein gene region was determined and confirmed from several different overlapping clones. The genome organization of GLRaV-2, shown in FIG. 2, includes nine open reading frames (e.g., ORF1a, 1b-8).
  • ORF1a and ORF1b: Analysis of the amino acid sequence of the N-terminal portion of GLRaV-2 ORF 1 a encoded product revealed two putative papain-like protease domains, which showed significant similarity to the papain-like leader protease of BYV (Agranovsky et al., “Beet Yellows Closterovirus: Complete Genome Structure and Identification of a Papain-like Thiol Protease,” Virology 198: 311-24 (1994), which is hereby incorporated by reference). Thus, it allowed prediction of the catalytic cysteine and histidine residues for the putative GLRaV-2 protease. Upon alignment of the sequence of the papain-like protease of BYV with that of GLRaV-2, the cleavage site at residues Gly-Gly (amino acid 588-589) of BYV aligned with the corresponding alanine-glycine (Ala-Gly) and Gly-Gly dipeptide of GLRaV-2 (FIG. 3A). Cleavage at this site would result in a leader protein and a 234 kDa (2090 amino acid) C-terminal fragment consisting of MT and HEL domains. However, the region upstream of the papain-like protease domain in GLRaV-2 did not show similarity to the corresponding region of BYV. In addition, variability in the residues located at the scissible bond (Gly in the BYV and Ala in the GLRaV-2) was present. Similar variability of the cleavage site residue in the P-PRO domain has been described in LChV (Jelkmann et al., “Complete Genome Structure and Phylogenetic Analysis of Little Cherry Virus, a Mealybug-Transmissible Closterovirus. J. General Virology 78: 2067-71 (1997), which is hereby incorporated by reference).
  • Database searching with the deduced amino acid sequence of the ORF1a/1b encoded protein revealed a significant similarity to the MT, HEL and RdRP domains of the other closteroviruses. The region downstream of the P-PRO cleavage site showed a significant similarity (57.% identity in a 266-residues alignment) to the putative methyltransferase domain of BYV and contained all the conserved motifs typical of positive-strand RNA viral type I MTs (FIG. 3B). The C-terminal portion of the ORF1a was identified as a helicase domain, the sequence of which showed a high similarity (57.1% identity in a 315-residues alignment) to the helicase domain of BYV and contained the seven conserved motifs characteristic of the Superfamily I helicase of positive-strand RNA viruses (FIG. 3C) (Hodgman, “A New Superfamily of Replicative Proteins,” Nature 333: 22-23 (1988); Koonin and Dolja, “Evolution and Taxonomy of Positive-strand RNA Viruses: Implications of Comparative Analysis of Amino Acid Sequences,” Crit. Rev. in Biochem. and Mol. Biol. 28: 375-430 (1993), both of which are hereby incorporated by reference).
  • ORF1b encoded a 460 amino acid polypeptide with a molecular mass of 52, 486 Da, counting from the frameshifting site. Database searching with the RdRP showed a significant similarity to the RDRP domains of positive strand RNA viruses. Comparison of the RdRP domains of GLRaV-2 and BYV showed the presence of the eight conserved motifs of RdRP (FIG. 3D).
  • As shown in FIG. 8, a tentative phylogenetic tree of the RdRP of GLRaV-2 with respect to other closteroviruses shows that it is closely related to the monopartite closteroviruses BYV, BYSV, and CTV.
  • In closteroviruses, a +1 ribosomal frameshift mechanism has been suggested to be involved in the expression of ORF1b as a large fusion protein with ORF1a (Agranovsky et al., “Beet Yellows Closterovirus: Complete Genome Structure and Identification of a Papain-like Thiol Protease,” Virology 198: 311-24 (1994); Karasev et al., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,” Virologv 208: 511-20 (1995); Klaassen et al., “Genome Structure and Phylogenetic Analysis of Lettuce Infectious Yellows Virus, a Whitefly-Transmitted, Bipartite Closterovirus,” Virology 208: 99-110 (1995); Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221: 199-207 (1996); Jelkmann et al., “Complete Genome Structure and Phylogenetic Analysis of Little Cherry Virus, a Mealybug-Transmissible Closterovirus,” J. General Virology 78: 2067-71 (1997), all of which are hereby incorporated by reference). In the overlapping ORF1a/1b region of BYV, the slippery sequence of GGGUUUA and two hairpins structure (stem-loop and pseudoknot) are believed to result in a +1 frameshift (Agranovsky et al., “Beet Yellows Closterovirus: Complete Genome Structure and Identification of a Papain-like Thiol Protease,” Virology 198: 311-24 (1994), which is hereby incorporated by reference). None of these features are conserved in CTV and BYSV (Karasev et al., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,” Virology 208: 511-20 (1995); Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virologv 221: 199-207 (1996), both of which are hereby incorporated by reference), in which a ribosomal pausing at a terminator or at a rare codon was suggested to perform the same function. Comparisons of the nucleotide sequence of the C-terminal region of the helicase and the N-terminal region of RdRP of GLRaV-2 with the same region of other closteroviruses revealed a significant similarity to BYV, BYSV, and CTV. As shown in FIG. 4, the terminator UAG at the end of C′-terminal helicase of GLRaV-2 aligned with the terminator UAG of BYV and BYSV, and arginine CGG codon of CTV.
  • ORF2 encodes a small protein consisting of 171 bp (57 amino acid) with a molecular mass of 6,297 Da. As predicted, the deduced amino acid sequence includes a stretch of nonpolar amino acids, which is presumed to form a transmembrane helix. A small hydrophobic analogous protein is also present in BYV, BYSV, CTV, LIYV, and LChV (Agranovsky et al. “Nucleotide Sequence of the 3′-Terminal Half of Beet Yellows Closterovirus RNA Genome Unique Arrangement of Eight Virus Genes,” J. General Virology 72: 15-24 (1991); Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221: 199-207 (1996); Pappu et al., “Nucleotide Sequence and Organization of Eight 3′ Open Reading Frames of the Citrus Tristeza Closterovirus Genome,” Virology 199: 35-46 (1994); Klaassen et al., “Partial Characterization of the Lettuce Infectious Yellows Virus Genomic RNAs, Identification of the Coat Protein Gene and Comparison of its Amino Acid Sequence With Those of Other Filamentous RNA Plant Viruses,” J. General Virology 75: 1525-33 (1994); Jelkmann et al., “Complete Genome Structure and Phylogenetic Analysis of Little Cherry Virus, a Mealybug-Transmissible Closterovirus,” J. General Virology 78: 2067-71 (1997), all of which are hereby incorporated by reference).
  • ORF3 encodes a 600 amino acid polypeptide with a molecular mass of 65,111 Da, which is homologous to the HSP70 cellular heat shock protein. HSP70 is highly conserved among closteroviruses and is probably involved in ATPase activity and the protein to protein interaction for chaperone activity (Agranovsky et al. “The Beet Yellows Closterovirus p65 Homologue of HSP70 Chaperones has ATPase Activity Associated with its Conserved N-terminal Domain but Interact with Unfolded Protein Chains,” J. General Virology 78: 535-42 (1997); Agranovsky et al., “Bacterial Expression and Some Properties of the p65, a Homologue of Cell Heat Shock Protein HSP70 Encoded in RNA Genome of Beet Yellows Closterovirus,” Doklady Akademii Nauk. 340: 416-18 (1995); Karasev et al., “HSP70-Related 65-kDa Protein of Beet Yellows Closterovirus is a Microtubule-Binding Protein,” FEBS Letters 304: 12-14 (1992), all of which are hereby incorporated by reference). As shown in FIG. 5, alignment of the complete ORF3 of GLRaV-2 with HSP70 homolog of BYV revealed the presence of the eight conserved motifs. The percentage similarity of the HSP70 between GLRaV-2 and that of BYV, BYSV, CTV, LIYV, and LChV is 47.8%, 47.2%, 38.6%, 20.9%, and 17.7%, respectively.
  • ORF4 encodes a 551 amino acid protein with a molecular mass of 63,349 Da. Database searching with the ORF4 protein product did not identify similar proteins except those of its counterparts in closteroviruses, BYV (P64), BYSV (P61), CTV (P61), LIYV (P59), and LChV (P61). This protein is believed to be a putative heat shock 90 protein. As shown in FIG. 9, two conserved motifs which were present in BYV (Agranovsky et al. “Nucleotide Sequence of the 3′-Terminal Half of Beet Yellows Closterovirus RNA Genome Unique Arrangement of Eight Virus Genes,” J. General Virology 72: 15-24 (1991), which is hereby incorporated by reference) and CTV (Pappu et al., “Nucleotide Sequence and Organization of Eight 3′ Open Reading Frames of the Citrus Tristeza Closterovirus Genome,” Virology 199: 35-46 (1994), which is hereby incorporated by reference) were also identified in the ORF4 of GLRaV-2.
  • ORF5 and ORF6 encode polypeptides with molecular mass of 24,803 Da and 21,661 Da, respectively. The start codon for both ORFs is in a favorable context for translation. ORF6 was identified as the coat protein gene of GLRaV-2 based on the sequence comparison with other closteroviruses. The calculated molecular mass of the protein product of ORF6 (21,662 Da) is in good agreement with the previously estimated 22˜26 kDa based on SDS-PAGE (Zimmermann et al., “Characterization and Serological Detection of Four Closterovirus-like Particles Associated with Leafroll Disease on Grapevine,” J. Phytopathology 130: 205-18 (1990); Boscia et al., “Nomenclature of Grapevine Leafroll-Associated Putative Closteroviruses,” Vitis 34: 171-75 (1995), both of which are hereby incorporated by reference).
  • Database searching with the deduced amino acid sequence of the ORF6 of GLRaV-2 showed a similarity with the coat proteins of closteroviruses, BYV, BYSV, CTV, LIYV, LChV, and GLRaV-3. At the nucleotide level, the highest percentage similarity was with the coat protein of BYSV (34.8%); at the amino acid level, the highest percentage similarity was with the coat proteins of BYV (32.7%) and BYSV (32.7%). As shown in FIG. 6A, alignment of the amino acid sequence of the coat protein and coat protein duplicate of GLRaV-2 with respect to other closteroviruses revealed that the invariant amino acid residues (N. R. G. D.) were present in both ORF5 and ORF6 of GLRaV-2. Two of these amino acid residues (R and D) are believed to be involved in stabilization of molecules by salt bridge formation and proper folding in the most conserved core region of coat proteins of all filamentous plant viruses (Dolja et al., “Phylogeny of Capsid Proteins of Rod-Shaped and Filamentous RNA Plant Viruses Two Families With Distinct Patterns of Sequence and Probably Structure Conservation,” Virology 184: 79-86 (1991), which is hereby incorporated by reference).
  • Identification of ORF6 as the coat protein gene was further confirmed by Western blot following expression of a fusion protein, consisting of a 22 kDa of ORF6 CP and a 42 kDa of maltose binding protein, produced by transformed E. coli as described in Example 5 supra. As shown in FIG. 6B, the putative phylogenetic tree of the coat protein and coat protein duplicate of GLRaV-2 with those of other closteroviruses showed that GLRaV-2 is more closely related to aphid transmissible closteroviruses (BYV, BYSV, and CTV) (Candresse, “Closteroviruses and Clostero-like Elongated Plant Viruses,” in Encyclopedia of Virology, pp. 24248, Webster and Granoff, eds., Academic Press, New York (1994), which is hereby incorporated by reference) than to whitefly (LIYV) or mealybug transmissible closteroviruses (LChV and GLRaV-3) (Raine et al., “Transmission of the Agent Causing Little Cherry Disease by the Apple Mealybug Phenacoccus aceris and the Dodder Cuscuta Lupuliformis,” Canadian J. Plant Pathology 8: 6-11 (1986); Jelkmann et al., “Complete Genome Structure and Phylogenetic Analysis of Little Cherry Virus, a Mealybug-Transmissible Closterovirus,” J. General Virology 78: 2067-71 (1997); Rosciglione and Gugerli, “Transmission of Grapevine Leafroll Disease and an Associated Closterovirus to Healthy Grapevine by the Mealybug Planococcus ficus,” Phytoparasitica 17: 63 (1989); Engelbrecht and Kasdorf, “Transmission of Grapevine Leafroll Disease and Associated Closteroviruses by the Vine Mealybug planococcus-ficus,” Phytophlactica, 22: 341-46 (1990); Cabaleiro and Segura, 1997; Petersen and Charles, “Transmission of Grapevine Leafroll-Associated Closteroviruses by Pseudococcus longispinus and P. calceolariae. Plant Pathology 46: 509-15 (1997), all of which are hereby incorporated by reference).
  • ORF7 and ORF8 encode polypeptides of 162 amino acid with a molecular mass of 18,800 Da and of 206 amino acid with a molecular mass of 23,659 Da, respectively. Database searching with the ORF7 and ORF8 showed no significant similarity with any other proteins. Nevertheless, these genes were of similar in size and location as those observed in the sequence of other closteroviruses, BYV (P20, P21), BYSV (P18, P22), and LChV (P21, P27) (FIG. 7). However, conserved regions were not observed between the ORF7 or ORF8 and its counterparts in BYV, BYSV, and LChV.
  • The 3′ terminal untranslated region (3′-UTR) consists of 216 nucleotides. Nucleotide sequence analysis revealed a long oligo(A) tract close to the end of the GLRaV-2 genome which is similar to that observed in the genome of BYV and BYSV (Agranovsky et al. “Nucleotide Sequence of the 3′-Terminal Half of Beet Yellows Closterovirus RNA Genome Unique Arrangement of Eight Virus Genes,” J. General Virology 72: 15-24 (1991); Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221: 199-207 (1996), both of which are hereby incorporated by reference). The genome of BYV ends in CCC, BYSV, and CTV ends in CC with an additional G or A in the double-stranded replicative form of BYSV (Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221: 199-207 (1996), which is hereby incorporated by reference) and CTV (Karasev et al., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,” Virology 208: 511-20 (1995), which is hereby incorporated by reference), respectively. GLRaV-2 had CGC at the 3′ terminus of the genome. Recently, a conserved 60 nt cis-element was identified in the 3′-UTR of three monopartite closteroviruses, which included a prominent conserved stem and loop structure (Karasev et al., 1996). As shown in FIG. 10, alignment of the 3′-UTR sequence of GLRaV-2 with the same regions of BYV, BYSV, and CTV showed the presence of the same conserved 60 nt stretch. Besides this cis-element, conserved sequences were not found in the 3′ UTRs of GLRaV-2, BYV, BYSV, and CTV.
  • The closteroviruses studied so far (e.g., BYV, BYSV, CTV, LIYV, LChV, and GLRaV-3) have apparent similarities in genome organization, which include replication associated genes that consist of MT, HEL, and RdRP conserved domains and a five-gene array unique for closteroviruses (Dolja et al. “Molecular Biology and Evolution of Closteroviruses: Sophisticated Build-up of Large RNA Genomes,” Annual Rev. Photopathology 32: 261-85 (1994); Agranovsky “Principles of Molecular Organization, Expression, and Evolution of Closteroviruses: Over the Barriers,” Adv. in Virus Res. 47: 119-218 (1996); Jelkmann et al., “Complete Genome Structure and Phylogenetic Analysis of Little Cherry Virus, a Mealybug-Transmissible Closterovirus,” J. General Virology 78: 2067-71 (1997); Ling et al., “Nucleotide Sequence of the 3′ Terminal Two-Thirds of the Grapevine Leafroll Associated Virus-3 Genome Reveals a Typical Monopartite Closterovirus,” J. General Virology 79(5): 1289-1301 (1998), all of which are hereby incorporated by reference).
  • The above data clearly shows that GLRaV-2 is a closterovirus. In the genome of GLRaV-2, two putative papain-like proteases were identified and an autoproteolytic cleavage process was predicted. The replication associated proteins consisting of MT, HEL, and RdRP conserved motifs were also identified, which were phylogenetically closely related to the replication associated proteins of other closteroviruses. A unique gene array including a small hydrophobic transmembrane protein, HSP70 homolog, HSP90 homolog, diverged CP and CP was also preserved in GLRaV-2. In addition, the calculated molecular mass (21,661 Da) of the coat protein (ORF6) of GLRaV-2 is in good agreement with that of the other closteroviruses (22 to 28 kDa) (Martelli and Bar-Joseph, “Closteroviruses: Classification and Nomenclature of Viruses,” Fifth Report of the International Committee on Taxonomy of Viruses, Francki et al., eds., Springer-Verlag Wein, New York, p. 345-47 (1991); Candresse and Martelli, “Genus Closterovirus,” in Virus Taxonomy, Report of the International Committee on Taxonomy of Viruses, Murphy et al., eds., Springer-Verlag., NY, p. 461-63 (1995), both of which are hereby incorporated by reference). Two ORFs downstream of the CP are of similar, in size and location, to those observed in the genome of BYV. Furthermore, lack of a poly(A) tail at the 3′ end of GLRaV-2 is also in good agreement with other closteroviruses. Like all other closteroviruses, the expression of ORF1b is suspected to occur via a +1 ribosomal frameshift and the 3′ proximal ORFs are probably expressed via formation of a nested set of subgenomic RNAs. Since the slippery sequence, stem-loop and pseudoknot structure involved in the frameshift of BYV were absent in GLRaV-2, the +1 frameshift of GLRaV-2 might be the same as proposed for CTV (Karasev et al., “Complete Sequence of the Citrus Tristeza Virus RNA Genome,” Virology 208: 511-20 (1995), which is hereby incorporated by reference) and BYSV (Karasev et al., “Organization of the 3′-Terminal Half of Beet Yellow Stunt Virus Genome and Implications for the Evolution of Closteroviruses,” Virology 221: 199-207 (1996), which is hereby incorporated by reference).
  • Overall, GLRaV-2 is more closely related to monopartite closteroviruses BYV, BYSV, and CTV than to GLRaV-3 (FIG. 7) (Ling et al., “Nucleotide Sequence of the 3′ Terminal Two-Thirds of the Grapevine Leafroll Associated Virus-3 Genome Reveals a Typical Monopartite Closterovirus,” J. General Virology 79(5): 1289-1301 (1998), which is hereby incorporated by reference), even though the latter causes similar leafroll symptoms in grapevine (Rosciglione and Gugerli, “Maladies de l'Enroulement et du Bois Strie de la Vigne: Analyse Microscopique et Serologique (Leafroll and Stem Pitting of Grapevine: Microscopical and Serological Analysis),” Rev Suisse Viticult Arboricult Horticulture 18: 207-11 (1986); Hu et al., “Characterization of Closterovirus-Like Particles Associated with Grapevine Leafroll Disease,” J. Phytopathology 128: 1-14 (1990), both of which are hereby incorporated by reference).
  • Closteroviruses are a diverse group with complex and heterogeneous genome organizations. So far, GLRaV-2 is the only closterovirus that matches with the genome organization of BYV, the type member of the genus Closterovirus. In addition, the genomic RNA of GLRaV-2 is about the same size as that of BYV; however, the transmission vector of GLRaV-2 is unknown. The genome organization of GLRaV-2 is more closely related to the aphid transmissible closteroviruses (BYV and CTV) than to whitefly (LIYV) or mealybug transmissible closteroviruses (LChV and GLRaV-3). Thus, it is possible that GLRaV-2 is transmitted by aphids. Aphid transmission experiments with GLRaV-2 should provide information that might help develop methods for further control of GLRaV-2.
  • A total of 15,500 nucleotides or over 95% of the estimated GLRaV-2 genome has been cloned and sequenced. GLRaV-2 and GLRaV-3 (Ling et al., “Nucleotide Sequence of the 3′ Terminal Two-Thirds of the Grapevine Leafroll Associated Virus-3 Genome Reveals a Typical Monopartite Closterovirus,” J. General Virology 79(5): 1289-1301 (1998), which is hereby incorporated by reference) are the first grapevine leafroll associated closteroviruses that have been almost completely sequenced. The above data clearly justify the inclusion of GLRaV-2 into the genus Closterovirus. In addition, the information regarding the genome of GLRaV-2 would provide a better understanding of this and related GLRaVs, and add fundamental knowledge to the group of closteroviruses.
    • Example 7 Construction of the CP Gene of GLRaV-2 in Plant Expression Vector
  • GLRaV-2 infected Vitis vinifera, cv Pinot Noir grapevines originated from a vineyard in central New York was used as the virus isolate, from which the cp gene of GLRaV-2 was identified. Based on the sequence information, two oligonucleotide primers have been designed. The sense primer CP-96F (SEQ. ID. No. 21) starts from the ATG initiation codon of the coat protein gene and the complementary primer CP-96R (SEQ. ID. No. 22) starts from 56 nucleotides downstream of the stop codon of the CP gene. A Nco I restriction site (11 bp in SEQ. ID. No. 21 and 13 bp in SEQ. ID. No. 22) is introduced in the beginning of both primers to facilitate the cloning. The coat protein gene of GLRaV-2 was amplified from dsRNA extracted from GLRaV-2 infected grapevine using reverse transcriptase polymerase chain reaction (RT-PCR). The PCR-amplified CP product was purified from low melting temperature agarose gel, digested with Nco I and cloned into the same enzyme digested plant expression vector pEPT8 (shown at FIG. 11). After screening, the orientation of recombinant construct was checked by using the internal restriction site of the CP gene and directly sequencing the CP gene. The recombinant construct with translatable (sense) full length coat protein gene, pEPT8CP-GLRaV2, was going through for the further cloning. The plant expression cassette, which consisted of a double cauliflower mosaic virus (CaMV) 35S-enhancer, a CaMV 35S-promoter, an alfalfa mosaic virus (ALMV) RNA4 5′ leader sequence, a coat protein gene of GLRaV-2 (CP-GLRaV-2), and a CaMV 35S 3′ untranslated region as a terminator, was cut using the EcoR I restriction enzyme, isolated from low melting point temperature agarose gel, and cloned into the same restriction enzyme treated binary vector pGA482GG or pGA482G (a derivative of pGA482 (An et al., “Binary Vectors,” in Plant Molecular Biology Manual, pp. A3: 1-19, Gelvin and Schilperoot, eds., Kinwer Academic Publishers, Dordrecht, Netherlands (1988), which is hereby incorporated by reference). The resulting recombinants constructs are pGA482GG/EPT8CP-GLRaV2 (shown at FIG. 11A), which contain both neomycin phosphotransferase (npt II) and P-glucuronidase (GUS) at the internal region of the T-DNA, and pGA482G/EPT8CP-GLRaV2 (shown at FIG. 11B) without GUS. These recombinants constructs were separately introduced by electroporation into disarmed avirulent Agrobacterium tumefaciens strain C58Z707. The Agrobacterium tumefaciens containing the vector was used to infect Nicotiana benthamiana wounded leaf disks according to the procedure essentially described by Horsch et al., “A Simple and General Method for Transferring Genes into Plants,” Science 277: 1229-1231 (1985), which is incorporated herein by reference.
    • Example 8 Analysis of Transgenic Nicotiana benthamiana Plants with the CP Gene of GLRaV-2
  • NPT II-ELISA: Double-antibody sandwich enzyme linked immuosorbent assay (DAS-ELISA) was used to detect the npt II enzyme with an NPT II-ELISA kit (5′ prime to 3′ prime, Inc., Boulder, Co.).
  • Indirect ELISA: Polyclonal antibodies to GLRaV-2, which were prepared from the coat protein expressed in E. coli, were used. Plates were coated with homogenized samples in extraction buffer (1:10, w/v) (phosphate buffered saline containing 0.05 % Tween 20 and 2% polyvinyl pyrrolidone) and incubated overnight at 4° C. After washing with phosphate buffered saline containing 0.05% Tween 20 (PBST), the plates were blocked with blocking buffer (phosphate buffered saline containing 2% BSA) and incubated at room temperature for 1 hr. The anti-GLRaV-2 IgG was added at 2 μg/ml after washing with PBST. After incubation at 30 C for 4 hr, the plates were washed with PBST, and the goat anti-rabbit IgG conjugate of alkaline phosphotase (Sigma) was added at 1:10,000 dilution. The absorbance was measured at 405 nm with a MicroELISA AutoReader. In addition, Western blot was also performed according to the method described by Hu et al., “Characterization of Closterovirus-like Particle Associated Grapevine Leafroll Disease,” J. Phytophathology 128: 1-14, (1990), which is incorporated herein by reference.
  • PCR analysis: Genomic DNA was extracted from leaves of putative transgenic and non-transgenic plants according to the method described by Cheung et al., “A Simple and Rapid DNA Microextraction Method for Plants, Animal, and Insect Suitable for RAPD and other PCR analysis,” PCR Methods and Applications 3: 69 (1996), which is incorporated herein by reference. The extracted total DNA served as the template for PCR reaction. The primers CP-96F and CP-96R (SEQ. ID. Nos. 21 and 22, respectively) for the CP gene of GLRaV-2, as well as npt II 5′- and 3′-primers were used for PCR analysis. PCR reaction was performed at the 94° C.×3 min for one cycle, followed by 30 cycles of 94° C.×1 min, 50° C.×1 min, and 72° C.×2:30 min with an additional extension at 72° C. for 10 min. The PCR product was analyzed on agarose gel.
  • After transformation, a total of 42 kanamycin resistant Nicotiana benthamiana lines (R0) were obtained, of which the leaf samples were tested by NPT II enzyme activity. Among them, 37 lines were NPT II positive by ELISA, which took about 88.0% of total transformants. However, some of NPT II negative plants were obtained among these selected kanamycin resistant plants. All of the transgenic plants were self-pollinated in a greenhouse, and the seeds from these transgenic lines were germinated for further analysis.
  • The production of GLRaV-2 CP in transgenic plants was detected by indirect ELISA prior to inoculation, and the results showed that GLRaV-2 CP gene expression was not detectable in all transgenic plants tested. This result was further confirmed with Western blot. Using the antibody to GLRaV-2, the production of the CP was not detected in the transgenic and nontransgenic control plants. However, a protein of expected size (˜22 kDa) was detected in GLRaV-2 infected positive control plants. This result was consistent with the ELISA result. The presence of the CP gene of GLRaV-2 in transgenic plants was detected from total genomic DNA extracted from plants tissue by PCR analysis (FIG. 12). The DNA product of expected size (653 bp) was amplified from twenty tested transgenic lines, but not in non-transgenic plants. The result indicated that the CP gene of GLRaV-2 was present at these transgenic lines, which was also confirmed by Northern blot analysis.
    • Example 9 R1 and R2 transgenic Nicotiana benthamiana Plants Are Resistant to GLRaV-2
  • Inoculation of transgenic plants: GLRaV-2 isolate 94/970, which was originally identified and transmitted from grapevine to Nicotiana benthamiana in South Africa (Goszczynski et al., “Detection of Two Strains of Grapevine Leafroll-Associated Virus 2,” Vitis 35: 133-35 (1996), which is incorporated herein by reference), was used as inoculum. The CP gene of isolate 94/970 was sequenced; and it is identical to the CP gene used in construction. Nicotiana benthamiana is an experimental host of GLRaV-2. The infection on it produces chlorotic and occasional necrotic lesions followed by systemic vein clearing. The vein clearing results in vein necrosis. Eventually the infected plants died, starting from the top to the bottom.
  • At five to seven leaf stage, two youngest apical leaves were challenged with GLRaV-2 isolate 94/970. Inoculum was prepared by grinding 1.0 g GLRaV-2 infected Nicotiana benthamiana leaf tissue in 5 ml of phosphate buffer (0.01 MK2HPO4, PH7.0). The tested plants were dusted with carborundum and rubbed with the prepared inoculum. Non-transformed plants were simultaneously inoculated as above. The plants were observed for symptom development every other day for 60 days after inoculation. Resistant R1 transgenic plants were carried on to R2 generation for further evaluation.
  • Transgenic progenies from 20 R0 lines were initially screened for the resistance to GLRaV-2 followed by inoculation with GLRaV-2 isolate 94/970. The seedlings of the transgenic plants (NPT II positive), and nontransformed control plants were inoculated with GLRaV-2. After inoculation, the reaction of tested plants were divided into three types: highly susceptible (i.e. typical symptoms were observed two to four weeks postinoculation); tolerant (i.e. no symptom was developed in the early stage and typical symptoms was shown four to eight weeks postinoculation); and resistant (i.e. the plants remained asymptomatic eight weeks postinoculation). Based on the plant reaction, the resistant plants were obtained from fourteen different lines (listed in Table 1 below). In each of these fourteen lines, there was no virus detected within these plants by ELISA at 6 weeks postinoculation. In contrast, GLRaV-2 was detected in symptomatic plants by indirect ELISA. In the other six lines, although there were a few plants with some kind of delay in symptom development, all the inoculated transgenic plants died at three to eight weeks postinoculation. Based on the initial screening results, five representative lines consisting of three resistant lines (1, 4, and 19) and two susceptible lines (12 and 13) were selected for the further analysis.
    TABLE 1
    Reaction of Tested Plants
    No. Line No. HS T HR
    line
    1 39 14 3 22
    line 2 36 7 6 23
    line 3 38 11 4 23
    line 4 31 4 5 22
    line 5 33 6 13 14
    line 6 36 4 16 16
    line 7 32 5 9 18
    line 8 37 22 9 6
    line 9 36 9 12 15
    line 10 14 13 1 0
    line 11 13 11 2 0
    line 12 17 16 1 0
    line 13 16 14 0 0
    line 14 17 17 0 0
    line 15 32 30 2 0
    line 16 33 6 13 14
    line 17 12 0 1 11
    line 19 15 0 0 15
    line 20 19 3 0 16
    line 21 14 1 3 10
    control 15 15 0 0

    No Line: include transgenic lines and nontransformed control;

    No: the number of transgenic and nontransformed plants;

    HS: highly susceptible, typical symptoms were observed two to four weeks after inoculation;

    T: tolerant, the symptoms were observed five to eight weeks after inoculation; and

    HR: plants remain without asymptoms after eight weeks inoculation.
  • Table 2 below shows the symptom development in transgenic plants relative to non-transgenic control plants in the five selected lines in separate experiments. Non-transgenic control plants were all infected two to four weeks after inoculation, which showed typical GLRaV-2 symptoms on Nicotiana benthamiana, including chlorotic and local lesions followed by systemic vein clearingand vein necrosis on the leaves. Three of the tested lines (1, 4, and 19) showed some resistance that was manifested by either an absence or a delay in symptom development. Two other lines, 12 and 13, developed symptoms at nearly the same time as the non-transformed control plants. From top to bottom, the leaves of infected plants gradually became yellow, wilted, and dried, and, eventually, the whole plants died. No matter when infection occurred, the eventual result was the same. Six weeks after inoculation, all non-transgenic plants and the susceptible plants were dead. Some tolerant plants started to die. In contrast, the asymptomatic plants were flowering normally and pollinating as the non-inoculated healthy control plants (FIG. 13).
    TABLE 2
    Reaction of Tested Plants
    No. Line No. HS T HR
    line
    1 19 5 6 8
    line 4 15 9 1 5
    line 12 16 14 2 0
    line 13 18 13 5 0
    line 19 13 10 0 3
    non-transgenic 24 23 1 0

    No. Line: include transgenic lines and nontransformed control;

    No.: Number of transgenic and nontransformed plants tested;

    HS: highly susceptible; typical symptoms were observed two to four weeks after inoculation;

    T: tolerant, the symptoms were observed five to eight weeks postinoculation; and

    HR: plants remain without asymptoms after eight weeks inoculation.
  • ELISA was performed at 6 weeks postinoculation to test the GLRaV-2 replication in the plants. Presumably, the increased level of CP reflected virus replication. The result showed that the absorbance value in symptomatic plants reached (OD) 0.7 to 3.2, compared to (OD) 0.10-0.13 prior to inoculation. In contrast, GLRaV-2 was not detected in asymptomatic plants, of which the absorbance value was the same or nearly the same as that of healthy nontransformed control plants. The data confirmed that virus replicated in symptomatic plants, but not in asymptomatic plants. The replication of GLRaV-2 was suppressed in asymptomatic plants. This result implicated that another mechanism other than the CP-mediated resistance was probably involved.
  • Three R2 progenies derived from transgenic resistant plants of lines 1, 4, and 19 were generated and utilized to examine the stable transmission and whether resistance was maintained in R2 generation. These results are shown in Table 3 below. NPT II analysis revealed that R2 progeny were still segregating. The CP expression in R2 progeny was still undetectable. After inoculation, all the nontransgenic plants were infected and showed GLRaV-2 symptoms on the leaves after 24 days postinoculation. In contrast, the inoculated transgenic R2 progeny showed different levels of resistance from those highly susceptible to highly resistant. The tolerant and resistant plants were manifested by a delay in symptom development and absence of symptoms, respectively. At 6 weeks postinoculation, GLRaV-2 was detected in the tolerant symptomatic infected plants by indirect ELISA; but not in asymptomatic plants. This result indicated that virus replication was suppressed in these resistant plants, which was confirmed by Western blot. These resistant plants remained asymptomatic eight weeks postinoculation, and they were flowering normally and pollinating.
    TABLE 3
    Reaction of
    NPT II Tested Plants
    No. Line No. Plants positive/negative HS T HR
    line
    1/22 12 12/20 3 3 6
    line 1/30 11 8/3 7 2 2
    line 1/31 11 10/1  6 3 2
    line 1/35 10 10/0  4 6 0
    line 1/41 8 7/1 2 2 4
    line 4/139 12 11/1  4 4 3
    line 4/149 10 7/3 4 5 1
    line 4/152 10 8/2 9 0 1
    line 4/174 9 8/1 4 0 4
    line 19/650 11 10/1  7 0 2
    line 19/657 12 12/0  6 2 4
    line 19/659 12 8/4 5 2 5
    line 19/660 10 8/2 3 6 1
    non-transformed 12  0/12 12 0 0
    CK

    HS: highly susceptible, typical symptoms were observed two to four weeks after inoculation;

    T: tolerant, the symptoms were observed five to eight weeks postinoculation; and

    HR: plants remain asymptomatic at eight weeks postinoculation.
    • Example 10 Evidence for RNA-Mediated Protection in Transgenic Plants
  • Northern blot analysis: Total RNA was extracted from leaves prior to inoculation following the method described by Napoli et al., Plant Cell 2: 279-89 (1990), which is hereby incorporatd by reference. The concentration of the extracted RNA was measured by spectrophotometer at OD 260. About 10 g of total RNA was used for each sample. The probe used was the 3′ one third of GLRaV-2 CP gene, which was randomly labeled with 32P (α-dATP) using Klenow fragment of DNA polymerase I.
  • Using a DNA corresponding to the 3′ one third CP gene sequence as probe, a single band was detected in the RNA extracted from susceptible plants from R1 progeny of lines 5, 12, and 13 by Northern hybridization. There was little or no signal detected in the transgenic plants from R1 progeny of line 1, 4, and 19. This RNA is not present in nontransformed control plants. The size of the hybridization signal was estimated to an approximately 0.9 kb nucleic acid, which was about the same as estimated (FIG. 14). In lines of 1, 4, and 19, the steady state level of RNA expression was also low in R2 progeny. This data showed that susceptible plants from lines 12 and 13 had high mRNA level and all transgenic plants from lines 1, 4, and 19 had low mRNA level.
    • Example 11 Transformation and Analysis of Transgenic Grapevines with the CP Gene of GLRaV-2
  • Plant materials: The rootstock cultivars Couderc 3309 (3309C) (V. riparia×V rupestris), Vitis riparia ‘Gloire de Montpellier’ (Gloire), Teleki 5C (5C) (V. berlandieri×V. riparia), Millardet et De Grasset 101-14 (101-14 MGT) (V. riparia×V. rupestris), and Richter 110 (10R) (V. rupestris×V. berlandieri) were utilized. Initial embryogenic calli of Gloire were provided by Mozsar and Süle (Plant Protection Institute, Hungarian Academy of Science, Budapest). All other plant materials came from a vineyard at the New York State Agricultural Experiment Station, Geneva, N.Y. Buds were removed from the clusters and surface sterilized in 70% ethanol for 1-2 min. The buds (from the greenhouse and the field) were transferred to 1% sodium hypochlorite for 15 min, then rinsed three times in sterile, double-distilled water. Anthers were excised aseptically from flower buds with the aid of a stereo microscope. The pollen was crushed on a microscope slide under a coverslip with a drop of acetocarmine to observe the cytological stage. This was done to determine which stage was most favorable for callus induction.
  • Somatic embryogenesis and regeneration: Anthers were plated under aseptic conditions at a density of 40 to 50 per 9 cm diameter Petri dish containing MSE. Plates were cultured at 28° C. in the dark. Callus was initiated, and, after 60 days, embryos were induced and were transferred to hormone-free HMG medium for differentiation. Torpedo stage embryos were then transferred from HMG to MGC medium to promote embryo germination. Cultures were maintained in the dark at 26-28° C. and transferred to fresh medium at 3-4 week intervals. Elongated embryos were transferred to rooting medium in baby food jars (5-8 embryos per jar). The embryos were grown in a tissue culture room at 25° C. with a daily 16 h photoperiod (76:mol. s) to induce shoot and root formation. After plants developed roots, they were transplanted to soil in the greenhouse.
  • Transformation: The protocols used for transformation were modified from those described by Scorza et.al., “Transformation of Grape (Vitis vinifera L.) Zygotic-derived Somatic Embryos and Regeneration of Transgenic Plants,” Plant Cell Rpt. 14: 589-92 (1995), which is hereby incorporated by reference. Overnight cultures of Agrobacterium strain C58Z707 or LBA4404 were grown in LB medium at 28° C. in a shaking incubator. Bacteria were centrifuged for 5 min at 3000-5000 rpm and resuspended in MS liquid medium (OD 1.0 at A600 nm). Calli with embryos were immersed in the bacterial suspension for 15-30 min blotted dry, and transferred to HMG medium with or without acetosyringone (100 μM).
  • Embryogenic calli were co-cultivated with the bacteria for 48 h in the dark at 28° C. Then, the plant material was washed in MS liquid plus cefotaxime (300 mg/ml) and carbenicillin (200 mg/ml) 2-3 times. To select transgenic embryos, the material was transferred to HMG medium containing either 20 or 40 mg/L kanamycin, 300 mg/L cefotaxime, and 200 mg/L carbenicillin. Alternatively, after co-cultivation, embryogenic calli were transferred to initiation MSE medium containing 25 mg/l kanamycin plus the same antibiotics listed above. All plant materials were incubated in continuous dark at 28° C. After growth on selection medium for 3 months, embryos were transferred to HMG or MGC without kanamycin to promote elongation of embryos. They were then transferred to rooting medium without antibiotics. Nontransformed calli were grown on the same media with and without kanamycin to verify the efficiency of the kanamycin selection process.
  • Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

Claims (20)

1. An isolated RNA molecule encoding protein or polypeptide of a grapevine leafroll virus (type 2).
2. The isolated RNA molecule according to claim 1, wherein the protein or polypeptide is selected from a group consisting of a polyprotein, an RNA-dependent RNA polymerase, a heat shock 70 protein, a heat shock 90 protein, a diverged coat protein, and a coat protein.
3. An isolated DNA molecule encoding a protein or polypeptide of a grapevine leafroll virus (type 2).
4. The isolated DNA molecule according to claim 3, wherein the protein or polypeptide is selected from a group consisting of a polyprotein, an RNA-dependent RNA polymerase, a heat shock 70 protein, a heat shock 90 protein, a diverged coat protein, and a coat protein.
5. An expression system comprising a DNA molecule according to claim 3 in a-vector heterologous to the DNA molecule.
6. The expression system according to claim 5, wherein the protein or polypeptide is selected from a group consisting of a polyprotein, an RNA-dependent RNA polymerase, a heat shock 70 protein, a heat shock 90 protein, a diverged coat protein, and a coat protein.
7. A host cell transformed with a heterologous DNA molecule according to claim 3.
8. The host cell according to claim 7, wherein the host cell is selected from the group consisting of Agrobacterium vitis and Agrobacterium tumefaciens.
9. The host cell according to claim 7, wherein the host cell is selected from a group consisting of a grape cell, a citrus cell, a beet cell, and a tobacco cell.
10. The host cell according to claim 7, wherein the protein or polypeptide is selected from a group consisting of a polyprotein, an RNA-dependent RNA-polymerase, a heat shock 70 protein, a heat shock 90 protein, a diverged coat protein, and a coat protein.
11. A transgenic plant cultivar comprising the DNA molecule according to claim 3.
12. The transgenic plant cultivar according to claim 11, wherein the plant cultivar is selected from a group consisting of a grape plant cultivar, a citrus plant cultivar, a beet plant cultivar, and a tobacco plant cultivar.
13. The transgenic plant cultivar according to claim 11, wherein the protein or polypeptide is selected from a group consisting of a polyprotein, an RNA-dependent RNA polymerase, a heat shock 70 protein, a heat shock 90 protein, a diverged coat protein, and a coat protein.
14. A method of imparting grapevine leafroll virus resistance to a Vitis scion or rootstock cultivar or a Nicotiana cultivar comprising the steps of:
(a) transforming of cells of a Vitis scion or rootstock cultivar or cells of a Nicotiana cultivar with an isolated DNA molecule encoding a protein or polypeptide of a grapevine leafroll virus (type 2); and
(b) regenerating a Vitis scion or rootstock cultivar or a Nicotiana cultivar from said transformed cells.
15. The method according to claim 14, wherein the protein or polypeptide is selected from a group consisting of a polyprotein, an RNA-dependent RNA polymerase, a heat shock 70 protein, a heat shock 90 protein, and a coat protein.
16. The method according to claim 14, wherein the protein or polypeptide is a grapevine leafroll virus (type 2) coat protein.
17. The method according to claim 14, wherein the protein or polypeptide is a grapevine leafroll virus (type 2) heat shock 70 protein.
18. The method according to claim 14, wherein the grapevine leafroll virus is GLRaV-2.
19. The method according to claim 14, wherein said transforming is Agrobacterium mediated.
20. The method according to claim 14, wherein said transforming comprises:
propelling particles at grape or tobacco plant cells under conditions effective for the particles to penetrate into the cell interior; and
introducing an expression vector comprising the DNA molecule into the cell interior.
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