EP1307572A2 - Materiels et methodes destines a l'administration et l'expression d'adn heterologue dans des cellules de vertebres - Google Patents

Materiels et methodes destines a l'administration et l'expression d'adn heterologue dans des cellules de vertebres

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Publication number
EP1307572A2
EP1307572A2 EP01962121A EP01962121A EP1307572A2 EP 1307572 A2 EP1307572 A2 EP 1307572A2 EP 01962121 A EP01962121 A EP 01962121A EP 01962121 A EP01962121 A EP 01962121A EP 1307572 A2 EP1307572 A2 EP 1307572A2
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European Patent Office
Prior art keywords
seq
polynucleotide
amepv
fragment
protein
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German (de)
English (en)
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Richard W. Moyer
Yi Li
Alison Louise Bawden
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University of Florida
University of Florida Research Foundation Inc
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University of Florida
University of Florida Research Foundation Inc
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Priority claimed from US09/662,254 external-priority patent/US6933145B1/en
Application filed by University of Florida, University of Florida Research Foundation Inc filed Critical University of Florida
Publication of EP1307572A2 publication Critical patent/EP1307572A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/24043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the subject invention was made with government support under a research project supported by U.S. Department of Agriculture Grant No. 97-35302-4431 and National Institute of Health Grant No. P50-HL59412-01. The government has certain rights in this invention.
  • Gene therapy is a powerful concept just now beginning to see applications designed to treat human diseases such as genetic disorders and cancer.
  • the introduction of genes into an organism can be achieved in a variety of ways, including virus-based vectors.
  • Viral gene therapy vectors can either be designed to deliver and express genes permanently (stable integration of a foreign gene into host chromosome) or transiently (for a finite period of time).
  • Current virus-based gene transfer vectors are typically derived from animal viruses, such as retroviruses, herpesviruses, adenoviruses, or adeno-associated viruses. Generally, these viruses are engineered to remove one or more genes of the virus. These genes may be removed because they are involved in viral replication and/or to provide the capacity for insertion and packaging of foreign genes. Each of these known vectors has some unique advantages as well as disadvantages.
  • One primary disadvantage is an inability to readily package and deliver large DNA inserts that are greater than 10 kb in size.
  • Adeno-associated virus is a parvovirus which consists of a 4.7 kb single stranded DNA genome
  • the viral genome consists of the family of rep genes responsible for regulatory function and DNA replication and the cap genes that encode the capsid proteins.
  • the AAV coding region is flanked by 145 nucleotide inverted terminal' repeat (ITR) sequences which are the minimum cis-acting elements essential for replication and encapsidation of the genome.
  • AAV recombinant AAV vectors
  • rAAV recombinant AAV vectors
  • the major advantages of recombinant AAV (rAAV) vectors include a lack of pathogenicity in humans (Berns, K.I. and R.A. Bohenzky [1987] "Adeno-associated viruses: an update" Adv. Virus Res. 32:243-306), the ability of wild-type AAV to integrate stably into the long arm of chromosome 19 (Kotin, R.M., R.M. Linden, K.I.
  • entomoviruses complex insect viruses
  • studies of entomoviruses have mainly concentrated on their use as biopesticides, expression systems or taxonomic novelties to compare to their mammalian virus counterparts.
  • the family Poxviridae comprises two subfamilies, the Chordopoxviridae (vertebrate) and the Entomopoxviridae (insect) viruses (EPVs).
  • EPVs were first discovered in the early 1960's, and have subsequently been shown to have a worldwide distribution.
  • the subfamily contains three genera; A, B and C, which infect beetles, moths (lepidoptera) and grasshoppers, and midge flies respectively (Moyer, R.W. [1994] Entomopoxviruses, p.392-397, Encyclopedia of Virology. R.G. Webster and A. Granoff (eds.), Academic Press Ltd, London).
  • EPVs are the most distant relatives of mammalian poxviruses and exhibit both similarities and differences to the more commonly studied chordopoxviruses, such as vaccinia virus (W). Similarities include morphology, a large linear double stranded genome (previously estimated at 225 kb for AmEPV, 190 kb for VV), common transcriptional regulation sequence motifs, non- spliced transcripts and a cytoplasmic site of replication. Differences include the G+C content of the viral DNA (a low 18% for AmEPV, 37% for W), optimal growth temperatures (28°C for AmEPV, 37°C for W), and host range.
  • W vaccinia virus
  • AmEPV does not replicate in vertebrate cells, and VV does not replicate in insect cells, although both viruses enter their respective non-permissive cells and initiate a replicative cycle (Langridge, W.H. [1983] "Detection of Amsacta moorei entomopoxvirus and vaccinia virus proteins in cell cultures restrictive for poxvirus multiplication" J. Invertebr. Pathol 42:77-82).
  • EPV promoters like those of vertebrate poxviruses
  • W contain a number of genes which are nonessential for growth in cell culture. Two examples are the thymidine kinase (TK) and spheroidin genes.
  • TK thymidine kinase
  • spheroidin gene can be viewed as a counterpart to the polyhedrin and A-type (ATI) occlusion genes of baculoviruses and cowpox viruses respectively.
  • W also contains an ATI gene, but it is defective.
  • Spheroidin is the most abundantly expressed AmEPV gene, and serves to "occlude" infectious virions within an environmentally resistant occlusion body. Both the AmEPV TK and spheroidin gene can readily serve as sites for insertion and expression of foreign genes by utilizing standard plasmid-mediated recombination.
  • Entomopoxvirus productively infect and kill only insects (Granados, R.R. [1981] "Entomopoxvirus infections in insects," in Pathogenesis of Invertebrate Microbial Disease, p.
  • AmEPV Amsacta moorei
  • the virus is cytoplasmic and does not normally enter the nucleus.
  • AmEPV promoters and those of the eucaryotic cell are completely different and cellular promoters are not recognized by the AmEPV transcription machinery nor are AmEPV viral promoters recognized by RNA polymerase II of the host cell.
  • the subject invention concerns a novel viral vector system for gene therapy based on an insect poxvirus designed to deliver genes for integration and stable, permanent expression in vertebrate cells.
  • a recombinant AmEPV vector was constructed that contains heterologous genes under the control of promoters that drive the expression of the heterologous genes in vertebrate cells.
  • the gfp gene and the gene encoding G418 resistance were used in an exemplified construct.
  • the recombinant AmEPV was used to infect vertebrate cells and following infection the cells were transferred to media containing G418. Cells expressing both GFP and G418 resistance were obtained.
  • the vectors of the subject invention can be used to deliver large DNA segments for the engineering of vertebrate cells.
  • the subj ect invention also concerns cells that have been infected with or transfonned with a recombinant vector of the present invention.
  • the subject invention also concerns methods for providing gene therapy for conditions or disorders of an animal requiring therapy, such as genetic deficiency disorders.
  • the subject invention concerns novel AmEVP polypeptides and the polynucleotide sequences which encode these polypeptides.
  • the AmEPV polynucleotide sequences of the subject invention encode a triacylglyceride lipase (SEQ ID NO: 1), a C ⁇ XlZrX superoxide dismutase (SOD) (SEQ ID NO: 2), a CPD photolyase (SEQ ID NO: 3), a baculovirus-like inhibitor of apoptosis (IAP) (SEQ ID NO: 4), two poly(A) polymerase small subunits (SEQ ID NOS: 5 and 6), two DNA polymerases (SEQ ID NOS: 7 and 8), an ABC transporter-like protein (SEQ ID NO: 9), a Kunitz-motif protease inhibitor (KIT) '(SEQ ID NO: 10), and a poly(A) polymerase large subunit (SEQ ED NO: 11).
  • the subject invention concerns isolated AmEPV polypeptides encoded by the polynucleotide sequences of the subject invention, including a triacylglyceride lipase (SEQ ID NO:
  • LAP baculovirus-like inhibitor of apoptosis
  • SEQ ID NO: 15 baculovirus-like inhibitor of apoptosis
  • SEQ ID NOS: 16 and 17 two poly(A) polymerase small subunits
  • SEQ ID NOS: 18 and 19 two DNA polymerases
  • the subject invention further pertains to other entomopoxvirus sequences.
  • Polynucleotides of the subject invention include, for example, sequences identified in the attached sequence listing, as well as the tables and figures and described by open reading frame position within the genome.
  • the subject invention includes polynucleotides which hybridize with other polynucleotides of the subject invention.
  • Polynucleotide sequences of this invention have numerous applications in techniques known to those skilled in the art of molecular biology having the benefit of the instant disclosure. These techniques include their use as insertion sites for foreign genes of interest, hybridization probes, for chromosome and gene mapping, in PCR technologies, and in the production of sense or antisense nucleic acids.
  • FIG 1 shows a physical map of an exemplified recombinant vector of the subject invention (pAmEPV TKUF5) in which a portion of the plasmid pTKUF5 has been cloned within the AmEPV TK gene flanking regions.
  • TR is the AAV terminal repeat;
  • pA is a polyadenylation site;
  • SD/SA is the SV40 late splice donor, splice acceptor sequence.
  • GFP the green fluorescent protein gene, is under the control of a CMV promoter.
  • Neo the neomycin resistance gene, is under the control of a herpes TK gene promoter.
  • Figure 2 shows an electrophoretic analysis of transformed mammalian cell lines. Each lane contains Hindlll digested genomic DNA.
  • Lane P contains genomic DNA from 293 cells and pTR-UF5 plasmid, as a positive control. Lanes Al through A5 contain DNA extracted from transformed cell lines made by recombinant AmEPV (AmEPVpTKUF5) infection. Lanes Bl through B6 contain DNA obtained from cell lines transfected with plasmid pTR-UF5. Figure 3 shows expression of lacL in recombinant AmEPV-infected mammalian cells.
  • CV-1 cells were mock infected (A) or infected with various AmEPV lacZ recombinants, where lacZ was under the control of the cowpox virus late ATI gene promoter (B), the late AmEPV spheroidin promoter (C), the M. melonontha early fits promoter (D) or the AmEPV early esp promoter (E).
  • B cowpox virus late ATI gene promoter
  • C late AmEPV spheroidin promoter
  • D M. melonontha early fits promoter
  • E AmEPV early esp promoter
  • Infection of human Huh-7 liver cells with the AmEPV TKesp-lacZ recombinant is also shown as an additional control (F).
  • the infected cell monolayers were stained with X-gal 24h postinfection.
  • Figure 4 shows the survival of mammalian cells following infection by recombinant AmEPV TKesp-gjp.
  • Subconfluent CV-1 cells were infected with AmEPV T esp-gfp at an m.o.i. of 1 PFU/cell.
  • the individual fluorescent cells were located and followed over a period of two to three days and periodically photographed with a fluorescent microscope.
  • Figure 5 shows AmEPV-mediated ⁇ -galactosidase expression in the muscle of mouse.
  • 2X10 6 PFU (100 ⁇ l) of recombinant AmEPV-espl ⁇ cZ was injected into the muscle of the hind leg of a mouse.
  • mice were injected with the same amount of recombinant AmEPV-
  • FIG. 6 shows transformed 293 cells (A) derived from the colony infected with recombinant AmEPV-TKUF5 which are G418 resistant showing that cells are GFP positive, as well as non-fluorescent, non-transformed 293 cells (B).
  • Figure 7 shows a linear map of the AmEPV genome, 0-139440(A) and 139441- 232392(B).
  • Predicted ORFs are numbered consecutively from left to right based upon the position of the initiating methionine codon. ORFs transcribed in a rightward direction are shown above the horizontal line designating the viral genome; ORFs transcribed to the left are below. ITRs are indicated by heavy black arrows. A distance of lkb is as shown. ChPV homologs are indicated with red numbers, additional MsEPV homologs are indicated with purple numbers. Some ORFs have been assigned function based upon BLAST data.
  • Figure 8 shows a comparison of the genomic organization of AmEPV, MsEPV and W.
  • AmEPV ITRs are positioned at the termini of the viral genome as indicated. AmEPV genes which have homology to W genes are depicted in (A). AmEPV genes which have homology to MsEPV are depicted in (B). Genes in the AmEPV genome common to both MsEPV and VV are in (C). Unique genes encoded by AmEPV are shown in (D).
  • Figure 9 shows a comparison of the spatial distribution of homologous genes between AmEPV, MsEPV and W.
  • a random sampling of genes conserved within the genomes of all three indicated viruses were plotted on the 119kb genome of W, the 232kb AmEPV genome, and the 236kb MsEPV genome. From left to right on the AmEPV genome, the genes shown and their BLAST-assigned function are: AMV016, thymidine kinase; AMV035, membrane protein;
  • Figure 10 shows residues shared between poxvirus poly(a) polymerase subunit homologs. Consensus shows the conservation between all five sequences. Insect consensus shows identity among the four EPV ORFs. AmEPV consensus displays identities between the two AmEPV subunits.
  • Figure 11 shows the transmembrane domains possessed by the AmEPV ABC transporter protein. This graphic was produced by the THAMM program (Sonnhammer, E. L. L., Hejne, G., and Krogh, A. [1998] "A hidden Markov model for predicting transmembrane helices inprotein sequences" Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology (J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen, Eds.), pp. 175-182. AAAI press,
  • FIG. 12 shows the amino acid sequence of the AmEPV serine protease inhibitor. Amino acid abbreviations are standard.
  • the Kunitz family signature (Prostite PS00280) is shown underlined and italicized from residues 55 to 73.
  • SEQ ID NO: 1 is the nucleotide sequence of the gene encoding AmEPV triacylglyceride lipase (AMV133).
  • SEQ ID NO: 2 is the nucleotide sequence of the gene encoding AmEPV CvXlZvX superoxide dismutase (SOD) (AMV255).
  • SEQ ED NO : 3 is the nucleotide sequence of the gene encoding AmEPV CPD photolyase
  • SEQ ED NO: 4 is the nucleotide sequence of the gene encoding AmEPV baculovirus-like inhibitor of apoptosis (JAP) (AMV021).
  • SEQ ED NO: 5 is the nucleotide sequence of the gene encoding a first AmEPV poly(A) polymerase small subunit (AMV060).
  • SEQ ED NO: 6 is the nucleotide sequence of the gene encoding a second AmEPV poly(A) polymerase small subunit (AMV115).
  • SEQ ED NO: 7 is the nucleotide sequence of the gene encoding a first AmEPV DNA polymerase (AMV050).
  • SEQ ID NO: 8 is the nucleotide sequence of the gene encoding a second AmEPV DNA polymerase (AMV210).
  • SEQ D3 NO: 9 is the nucleotide sequence of the gene encoding AmEPV ABC transporterlike protein (AMV130).
  • SEQ ID NO: 10 is the nucleotide sequence of the gene encoding AmEPV Kunitz-motif inhibitor (KPI) (AMV007).
  • SEQ ID NO: 11 is the nucleotide sequence of the gene encoding AmEPV poly(A) polymerase large subunit (AMV038).
  • SEQ ID NO: 12 is the amino acid sequence for the AmEPV triacylglyceride lipase (AMV133).
  • SEQ ID NO: 13 is the amino acid sequence for the AmEPV Cu ++ /Zn ++ superoxide dismutase (SOD) (AMV255).
  • SEQ ED NO: 14 is the amino acid sequence for the AmEPV CPD photolyase (AMV025).
  • SEQ ED NO: 15 is the amino acid sequence for the AmEPV baculovirus-like inhibitor of apoptosis (IAP) (AMV021).
  • SEQ ED NO: 16 is the amino acid sequence for the first AmEPV poly(A) polymerase small subunit (AMV060).
  • SEQ ED NO: 17 is the amino acid sequence for the second AmEPV poly(A) polymerase small subunit (AMI 15).
  • SEQ ED NO: 18 is the amino acid sequence for the first AmEPV DNA polymerase
  • SEQ ED NO: 19 is the amino acid sequence for the second AmEPV DNA polymerase (AMV210).
  • SEQ ED NO: 20 is the amino acid sequence for the AmEPV ABC transporter-like protein (AMV130).
  • SEQ ED NO: 21 is the amino acid sequence for the AmEPV Kunitz-motif inhibitor (KPI) (AMV007).
  • SEQ ED NO: 22 is the amino acid sequence for the AmEPV poly(A) polymerase large subunit (AMV038).
  • SEQ ED NOS: 23-27 is the nucleotide sequence of the AmEPV genome.
  • SEQ ED NO: 28 is the nucleotide sequence and amino acid sequence for an AmEPV enhancin protein (AMVITR10).
  • SEQ ID NO: 29 is the nucleotide sequnece and amino acid sequence for an AmEPV dUTPase (AMV002).
  • SEQ ED NO: 30 is the nucleotide sequence and amino acid sequence for an AmEPV very late transcription factor-2 (VLTF-2) (AMV047).
  • SEQ ED NO: 31 is the nucleotide sequence and amino acid sequence for a first AmEPV RNA polymerase (AMV051).
  • SEQ ED NO: 32 is the nucleotide sequence and amino acid sequence for a second AmEPV RNA polymerase (AMV054).
  • SEQ ED NO: 33 is the nucleotide sequence and amino acid sequence for an AmEPV DNA helicase (AMV059).
  • SEQ ED NO: 34 is the nucleotide sequence and amino acid sequence for an AmEPV 3 OK virion protein (AMV061).
  • SEQ ED NO: 35 is the nucleotide sequence and amino acid sequence for a third AmEPV
  • RNA polymerase AMV0666
  • SEQ ED NO: 36 is the nucleotide sequence and amino acid sequence for an AmEPV protein tyrosine phosphatase (AMV078).
  • SEQ ID NO: 37 is the nucleotide sequence and amino acid sequence for an AmEPV thioredoxin protein (AMV079).
  • SEQ ED NO: 38 is the nucleotide sequence and amino acid sequence for an AmEPV RNA helicase (AMV081).
  • SEQ ED NO: 39 is the nucleotide sequence and amino acid sequence for a first AmEPV serine/threonine protein kinase (AMV084).
  • SEQ ED NO: 40 is the nucleotide sequence and amino acid sequence for an AmEPV NTPase (AMV087).
  • SEQ ED NO: 41 is the nucleotide sequence and amino acid sequence for an AmEPV transcription factor (AMV091).
  • SEQ ED NO: 42 is the nucleotide sequence and amino acid sequence for an AmEPV mRNA capping small subunit (AMV093).
  • SEQ ED NO: 43 is the nucleotide sequence and amino acid sequence for an AmEPV very early transcription factor-large protein (VETF-L) (AMV105).
  • SEQ ED NO: 44 is the nucleotide sequence and amino acid sequence for an AmEPV redox protein (AMV114).
  • SEQ ED NO: 45 is the nucleotide sequence and amino acid sequence for an AmEPV rifampicin resistance protein (AMV122).
  • SEQ ED NO: 46 is the nucleotide sequence and amino acid sequence for an AmEPV mRNA capping large subunit (AMV135).
  • SEQ ED NO: 47 is the nucleotide sequence and amino acid sequence for an AmEPV P4a core protein (AMV139).
  • SEQ ED NO: 48 is the nucleotide sequence and amino acid sequence for an AmEPV P4b core protein (AMV147).
  • SEQ ED NO: 49 is the nucleotide sequence and amino acid sequence for an AmEPV
  • ATP/GTP binding protein (AMV150).
  • SEQ ED NO: 50 is the nucleotide sequence and amino acid sequence for a second AmEPV serine threonine protein kinase (AMV153).
  • SEQ ED NO: 51 is the nucleotide sequence and amino acid sequence for a fourth AmEPV RNA polymerase (AMV166).
  • SEQ ED NO: 52 is the nucleotide sequence and amino acid sequence for an AmEPV polyubiquitin protein (AMV167).
  • SEQ ED NO: 53 is the nucleotide sequence and amino acid sequence for AmEPV very small transcription factor-short protein (VETF-s) (AMV174).
  • SEQ ED NO: 54 is the nucleotide sequence and amino acid sequence for AmEPV core protein (AMV181).
  • SEQ ED NO: 55 is the nucleotide sequence and amino acid sequence for an AmEPV nucleoside triphosphate phosphorylase I (NPH I) (AMV192).
  • SEQ ED NO: 56 is the nucleotide sequence and amino acid sequence for an AmEPV apoptosis-associated protein (AMV193).
  • SEQ ED NO: 57 is the nucleotide sequence and amino acid sequence for a third AmEPV serine/threonine protein kinase (AMV 197).
  • SEQ ED NO: 58 is the nucleotide sequence and amino acid sequence for an AmEPV NAD+ dependent DNA ligase (AMV 199).
  • SEQ ED NO: 59 is the nucleotide sequence and amino acid sequence for an AmEPV very late transcription factor-3 (VLTF-3) (AMV205).
  • SEQ ED NO: 60 is the nucleotide sequence and amino acid sequence for a fifth AmEPV
  • RNA polymerase (AMV221).
  • SEQ ED NO: 61 is the nucleotide sequence and amino acid sequence for an AmEPV Ca + binding protein (AMV228).
  • SEQ ED NO: 62 is the nucleotide sequence and amino acid sequence for a sixth AmEPV RNA polymerase (AMV230).
  • SEQ ED NO: 63 is the nucleotide sequence and amino acid sequence for an AmEPV DNA glycosylase (AMV231).
  • SEQ ED NO: 64 is the nucleotide sequence and amino acid sequence for an AmEPV phosphatase (AMV234).
  • SEQ ED NO: 65 is the nucleotide sequence and amino acid sequence for an AmEPV phosphotyrosine kinase (AMV246).
  • SEQ ED NO: 66 is the nucleotide sequence and amino acid sequence for an AmEPV glycosyl transferase (AMV248).
  • SEQ ED NO: 67 is the nucleotide sequence and amino acid sequence for an AmEPV metalloprotease (AMV256).
  • SEQ ID NO: 68 is the nucleotide sequence and amino acid sequence for an AmEPV myristylated membrane protein (AMV217).
  • SEQ ID NO: 69 is the nucleotide sequence and amino acid sequence for an AmEPV NTP pyrophosphohydrolase (AMV058).
  • SEQ ED NO: 70 is the nucleotide sequence and amino acid sequence for an AmEPV DNA topoisomerase (AMV052).
  • SEQ ED NO: 71 is the nucleotide sequence and amino acid sequence for a first AmEPV membrane protein (AMV 118).
  • SEQ ED NO: 72 is the nucleotide sequence and amino acid sequence for a second AmEPVmembrane protein (AMV232).
  • SEQ ED NO: 73 is the nucleotide sequence and amino acid sequence for a third AmEPV membrane protein (AMV243).
  • SEQ ED NO: 74 is the nucleotide sequence and amino acid sequence for a fourth AmEPV membrane protein (AMV035).
  • the subject invention concerns three aspects of entomopoxviruses (EPVs) as novel recombinant vectors: (1) As a system for the expression of high levels of foreign proteins, (2) for the transient expression of foreign genes in mammalian cells and (3) for the stable transformation of vertebrate cells for the long term expression of foreign proteins.
  • the subject invention provides the nucleotide sequence of the entire genome of genus B entomopoxvirus from Amsacta moorei (AmEPV). Accordingly, the subject invention also concerns isolated polynucleotides encoding AmEPV proteins.
  • the subject invention concerns novel recombinant vectors and methods for delivery and expression of heterologous polynucleotides in vertebrate cells.
  • the recombinant vectors of the subject invention provide for stable integration and expression of heterologous DNA in the host cell.
  • the vectors of the invention are adapted for accepting large heterologous polynucleotide inserts which can be delivered in an infected or transformed cell and expressed in a stable fashion.
  • the subject invention can be used to provide gene therapy for conditions or disorders of vertebrate animals, such as a mammal or human, that is in need of such therapy.
  • One aspect of the subject invention concerns a recombinant EPV vector which can optionally include heterologous DNA which can be expressed in a cell infected or transformed with the subject vector.
  • the EPV vector is derived from AmEPV.
  • the recombinant EPV vectors of the present invention can optionally include inverted terminal repeat (ITR) sequences of a virus, such as, for example, adeno-associated virus, that flank the heterologous DNA insertion site on the vector.
  • ITR inverted terminal repeat
  • the subject vectors comprise heterologous DNA inserted within the vector.
  • the heterologous DNA contained within the recombinant vectors of the invention can include polynucleotide sequences which encode a biologically functional protein.
  • the polynucleotides encode proteins which can provide therapeutic replacement or supplement in animals afflicted with disorders which result in the animal expressing abnormal or deficient levels of the protein that are required for no ⁇ nal biological function.
  • Proteins encoded by the heterologous DNA can include, but are not limited to interleukins, cytokines, growth factors, interferons, enzymes, and structural proteins. Proteins encoded by the heterologous DNA can also include proteins that provide a selectable marker for expression, such as antibiotic resistance in eukaryotes.
  • heterologous DNA within the subject vectors is operably linked with and under the control of regulatory sequences, such as promoters.
  • the recombinant vectors of the invention preferably comprises a constitutive or regulatable promoter capable of promoting sufficient levels of expression of the heterologous DNA contained in the viral vector in a vertebrate cell.
  • Promoters useful with the subject vectors include, for example, the cytomegalovirus (CMV) promoters and the herpes TK gene promoter.
  • CMV cytomegalovirus
  • the vectors can also include other regulatory elements such as introns inserted into the polynucleotide sequence of the vector.
  • the strategy for generation of recombinant viruses is identical to that used for VV virus and takes advantage of the high levels of recombination with transfected plasmids mediated by these viruses.
  • the basic procedure utilizes transfection of AmEPV-infected cells with an appropriately designed shuttle vector. Insertion of foreign genes occurs within a non-essential gene (e.g., spheroidin or TK). Because of the cytoplasmic nature of AmEPV, it is necessary to place all foreign genes under control of an AmEPV (early or late) poxvirus promoter. Recombinants are selected and subjected to three rounds of plaque purification before use.
  • the subject invention also concerns cells containing recombinant vectors of the present invention.
  • the cells can be, for example, vertebrate cells such as mammalian cells.
  • the cells are human cells.
  • Cell lines infected or transformed with the recombinant vectors of the present invention are also within the scope of the invention.
  • the recombinant vectors of the present invention can be introduced into suitable cells or cell lines by methods known in the art. If the recombinant vectors are packaged in viral particles then cells or cell lines can be infected with the virus containing the recombinant vector. Methods contemplated for introducing recombinant vector into cells or cell lines also include transfection, transduction and injection. For example, vectors can be introduced into cells using liposomes containing the subject recombinant vectors. Recombinant viral particles and vectors ofthe present invention can be introduced into cells by in vitro or in vivo means. Infection of vertebrate cells is non-permissive, in that early but not late AmEPV gene expression occurs (Li, Y., R.L.
  • Melolontha melolontha EPV fusolin gene promoter (Gauthier, L., F. Coussrans, J.C. Veyrunes, M. Bergoin [1995] "The Melolontha melolontha entomopoxvirus (MmEPV) fusolin is related to the fusolins of lepidoptera EPVs and to the 37 K baculovirus glycoprotein" Virology 208:427-436) or the 42 kDa early AmEPV protein (Li et al. [1997] supra), high levels of galactosidase in the recombinant AmEPV infected vertebrate cells are observed. These results provide clear evidence of AmEPV entry into vertebrate cells followed by early, but not late, viral gene expression.
  • AmEPV vectors ofthe subject invention have the ability to stably transform cells and express genes in a long term fashion as well.
  • the data presented within the Examples (e.g., Example 2) and accompanying Figures (e.g., Figure 2) confirm that the AmEPV vectors ofthe subject invention can be used to deliver DNA which subsequently integrates into DNA ofthe mammalian cell nucleus.
  • the ability of AmEPV to deliver DNA to mammalian cells creates endless opportunity for use ofthe vector in the stable transformation and engineering of vertebrate cells.
  • the Examples describe methodology for growth, titration and preparation of recombinant AmEPV, as well as transient expression of AmEPV in vertebrate cells, the use of AmEPV to stably transform mammalian cells, and potential uses of AmEPV vectors.
  • the subj ect invention provides the nucleotide sequence ofthe entire genome ofthe genus
  • AmEPV Amsacta moorei
  • the AmEPV polynucleotide sequences ofthe subject invention include polynucleotides encoding a triacylglyceride lipase (SEQ ED NO: 1), a Cu + 7Zn H" superoxide dismutase (SOD) (SEQ ID NO: 2), a CPD photolyase (SEQ ID NO: 3), a baculovirus-like inhibitor of apoptosis (LAP) (SEQ ID NO: 4), two poly(A) polymerase small subunits (SEQ ID NOS: 5 and 6), two DNA polymerases (SEQ ED NOS: 7 and 8), an ABC transporter-like protein (SEQ ID NO: 9), a Kunitz-motif protease inhibitor (KPI) (SEQ ID NO: 10), and a poly(A) polymerase large subunit (SEQ ID NO: 11) and other polynucleotides.
  • SEQ ED NO: 1 triacylglyceride lipa
  • the subject invention concerns isolated AmEPV polypeptides encoded by the polynucleotide sequences ofthe subject invention, including a triacylglyceride lipase (SEQ ID NO: 12), a Cu ⁇ /Zn ⁇ superoxide dismutase (SOD) (SEQ ID NO: 13), a CPD photolyase (SEQ ED NO:
  • LAP baculovirus-like inhibitor of apoptosis
  • SEQ ID NO: 15 two poly(A) polymerase small subunits
  • SEQ JD NOS: 16 and 17 two DNA polymerases
  • SEQ ED NOS: 18 and 19 an ABC transporter-like protein
  • SEQ ED NO: 20 a Kunitz-motif protease inhibitor
  • KPI Kunitz-motif protease inhibitor
  • SEQ ID NO: 22 poly(A) polymerase large subunit
  • the subject invention includes other AmEPV sequences, as described in Table 1, for example.
  • the subject invention includes polynucleotides which hybridize with other polynucleotides ofthe subject invention.
  • the genome ofthe genus B entomopoxvirus from Amsacta moorei (SEQ JD NOS: 23-27) was sequenced and found to contain 232,392 bases with 279 unique open reading frames (ORFs) of greater than 60 amino acids.
  • the central core of the viral chromosome is flanked by 9.4kbp inverted terminal repeats (ITRs), each ofwhich contain 13 ORFs, raising the total number of ORFs within the viral chromosome to 292.
  • Default E (EXPECT) values of ⁇ 0.01 were used to define homology to sequences in current databases. ORFs lacking homology to other poxvirus genes were shown to comprise 33.6% ofthe viral genome.
  • AmEPV ORFs Approximately 28.6% ofthe AmEPV genome (52 AmEPV ORFs) encodes homologues ofthe mammalian poxvirus co-linear core genes, which are found dispersed throughout the AmEPV chromosome. There is also no significant gene order conservation between AmEPV and the orthopteran genus B poxvirus of Melanoplus sanguinipes (MsEPV). Novel AmEPV genes include those encoding an ABC transporter and a Kunitz motif protease inhibitor. The most unusual feature of the AmEPV genome relates to the viral encoded poly(A) polymerase. In all other poxviruses this heterodimeric enzyme consists of a single large and small subunit. However, AmEPV appears to encode one large and two distinct small poly (A) polymerase subunits. AmEPV is one of the few entomopoxviruses which can be grown and manipulated in cell culture.
  • Table 1 lists all the ORFs encoded by the AmEPV genome, and functions assigned to the encoded proteins. Default E (EXPECT) values of ⁇ 0.01 were used to define homology to sequences in current databases. 52 AmEPV ORFs (28.6% ofthe genome) show homology to
  • ChPV genes, and 91 ORFs have homologs in EPVs or other insect viruses.
  • the terminal regions of AmEPV contain few genes homologous to any other gene.
  • Figure 8 illustrates this phenomenon, as well as the observation that AmEPV homologs of both vaccinia and MsEPV genes (which we have used as available examples for the ChPV and EPV) are positioned more towards the centre of the AmEPV genome. In contrast, novel AmEPVgenes are easily identified as occurring more often towards the genomic termini.
  • AMV041 40203-40841 213 AF063866 MSV039 (G6R) 1.00E-43 193 X X L
  • AMV062 62518-63009 164 AF022176 HaEPV orf4 [160] 1.00E-61 166 X E?,L?
  • Vertebrate poxviruses have been shown to generally share a co-linear arrangement of core genes (Goebel, S. J., Johnson, G. P., Perkus, M. E., Davis, S. W., Winslow, J. P., and Paoletti, E. [1990] "The complete DNA sequence of vaccinia virus" Virology 179, 247-66, 517-63; Massung, R. F., Liu, L. I., Qi, J., Knight, J. C, Yuran, T. E., Kerlavage, A. R., Parsons, J. M., Venter, J. C, and Esposito, J. J.
  • HaEPV Heliothis armigera entomopoxvirus
  • AmEPV contains promoter elements which govern gene expression. 133 AmEPV genes are considered to be early, or potentially early. 158 genes possess motifs which result in late, or potentially late promoters. Only 15 genes from the entire 279 gene genome have no recognizable promoter or regulatory elements. Genes that contain the sequences TGAAAXXXXA or TGAATXXXXA within 100 bases of their translational start codons were considered early (E) or potentially early (E?), respectively (Table 1). This motif resembles the ChPV early promoter core consensus sequence (Moss, B. [1996] Poxviridae: The viruses and their replication. In "Fields Virology" (B. N. Fields, D. M. Knipe, and P. M.
  • MmEPV fusolin is related to the fusolins of lepidopteran EPVs and to the 37K baculovirus glycoprotein" Virology 208:427-
  • HaEPV Heliothis armigera entomopoxvirus
  • AmEPV ORFs that contained the sequence TAAATG at the translational start site were considered late genes (L) (Bertholet, C, Stocco, P., Van Meir, E., and Wittek, R. [1986]
  • AmEPV is one ofthe few entompoxviruses which can be easily and reliably replicated in tissue culture (Winter, J., Hall, R. L., and Moyer, R. W. [1995] "The effect of inhibitors on the growth ofthe entomopoxvirus from Amsacta moorei in Lymantria dispar (gypsy moth) cells" Virology 211: 462-473; Hall, R. L., Li, Y., Feller, J. A., and Moyer, R. W.
  • Poxvirus ITRs can vary considerably in size. The smallest ITRs are those of variola Bangladesh which are only 725 bp (Massung, R. F., Esposito, J. J., Liu, L. I., Qi, J., Utterback, T. R., Knight, j. C, Aubin, L., Yuran, T. E., Parsons, J. M., Loparev, V. N., Selivanov, N. A., Cavallaro, K. F., Kerlavage, A. R., Mahy, B. W. J., and Venter, J. C. [1993] "Potential virulence determinants in terminal regions of variola smallpox virus genome" Nature 366:748-751;
  • Spontaneous DNA arrangements occur with an increased frequency at or near the terminal inverted repeat sequences of poxviral genomes (Moyer, R. W., Graves, R. L., and Rothe, C. T. [1980] "The white pock (mu) mutants of rabbit poxvirus. in. Terminal DNA sequence duplication and transposition in rabbit poxvirus" Cell 22:545-553). Indeed, the majority of novel and non- essential genes are generally found within poxviral ITRs or toward the genomic termini.
  • the complete genomic sequences of vaccinia and variola viruses from the orthopoxvirus genus, myxoma and Shope fibroma viruses from the leporipoxvirus genus, fowlpox from the avipoxvirus genus, the molluscipoxvirus molluscum contagiosum and the genus B EPV, MsEPV have allowed definition of conserved poxvirus genes present in most, if not all, poxviruses. Inclusion ofthe AmEPV genomic sequence extends that concept.
  • the ORFs AMV038, AMV060 and AMVl 15 are predicted to represent one large and two small poly(A) polymerase subunits respectively. This unusual feature will be discussed in a subsequent section.
  • neither AmEPV nor MsEPV encode a homolog ofthe vaccinia I3L protein.
  • the I3L protein is a DNA binding protein and is presumably involved in DNA replication (Davis, R. E. and Mathews, C. K.
  • ChPV homolog ORF's shown are from VV. Where no homolog exists, *SFV, and **CPV.
  • Several ofthe genes included in Table 2 showing AmEPV vertebrate poxvirus homologs are not universally conserved, but are nevertheless present in many poxviruses.
  • One example is the thymidine kinase (TK) gene.
  • AmEPV encodes a TK gene, as do most ChPV and most other genus B EPVs investigated to date (Lytvyn, V., Fortin, Y., Banville, M., Arif, B., and Richardson, C. [1992] "Comparison ofthe thymidine kinase genes from three entomopoxviruses" J. Gen. Virol
  • MsEPV ORF237 which is homologous to vaccinia virus B2R (Afonso et al. [1999] supra; Goebel, S. J., Johnson, G. P., Perkus, M. E., Davis, S. W., Winslow, J.
  • the A21L protein has been shown to interact with the A6L protein using the two hybrid system (McCraith, S., Holtzman, T., Moss, B., and Fields, S. [2000] "Genome-wide analysis of vaccinia virus protein- protein interactions" Proc. Natl. Acad. Sci. U.S.A 97:4879-4884).
  • TK TK
  • SOD protein tyrosine phosphatase
  • W B2R protein tyrosine phosphatase
  • AmEPV and MsEPV share the same suite of ChPV virus homologs.
  • MsEPV and AmEPV genes may be present as the last remnants of divergence from a common ancestor. Alternatively, small clusters might have remained in close proximity to each other due to a more recent acquisition or for functional or regulatory reasons.
  • the homologs of MsEPV genes MSV085-MSV089 are also non-sequentially grouped within a 9 gene assembly within AmEPV (AMV079-AMV087).
  • HaEPV PAP2, 3 OK and ORF4 genes are also immediately adjacent and co-linear in AmEPV, with ORF direction preserved (AMV060, AMV061 and AMV062)(Crnov and Dall 1999). Comparative alignments have also highlighted differences between these two more closely related genus B lepidopteran EPVs. For example, the large RNA polymerase of HaEPV is located toward the leftmost end of the genome, whereas it is positioned at the right end of AmEPV. Likewise the "17K" ORF of HaEPV is duplicated and teminally located within its ITRs, but homologous regions within
  • AmEPV are not repeated, and are positioned approximately one hundred genes from the genomic termini. Whether or not a generally co-linear arrangement of genes emerges for the lepidopteran EPVs, it is obvious that EPVs in general have not followed the evolutionary direction of ChPV which has enabled them to retain a common co-linear gene core. Clearly, genes shared between AmEPV and MsEPV are not arranged in a co-linear fashion and based on overall gene organization, MsEPV and AmEPV may be far more distantly related than the current common morphologically based classification as genus B EPVs would suggest. There are two possibilities to explain this divergence in gene order between AmEPV (lepidopteron) and MsEPV (orthopteran) viruses.
  • a second model is based on intrinsic genomic plasticity and generalized movement of genes within the viral chromosome of EPVs. Comparative homologies among essential genes; e.g. RNA polymerase subunits, suggests MsEPV and AmEPV are more closely related to each other than either is to ChPVs homologs. Therefore, it may well be that plasticity or position independent location of genes within EPVs plays a significant role in the creation of divergent gene orders.
  • AmEPV encodes an additional 27 gene homologs not found within ChPV or MsEPV, but which are present in other insect viruses including baculoviruses (AcNPV, XcGV, SeNPV, CpGV, LdNPV and TnGV) and an iridovirus (Chilo iridescent virus). The majority of these genes have previously been assigned functions, and a number are not specific to insect viruses alone (see Table 1). AmEPV gene families
  • MsEPV was found to encode 43 novel ORFs which could be grouped into five gene families of varying stringency. Examination of the AmEPV genomic sequence revealed the presence of 23 genes which can be grouped into six gene families (Table 3). Table 3. AmEPV Gene Families.
  • the AMV 176 gene family has no homology to any proteins within current databases. Each of these 86 residue proteins is identical, except for a single nucleotide substitution in AMV056 which results in an isoleucine codon at residue 37, instead ofthe leucine coded by both AMV176 and AMV178. It is unusual to observe perfect copies of genes within a gene family. All members ofthe family are predicted to contain a transmembrane domain.
  • the five member ALI-like (alanine-leucine-isoleucine) gene family largely comprises ORFs related to the AMV 176 gene family discussed above.
  • the ORFs do not possess any motifs indicative of transmembrane domains or signal sequences.
  • AMV055 appears to be a carboxy terminal truncated member of this family. This 133 residue ORF shares a large number of residue identities with the other family members.
  • the final member ofthe family, AMV257 appears to be truncated at the N-terminus, and is less related to the other members of this family. Nevertheless, its homology to MSV196 warrants its inclusion in this group.
  • a third MTG-like gene family has three members; AMV194, AMV207 and AMV209. There is a 69% identity between AJVTV207 and AMV209. AMV 194 is somewhat less related to the other family members.
  • Each gene was identified independantly based on its homology to the MTG gene family ORF MSV198 found in MsEPV. However, the invariant signature MTG (methionine-threonine-glycine) motif is absent from all AmEPV proteins, and an expected internal motif found within the MsEPV proteins was found to be degenerate.
  • a fourth family comprising only AMV029 and AMV254 shows homology to MsEPV ORF MSV027, which is a member ofthe tryptophan repeat gene family. Both AmEPV ORFs contain the expected motifs, although AMV029 does show degeneracy.
  • the fifth, 17K ORF gene family contains five members which do not show any homology to MsEPV proteins, but are instead related to the 17K ORF of HaEPV.
  • AMV024, AMVl 10 and AMVl 12 show excellent conservation at both their amino and carboxy termini, with a 60 residue internal portion of lesser similarity. Interestingly, these three genes also show homology with the
  • NlR/p28 gene family of FPV (FPV124).
  • AMVIOO and AMV132 are also homologous to the
  • the sixth gene family is the LRR (leucine-rich repeat) gene family which contains five AmEPV genes based upon the position of a motif containing regularly spaced leucine residues.
  • AMVLTRl and AMV005 are 63% identical, and very well conserved at their amino terminus.
  • AMV014 and AMV134 share regions of homology along their lengths.
  • AMV076 is significantly smaller than other LRR-like gene family members (varying from 350 to 535 residues).
  • an internal conserved motif emerges which includes seven leucine or isoleucine residues.
  • AMV 133 (SEQ ID NO: 1) encodes a AmEPV triacylglyceride lipase gene which could conceivably function as a virulence gene through lipid hydrolysis.
  • AmEPV has been shown to launch a promiscuous infection within the insect, including the fat body (Arif, B. M. and Kurstak, E. [1991]. The Entomopoxviruses. In "Viruses of Invertebrates,” E. Kurstak, Ed. together pp. 175-195. Marcel Dekker, Inc., New York), which is the major site of lipid storage (Chapman, R. F. [1998] Circulatory system, blood and immune systems. In "The Insects" pp.
  • AmEPV AMV255 (SEQ ID NO: 2) encodes a Cu ++ /Zn ++ superoxide dismutase homolog. These proteins are widespread in nature and are recognized as a primary defense against the damage of superoxide radicals (Fridovich, I. [1997] "Superoxide anion radical (02-.), superoxide dismutases, and related matters" J. Biol. Chem. 272:18515-18517). Although the SOD homolog was initially discovered in a baculovirus (Tomalski, M. D., Eldridge, R., and Miller, L. K.
  • apoptosis is controlled in part by serpins (Petit, F., Bergagnoli, S., Gelfi, J., Fassy, F., Boucraut-Baralon, C, and Milon, A. [1996] "Characterization of a myxoma virus-encoded serpin-like protein with activity against interleukin-lb converting enzyme" J. Virol. 70:5860-5866; Ray, C. A., Black, R. A., Kronheim, S. R., Greenstreet, T. A., Sleath, P. R.,
  • cowpox virus encodes an inhibitor ofthe interleukin-1 beta converting enzyme
  • Cell 69:597-604 Spriggs, M. K., Hruby, D. E., Maliszeswki, C. R., Pickup, D. J., Sims, J. E., Buller, R. M. L., and VanSlyke, J.
  • Vaccinia and Cowpox viruses encode a novel secreted interleukin-1 binding protein" Cell 71 : 145- 152; Ray, C. A. and Pickup, D. J.
  • AMV021 (SEQ ID NO: 4) encodes one such inhibitor of apoptosis protein (LAP), and contains two typical baculovirus LAP repeats and a C-terminal RLNG finger motif.
  • the AMV021 ORF shows significant identity to the IAP of Cydia pomonella granulosis virus (47%), which has previously been shown to be functionally active (Crook, N. E., Clem, R. J., and Miller, L. K. [1993] "An apoptosis-inhibiting baculovirus gene with a zinc finger-like motif J Virol. 67:2168-2174).
  • AmEPV and MsEPV are the only poxviruses found to encode IAPs. These proteins have only been noted in the genomes of viruses which infect insect or arthropod hosts.
  • the cytoplasmic synthesis of poxvirus mRNAs involves not only transcription of a given gene by the viral RNA polymerase, but also post-transcriptional modification ofthe transcripts, including 3' poly(A) addition and 5'capping as well as 2'0-methylation.
  • the AmEPV genomic sequence has revealed an unusual feature ofthe poly(A) polymerase in this entomopoxvirus. Like other poxviruses, there is a single, large subunit (AMV038) (SEQ LD NO: 11) of approximately 570 amino acids. This is similar in size to the large VV poly(A) polymerase subunit (VP55). However, unlike any other poxvirus (Afonso et al. [2000] supra; Afonso et al. [1999] supra; Cameron et al. [1999] supra; Wilier et al. [1999] supra; Senkevich etal. [1997] supra; Antoine etal. [1998] supra; Goebel etal.
  • AmEPV may encode two small subunits (AMV060 and AMVl 15).
  • the two small subunits are somewhat smaller than the 333 amino acid VV small subumit (295 and 293 amino acids respectively) ( Figure 10) and related throughout their length.
  • Both AmEPV small subunits contain a highly conserved poly(A) polymerase regulatory structural motif encompassing amino acids 1-281 within AMV060 and amino acids 8- 271 withinAMV115.
  • the AMV060 subunit is more related to VP39 than is AMVl 15.
  • MSV041 single poly(A) polymerase small subunit of MsEPV
  • the comparable W small subunit contains a C- terminal extension.
  • the W 36-43 amino acid C-terminal tail is non-essential for activity (Shi, X., Yao, P., Jose, T., and Gershon, P. [1996] "Methyltransferase-specific domains within VP-39, a bifunctional protein that participates in the modification of both mRNA ends" RNA 2:88-101), and is probably retained because the C-terminal region of the W subunit overlaps the next open reading frame (J4R) which encodes a 22kDa subunit ofthe W RNA polymerase (Goebel et al. [1990] supra).
  • the second, distinct activity, mediated by VP39 alone, is an mRNA cap-specific 2'-0-methyltransferase (Schnierle et al. [1992] supra).
  • the third activity is an associated transcription elongation factor (Latner, D. R., Xiang, Y., Lewis, J. I., Condit, J., and Condit, R. C. [2000] "The vaccinia virus bifunctional gene J3 (nucleoside-2 '-O-)- methyltransferase and poly(A) polymerase stimulatory factor is implicated as a positive transcription elongation factor by two genetic approaches" Virology 269:345-355). It is possible that these various activities have been distributed amongst the two subunits. Alternatively, one ofthe subunits may have evolved to fulfill an entirely unrelated function.
  • AMV050 (SEQ LD NO: 7) and AMV210 (SEQ LD NO: 8) encode AmEPV DNA polymerases.
  • African Swine Fever virus Oliveros, M., Yanez, R. J., Salas, M. L., Salas, J., Vinuela, E., and Blanco, L. [1997] "Characterization of an African swine fever virus 20-kDa DNA polymerase involved in DNA repair" J Biol. Chem. 272:30899-30910) and later in MsEPV (Afonso etal. [1999] supra), which has also been found in AmEPV.
  • the 1105 residue AmEPV ORF AMV050 is similar in length, and homologous to typical poxvirus encoded DNA polymerases.
  • AmEPV encoded ORF AMV210
  • AMV210 shares a 460 amino acid region of clear homology with AMV050, although both proteins possess completely unique regions; i.e., the N-terminus of AMV050 (residues 1-645) and C-terminus of AMV210 (residues 463-612). Both proteins have been found to contain DNA polymerase motifs (Table 1).
  • AMV130 SEQ LD NO: 9 encodes an AmEPV ABC transporter-like protein.
  • AMV130 represents the largest ORF in AmEPV.
  • the 1384 residue protein shows homology to the ATP- binding cassette (ABC) proteins. These are a large gene family found from bacteria to man, and have a variety of functions (van Veen, H. W.
  • All ABC proteins share a common molecular architecture consisting of at least one 200-250 amino acid ABC cassette and several predicted ⁇ -helical membrane spanning segments (TMS or TMD). The minimum structural requirement is considered to be 2 ABC and 2 TMD regions, present in either 1 (full transporter) or 2 (half transporter) polypeptide chains.
  • the AmEPV ABC protein consists of TMD-ABC-TMD-ABC domains, one ofthe structures of active ABC transporters.
  • AMV130 domains is also found in the MDR TAP, MRP, CFTR and ABCl subfamilies and is associated with activities ranging from control of sex (yeast), drug resistance (humans, bacteria), ion channels (human CFTR gene) and engulfment of dead cells (C. elegans) (Bauer etal. [1999] supra; Klein et al. [1999] supra; Abele and Tampe [1999] supra).
  • Each AmEPV TMD contains 6 or 7 transmembrane helices
  • AMV007 (SEQ LD NO: 10) encodes an AmEPV Kunitz-motif protease inhibitor (KPI).
  • AmEPV ORF AMV007 is located near the left end ofthe AmEPV genome, and encodes a small protein of 79 amino acids.
  • a Prosite search revealed the presence of a Kunitz family signature (Prosite PS00280), a motif associated with protease inhibitors ( Figure 12).
  • Kunitz- type pancreatic trypsin inhibitors represent one ofthe most common families of serine protease inhibitors.
  • Kunitz-type inhibitors found within insects are typically less than 100 amino acids in length. All contain certain five invariant cysteine residues.
  • AMV007 has all five cysteines and the alignment allows prediction of an arginine PI .
  • the inducible serine protease inhibitor (ISP-2) of Galleria mellonella (Frobius, A. C, Kanost, M. R., Gotz, P., and Vilcinskas, A. [2000] "Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella" Eur.
  • Kunitz-type inhibitors are comprised of short alpha/beta proteins with little secondary structure. Although widespread in nature, there are no reports ofthe presence of a Kunitz-type protease inhibitor (KPI) from this family in any viral genome. It is interesting to note that vertebrate poxviruses do encode protease inhibitors, but they are members of a different family (the serine protease inhibitor, serpin) family. The vertebrate poxvirus serpins have been shown to have an immunoregulatory role in the infected vertebrate host (Turner, S., Kenshole, B., and Ruby, j.
  • AmEPV KPI protein may fulfill a similar immunoregulatory role in the infected invertebrate host, but may target different pathways than do the serpins which control inflammation, apoptosis and the host immune response (Turner and Moyer [1998] supra; Turner, P. C, Musy, P. Y., and Moyer, R. W. [1995] Poxvirus Serpins, In "Viroceptors, Virokines and related immune modulators encoded by DNA viruses," G. McFadden, ed., pp. 67-88. R. G. Landes, Galveston, TX).
  • KPI protein may possess a variety of protease inhibitors from several different gene families (Kanost, M. R. [1999] "Serine proteinase inhibitors in arthropod immunity” Developmental and Comparative Immunology 23:291-301; Jiang, H. B. and Kanost, M. R. [1997] "Characterization and functional analysis of 12 naturally occurring reactive site variants of serpin-1 from Manduca sexta” J. Biol. Chem. 272:1082-1087).
  • Protease inhibitors from the Kunitz family have been identified as haemolymph proteins from lepidopteran insect species (Sugumaran, M., Saul, S. J., and Ramesh, N. [1985] "Endogenous protease inhibitors prevent undesired activation of prophenolase in insect hemolymph” Biochem. Biophys. Res. Common. 132:1124-1129; Sasaki, T. [1984] "Amino acid sequence of a novel Kunitz-type chymotrysin inhibitor from hemolymph of silkworm larvae" Bombyx moorei. FEBS Lett. 168:230), which function as inhibitors of trypsin or chymotrypsin.
  • This enzyme is an early component ofthe cascade required by the insect immune system to produce melanin, which is used to engulf and overcome invading foreign objects (Gillespie, J. P., Kanost, M. R., and Trenczek, T. [1997] "Biological mediators of insect immunity” Annu. Rev. Entomol. 42:611-43, 611-643; Vilmos, P. and Kurucz, E. [1998] "Insect immunity: evolutionary roots ofthe mammalian imiate immune system” Immunol. Lett. 62:59-66). Production of such a protein by an infecting virus may therefore lessen the amount of prophenyloxidase induced by the insect immune system during infection.
  • Polynucleotides of the subject invention include sequences identified in the attached sequence listing as well as the tables and figures and described by open reading frame (ORF) position within the genome.
  • the subject invention includes polynucleotides which hybridize with other polynucleotides ofthe subject invention.
  • polynucleotide sequences exemplified herein can be used in a variety of ways, having numerous applications in techniques known to those skilled in the art of molecular biology having the instant disclosure. These techniques include their use as hybridization probes, for chromosome and gene mapping, in PCR technologies, and in the production of sense or antisense nucleic acids.
  • polynucleotides can be used in assays for additional polynucleotides and additional homologous genes, and can be used in tracking the quantitative and temporal expression of these genes in cells and organisms.
  • Polynucleotides ofthe subject invention may be used as insertion sites for foreign genes of interest.
  • Antisense technology can also be used to interfere with expression of the disclosed polynucleotides. For example, the transformation of a cell or organism with the reverse complement of a gene encoded by a polynucleotide exemplified herein can result in strand co- suppression and silencing or inhibition of a target gene, e.g., one involved in the infection process.
  • Polynucleotides disclosed herein are useful as target genes for the synthesis of antisense
  • RNA or dsRNA useful for RNA-mediated gene interference.
  • the ability to specifically inhibit gene function in a variety of organisms utilizing antisense RNA or ds RNA-mediated interference is well known in the fields of molecular biology (see for example C.P. Hunter, Current Biology [1999] 9:R440-442; Hamilton et al., [1999] Science, 286:950-952; and S.W. Ding, Current Opinions in Biotechnology [2000] 11:152-156, hereby incorporated by reference in their entireties).
  • dsRNA typically comprises a polynucleotide sequence identical or homologous to a target gene (or fragment thereof) linked directly, or indirectly, to a polynucleotide sequence complementary to the sequence of the target gene (or fragment thereof).
  • the dsRNA may comprise a polynucleotide linker sequence of sufficient length to allow for the two polynucleotide sequences to fold over and hybridize to each other; however, a linker sequence is not necessary.
  • the linker sequence is designed to separate the antisense and sense strands of RNAi significantly enough to limit the effects of steric hindrances and allow for the formation of dsRNA molecules and should not hybridize with sequences within the hybridizing portions ofthe dsRNA molecule.
  • the specificity of this gene silencing mechanism appears to be extremely high, blocking expression only of targeted genes, while leaving other genes unaffected. Accordingly, one method for controlling gene expression according to the subject invention provides materials and methods using double-stranded interfering RNA (dsRNAi), or RNA-mediated interference (RNAi).
  • dsRNAi double-stranded interfering RNA
  • RNAi RNA-mediated interference
  • RNA containing a nucleotide sequence identical to a fragment of the target gene is preferred for inhibition; however, RNA sequences with insertions, deletions, and point mutations relative to the target sequence can also be used for inhibition.
  • Sequence identity may optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g.,
  • the duplex region ofthe RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a fragment ofthe target gene transcript.
  • RNA may be synthesized either in vivo or in vitro. Endogenous RNA polymerase ofthe cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro.
  • a regulatory region e.g., promoter, enhancer, silencer, splice donor and acceptor, polyadenylation
  • the promoters may be known inducible promoters such as baculovirus. Inhibition may be targeted by specific transcription in an organ, tissue, or cell type.
  • the RNA strands may or may not be polyadenylated; the RNA strands may or may not be capable of being translated into a polypeptide by a cell's translational apparatus.
  • RNA may be chemically or enzymatically synthesized by manual or automated reactions.
  • the RNA may be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase
  • RNA may be purified prior to introduction into the cell.
  • RNA can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof.
  • the RNA may be used with no or a minimum of purification to avoid losses due to sample processing.
  • RNA may be dried for storage or dissolved in an aqueous solution.
  • the solution may contain buffers or salts to promote annealing, and/or stabilization ofthe duplex strands.
  • dsRNAi can be targeted to an entire polynucleotide sequence set forth herein.
  • Preferred RNAi molecules of the instant invention are highly homologous or identical to the polynucleotides ofthe sequence listing. The homology may be greater than 70%, preferably greater than 80%, more preferably greater than 90% and is most preferably greater than 95%. Fragments of genes can also be utilized for targeted suppression of gene expression.
  • fragments are typically in the approximate size range of about 20 nucleotides.
  • targeted fragments are preferably at least about 15 nucleotides.
  • the gene fragment targeted by the RNAi molecule is about 20-25 nucleotides in length. In a more preferred embodiment, the gene fragments are at least about 25 nucleotides in length. In an even more preferred embodiment, the gene fragments are at least 50 nucleotides in length.
  • RNAi molecules ofthe subject invention are not limited to those that are targeted to the full-length polynucleotide or gene.
  • Gene product can be inhibited with a RNAi molecule that is targeted to a portion or fragment ofthe exemplified polynucleotides; high homology (90-95%) or greater identity is also preferred, but not necessarily essential, for such applications.
  • the dsRNA molecules of the invention may be introduced into cells with single stranded (ss) RNA molecules which are sense or anti-sense RNA derived from the nucleotide sequences disclosed herein.
  • Methods of introducing ssRNA and dsRNA molecules into cells are well-known to the skilled artisan and includes transcription of plasmids, vectors, or genetic constructs encoding the ssRNA or dsRNA molecules according to this aspect ofthe invention; electroporation, biolistics, or other well-known methods of introducing nucleic acids into cells may also be used to introduce the ssRNA and dsRNA molecules of this invention into cells.
  • nucleotide sequences as disclosed herein may be used to produce an amino acid sequence using well known methods of recombinant DNA technology.
  • Goeddel Gene Expression Technology, Methods and Enzymology [1990] Vol 185, Academic Press, San Diego, CA
  • the amino acid or peptide may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species from which the nucleotide sequence was derived or from a different species.
  • Still further aspects ofthe invention use these purified peptides to produce antibodies or other molecules able to bind to the peptides. These antibodies or binding agents can then be used for the screening of cells in order to localize the cellular distribution ofthe peptides or proteins. The antibodies are also useful for the affinity purification of recombinantly produced peptides or proteins.
  • the disclosed nucleotide sequences can be used individually, or in panels, in tests or assays to detect levels of peptide, polypeptide, or protein expression.
  • the form of such qualitative or quantitative methods may include northern analysis, dot blot or other membrane based technologies, dip stick, pin or chip technologies, PCR, ELISAs or other multiple sample format technologies.
  • the subject invention also provides polynucleotides identified as control elements or regulatory sequences, such as gene promoters, enhancers, introns and untranslated regions which interact with cellular components to carry out regulatory functions such as replication, transcription, and translation.
  • the invention further comprises the use of the disclosed polynucleotide sequences, or fragments thereof, in assays to characterize and/or identify sequences having promoter or other regulatory activity. Also contemplated according to the subject invention is the use of oligomers from these sequences in kits which can be used to identify promoters or other regulatory sequences. As used herein, the following definitions apply:
  • oligonucleotide or “oligomer” is a stretch of nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR). These short sequences are based on (or designed from) genomic or cDNA sequences and are used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides. They can be chemically synthesized and may be used as probes.
  • Probes are nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use. They are used in the detection of identical, similar, or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. They may be single- or double-stranded and designed to have specificity in PCR, hybridization membrane-based, or ELISA-like technologies.
  • Reporter molecules are chemical moieties used for labeling a nucleic or amino acid sequence. They include, but are not limited to, radionuclides, enzymes, fluorescent, chemi-luminescent, or chromogenic agents. Reporter molecules associate with, establish the presence of, and may allow quantification of a particular nucleic or amino acid sequence.
  • a "portion" or “fragment” of a polynucleotide or nucleic acid comprises all or any part of the nucleotide sequence having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb which can be used as a probe.
  • probes may be labeled with reporter molecules using nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. After pretesting to optimize reaction conditions and to eliminate false positives, nucleic acid probes may be used in Southern, northern or in situ hybridizations to determine whether target DNA or RNA is present in a biological sample, cell type, tissue, organ or organism.
  • Recombinant nucleotide variants are alternate polynucleotides which encode a particular protein. They may be synthesized, for example, by making use ofthe “redundancy” in the genetic code. Various codon substitutions, such as the silent changes which produce specific restriction sites or codon usage-specific mutations, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic host system, respectively.
  • Linkers are synthesized palindromic nucleotide sequences which create internal restriction endonuclease sites for ease of cloning the genetic material of choice into various vectors.
  • Polylinkers are engineered to include multiple restriction enzyme sites and provide for the use of both those enzymes which leave 5' and 3' overhangs such as BamHI, EcoRI, Pstl, Kpnl and Hind III or which provide a blunt end such as EcoRV, SnaBI and Stul.
  • Control elements are regions ofthe gene or DNA such as enhancers, promoters, introns and 3' untranslated regions which interact with cellular proteins to carry out replication, transcription, and translation. Typically, these regions are nontranslated.
  • Chimeric molecules are polynucleotides or polypeptides which are created by combining one or more nucleotide peptide sequences (or their parts). In the case of nucleotide sequences, such combined sequences may be introduced into an appropriate vector and expressed to give rise to a chimeric polypeptide which may be expected to be different from the native molecule in one or more ofthe following characteristics: cellular location, distribution, ligand-binding affinities, interchain affinities, degradation/turnover rate, signaling, etc.
  • Active is that state which is capable of being useful or of carrying out some role. It specifically refers to those forms, fragments, or domains of an amino acid sequence which display the biologic and/or immunogenic activity characteristic of the naturally occurring peptide, polypeptide, or protein.
  • Naturally occurring refers to a polypeptide produced by cells which have not been genetically engineered or which have been genetically engineered to produce the same sequence as that naturally produced.
  • Derivative refers to those polypeptides which have been chemically modified by such techniques as ubiquitination, labeling, pegylation (derivatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in proteins.
  • Recombinant polypeptide variant refers to any polypeptide which differs from naturally occurring peptide, polypeptide, or protein by amino acid insertions, deletions and/or substitutions.
  • substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.
  • Amino acid "insertions” or “deletions” are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. The variation allowed in a particular amino acid sequence may be experimentally determined by producing the peptide synthetically or by systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.
  • a “signal or leader sequence” is a short amino acid sequence which can be used, when desired, to direct the polypeptide through a membrane of a cell. Such a sequence may be naturally present on the polypeptides ofthe present invention or provided from heterologous sources by recombinant DNA techniques. Such sequences include nuclear localization sequences (NLS) known in the art.
  • NLS nuclear localization sequences
  • oligopeptide is a short stretch of amino acid residues and may be expressed from an oligonucleotide. Such sequences comprise a stretch of amino acid residues of at least about 5 amino acids and often about 17 or more amino acids, typically at least about 9 to 13 amino acids, and of sufficient length to display biologic and/or immunogenic activity.
  • inhibitor is a substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, antagonists and their derivatives.
  • a "standard” is a quantitative or qualitative measurement for comparison. Preferably, it is based on a statistically appropriate number of samples and is created to use as a basis of comparison when performing diagnostic assays, running clinical trials, or following patient treatment profiles. The samples of a particular standard may be normal or similarly abnormal.
  • DNA possesses a fundamental property called base complementarity.
  • DNA ordinarily exists in the form of pairs of anti-parallel strands, the bases on each strand projecting from that strand toward the opposite strand.
  • the base adenine (A) on one strand will always be opposed to the base thymine (T) on the other strand, and the base guanine (G) will be opposed to the base cytosine (C).
  • the bases are held in apposition by their ability to hydrogen bond in this specific way. Though each individual bond is relatively weak, the net effect of many adjacent hydrogen bonded bases, together with base stacking effects, is a stable joining ofthe two complementary strands.
  • polynucleotides ofthe subject invention can themselves be used as probes. Additional polynucleotide sequences can be added to the ends of (or internally in) the exemplified polynucleotide sequences so that polynucleotides that are longer than the exemplified polynucleotides can also be used as probes. Thus, isolated polynucleotides comprising one or more ofthe exemplified sequences are within the scope ofthe subject invention. Polynucleotides that have less nucleotides than the exemplified polynucleotides can also be used and are contemplated within the scope ofthe present invention.
  • polynucleotides ofthe subject invention can be used to find additional, homologous (wholly or partially) genes.
  • Hybridization probes of the subject invention may be derived from the open reading frames specifically exemplified in the sequence listing, figures, and tables as well as from surrounding or included genomic sequences comprising untranslated regions such as promoters, enhancers and introns.
  • Probes ofthe subject invention may be composed of DNA, RNA, or PNA (peptide nucleic acid).
  • the probe will normally have at least about 10 bases, more usually at least about 17 bases, and may have up to about 100 bases or more. Longer probes can readily be utilized, and such probes can be, for example, several kilobases in length.
  • the probe sequence is designed to be at least substantially complementary to a portion of a gene encoding a protein of interest. The probe need not have perfect complementarity to the sequence to which it hybridizes.
  • the probes may be labeled utilizing techniques that are well known to those skilled in this art.
  • One approach for the use ofthe subject invention as probes entails first identifying DNA segments that are homologous with the disclosed nucleotide sequences using, for example, Southern blot analysis of a gene bank.
  • Southern blot analysis of a gene bank.
  • One hybridization procedure useful according to the subject invention typically includes the initial steps of isolating the DNA sample of interest and purifying it chemically. Either lysed cells or total fractionated nucleic acid isolated from cells can be used. Cells can be treated using known techniques to liberate their DNA (and/or RNA). The DNA sample can be cut into pieces with an appropriate restriction enzyme. The pieces can be separated by size through electrophoresis hi a gel, usually agarose or acrylamide. The pieces of interest can be transferred to an immobilizing membrane.
  • the particular hybridization technique is not essential to the subject invention. As improvements are made in hybridization techniques, they can be readily applied.
  • the probe and sample can then be combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs. Thereafter, the membrane is washed free of extraneous materials, leaving the sample and bound probe molecules typically detected and quantified by autoradiography and/or liquid scintillation counting.
  • the probe molecule and nucleic acid sample hybridize by forming a strong non-covalent bond between the two molecules, it can be reasonably assumed that the probe and sample are essentially identical or very similar.
  • the probe's detectable label provides a means for determining in a known manner whether hybridization has occurred.
  • the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels.
  • Typical radioactive labels include 32 P, 35 S, or the like.
  • Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or peroxidases, or the various chemiluminescers such as luciferin, or fluorescent compounds like fluorescein and its derivatives.
  • the probes can be made inherently fluorescent as described in International Application No. WO 93/16094.
  • hybridization is conducted under moderate to high stringency conditions by techniques well known in the art, as described, for example, in Keller, G.H., M.M. Manak (1987) DNA Probes, Stockton Press, New York, NY., pp. 169-170.
  • moderate to high stringency conditions for hybridization refers to conditions that achieve the same, or about the same, degree of specificity of hybridization as the conditions "as described herein.” Examples of moderate to high stringency conditions are provided herein. Specifically, hybridization of immobilized DNA on Southern blots with 32 P-labeled gene- specific probes was performed using standard methods (Maniatis et al). In general, hybridization and subsequent washes were carried out under moderate to high stringency conditions that allowed for detection of target sequences with homology to sequences exemplified herein.
  • Tm melting temperature
  • Washes are typically carried out as follows:
  • Tm (°C) 2(number T/A base pairs) +4(number G/C base pairs) Washes were typically carried out as follows:
  • salt and/or temperature can be altered to change stringency.
  • a labeled DNA fragment of greater than about 70 or so bases in length the following conditions can be used: Low: 1 or 2X SSPE, room temperature
  • polynucleotide sequences ofthe subject invention include mutations (both single and multiple), deletions, and insertions in the described sequences, and combinations thereof, wherein said mutations, insertions, and deletions permit formation of stable hybrids with a target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence using standard methods known in the art. Other methods may become known in the future.
  • the mutational, insertional, and deletional variants ofthe polypeptide sequences ofthe invention can be used in the same manner as the exemplified polynucleotide sequences so long as the variants have substantial sequence similarity with the original sequence.
  • substantial sequence similarity refers to the extent of nucleotide similarity that is sufficient to enable the variant polynucleotide to function in the same capacity as the original sequence.
  • this similarity is greater than 50%; more preferably, this similarity is greater than 75%; and most preferably, this similarity is greater than 90%.
  • the degree of similarity needed for the variant to function in its intended capacity will depend upon the intended use ofthe sequence. It is well within the skill of a person trained in this art to make mutational, insertional, and deletional mutations that are designed to improve the function of the sequence or otherwise provide a methodological advantage.
  • the genes ofthe subject invention have at least one ofthe following characteristics: said gene is encoded by a nucleotide sequence which hybridizes under stringent conditions with a nucleotide sequence selected from the group consisting of: DNA which encodes
  • SEQ LD NO: 1 DNA which encodes SEQ LD NO: 2
  • DNA which encodes SEQ LD NO: 3 DNA which encodes SEQ LD NO: 4
  • DNA which encodes SEQ LD NO: 6 or SEQ LD NO: 8 DNA which encodes SEQ
  • the subject invention also includes polynucleotides that hybridize with other polynucleotides ofthe subject invention.
  • PCR Polymerase Chain Reaction
  • PCR is a repetitive, enzymatic, primed synthesis of a nucleic acid sequence. This procedure is well known and commonly used by those skilled in this art (see U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al, 1985).
  • PCR is based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize to opposite strands ofthe target sequence. The primers are oriented with the 3 ' ends pointing towards each other.
  • thermostable DNA polymerase such as Taq polymerase, which is isolated from the thermophilic bacterium Thermus aquaticus, the amplification process can be completely automated.
  • Other enzymes that can be used are known to those skilled in the art.
  • polynucleotide sequences of the subject invention can be used as, and/or used in the design of, primers for PCR amplification.
  • a certain degree of mismatch can be tolerated between primer and template. Therefore, mutations, deletions, and insertions (especially additions of nucleotides to the
  • PCR as a direct method which uses universal primers to retrieve unknown sequence adjacent to a known locus.
  • genomic DNA is amplified in the presence of primer to linker and a primer specific to the known region.
  • the amplified sequences are subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR can be used to acquire unknown sequences starting with primers based on a known region (Triglia T. et al. (1988) Nucleic Acids Res 16:8186).
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. The multiple rounds of restriction enzyme digestions and ligations that are necessary prior to PCR make the procedure slow and expensive (Gobinda et al. [1993] supra). Capture PCR (Lagerstrom M. et al.
  • PCR Methods Applic 1 111-19 is a method for PCR amplification of DNA fragments adjacent to a known sequence in eucaryotic and YAC DNA.
  • capture PCR also requires multiple restriction enzyme digestions and ligations to place an engineered double-stranded sequence into an unknown portion ofthe DNA molecule before PCR.
  • restriction and ligation reactions are carried out simultaneously, the requirements for extension, immobilization and two rounds of PCR and purification prior to sequencing render the method cumbersome and time consuming.
  • PromoterFinderTM is a kit available from Clontech Laboratories, Lnc. (Palo Alto, CA) which uses PCR and primers derived from p53 to walk in genomic DNA. Nested primers and special PromoterFinderTM libraries are used to detect upstream sequences such as promoters and regulatory elements. This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • a new PCR method replaces methods which use labeled probes to screen plasmid libraries and allow one researcher to process only about 3-5 genes in 14-40 days.
  • the first step which can be performed in about two days, any two of a plurality of primers are designed and synthesized based on a known partial sequence.
  • step 2 which takes about six to eight hours, the sequence is extended by PCR amplification of a selected library.
  • Steps 3 and 4 which take about one day, are purification ofthe amplified cDNA and its ligation into an appropriate vector.
  • Step 5 which takes about one day, involves transforming and growing up host bacteria.
  • step 6 which takes approximately five hours, PCR is used to screen bacterial clones for extended sequence.
  • the final steps which take about one day, involve the preparation and sequencing of selected clones.
  • the preferred library may be one that has been size-selected to include only larger cDNAs or may consist of single or combined commercially available libraries, e.g., from Clontech Laboratories, Lnc. (Palo Alto, CA).
  • the cDNA library may have been prepared with oligo (dT) or random priming. Random primed libraries are preferred in that they will contain more sequences which contain 5' ends of genes. A randomly primed library may be particularly useful if an oligo (dT) library does not yield a complete gene. It must be noted that the larger and more complex the protein, the less likely it is that the complete gene will be found in a single plasmid.
  • CLONTECH PCR-SelectTM cDNA Subtraction (Clontech Laboratories, Inc., Palo Alto, CA) is yet another means by which differentially expressed genes may be isolated. The procedure allows for the isolation of transcripts present in one mRNA population which is absent, or found in reduced numbers, in a second population of mRNA. Rare transcripts may be enriched 1000- fold.
  • a new method for analyzing either the size or the nucleotide sequence of PCR products is capillary electrophoresis.
  • Systems for rapid sequencing are available from Perkin Elmer (Foster City CA), Beckman Instruments (FuUerton, CA), and other companies.
  • Capillary sequencing employs flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection ofthe emitted wavelengths by a charge coupled devise camera.
  • Output/light intensity is converted to electrical signal using appropriate software (eg. GenotyperTM and Sequence NavigatorsTM from Perkin Elmer) and the entire process from loading of samples to computer analysis and electronic data display is computer controlled.
  • Capillary electrophoresis provides greater resolution and is many times faster than standard gel based procedures. It is particularly suited to the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample. The reproducible sequencing of up to 350 bp of M13 phage DNA in 30 min has been reported (Ruiz-Martinez M. C. et al. [1993] Anal Chem 65:2851-8).
  • Polynucleotides and proteins Polynucleotides and proteins. Polynucleotides ofthe subject invention can be defined according to several parameters. One characteristic is the biological activity of the protein products as identified herein.
  • the proteins and genes of the subject invention can be further defined by their amino acid and nucleotide sequences.
  • the sequences ofthe molecules can be defined in terms of homology to certain exemplified sequences as well as in terms ofthe ability to hybridize with, or be amplified by, certain exemplified probes and primers. Additional primers and probes can readily be constructed by those skilled in the art such that alternate polynucleotide sequences encoding the same amino acid sequences can be used to identify and/or characterize additional genes.
  • the proteins of the subject invention can also be identified based on their immunoreactivity with certain antibodies.
  • polynucleotides and proteins or polypeptides ofthe subject invention include portions, fragments, variants, and mutants ofthe full-length sequences as well as fusions and chimerics, so long as the encoded protein retains the characteristic biological activity ofthe proteins identified herein.
  • variants or variantations refer to nucleotide sequences that encode the same proteins or which encode equivalent proteins having equivalent biological activity.
  • equivalent proteins refers to proteins having the same or essentially the same biological activity as the exemplified proteins.
  • genes may be readily constructed using standard techniques such as site- directed mutagenesis and other methods of making point mutations and by DNA shuffling, for example.
  • gene and protein fragments can be made using commercially available exonucleases, endonucleases, and proteases according to standard procedures.
  • enzymes such as BaB 1 can be used to systematically cut off nucleotides from the ends of genes.
  • genes that encode fragments may be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these proteins.
  • molecular techniques for cloning polynucleotides and producing gene constructs of interest are also well known in the art. In vitro evaluation techniques, such as MAXYGEN's "Molecular Breeding" can also be applied to practice the subject invention.
  • DNA sequences can encode the amino acid sequences encoded by the polynucleotide sequences disclosed herein. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding proteins having the same, or essentially the same, amino acid sequence. These variant DNA sequences are within the scope of the subject invention. As used herein, reference to "essentially the same" sequence refers to sequences that have amino acid substitutions, deletions, additions, or insertions that do not materially affect biological activity. Fragments retaining the characteristic biological activity are also included in this definition.
  • a further method for identifying genes and polynucleotides (and the proteins encoded thereby) ofthe subject invention is through the use of oligonucleotide probes.
  • Probes provide a rapid method for identifying genes ofthe subject invention.
  • the nucleotide segments that are used as probes according to the invention can be synthesized using a DNA synthesizer and standard procedures.
  • the subject invention comprises variant or equivalent proteins (and nucleotide sequences coding for equivalent proteins) having the same or similar biological activity of proteins encoded by the exemplified polynucleotides.
  • Equivalent proteins will have amino acid similarity with an exemplified protein (or peptide). The amino acid identity will typically be greater than 60%.
  • the amino acid identity will be greater than 75%. More preferably, the amino acid identity will be greater than 80%, and even more preferably greater than 90%. Most preferably, amino acid identity will be greater than 95%.
  • the polynucleotides that encode the subject polypeptides will also have corresponding identities in these preferred ranges.) These identities are as determined using standard alignment techniques for determining amino acid identity.
  • the amino acid identity/similarity/ homology will be highest in critical regions ofthe protein including those regions that account for biological activity or that are involved in the determination of three-dimensional configuration that is ultimately responsible for the biological activity.
  • amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid ofthe same type fall within the scope ofthe subject invention so long as the substitution does not materially alter the biological activity ofthe compound.
  • Table 4 provides a listing of examples of amino acids belonging to each class.
  • non-conservative substitutions can also be made.
  • the critical factor is that these substitutions must not significantly detract from the biological activity of the polypeptide.
  • nucleic acid molecule or polynucleotide is a polynucleotide that is substantially separated from other polynucleotide sequences which naturally accompany a nucleic acid molecule.
  • the term embraces a polynucleotide sequence which was removed from its naturally occurring environment by the hand of man. This includes recombinant or cloned DNA isolates, chemically synthesized analogues and analogues biologically synthesized by heterologous systems.
  • An "isolated” or “purified” protein or polypeptide likewise, is a one removed from its naturally occurring environment.
  • TK negative cell line designated C11.3 was selected by a process of adaption of TK(+) LD652 cell to increasing levels, 10 ⁇ g/ml every 5 weeks, of 5-bromo-2'-deoxyuridine (BudR) over one year up to 100 ⁇ g/ml BudR and maintained in TE medium containing BudR (100 A ⁇ g/ml). 293 cells were grown in DMEM medium supplemented with 5% fatal bovine serum.
  • pTR-UF5 contains GFP and NeoR genes under control CMV promoter and herpes virus TK promoter respectively and flanked by ITR sequences of AAV.
  • the Pst I fragment which contains
  • GFP and NeoR markers was inserted into Pst I site of pTKDU (Li, Y., R.L. Hall, S.L. Yuan, R.W. Moyer [1998] "High level expression of Amsacta moorei entomopoxvirus Spheroidin depends on sequences within the gene" J Gen. Virol. 79:613-622) to produce pTKUF5.
  • AmEPV recombinant with an insert in the TK gene was obtained as described previously (Li et al [1998] supra).
  • Infections (cells and medium) were harvested 6 days post-infection and centrifuged at 500 x g for 15 minutes to remove cells. The supernatant was centrifuged at 40,000 x g for 30 minutes to pellet virus. The pellet was resuspended in dH 2 0 (100 ⁇ L for each initial 30 mL of supernatant). DNase free RNase was added to a final concentration of 50 ⁇ g/mL and incubated at 37 °C for 30 min.
  • the sample and lysis buffer (lOOmM Tris pH 8.0, 10 mM EDTA, 54% sucrose, 2% SDS, lOmM ⁇ - mercaptoethanol) were brought to 50°C, and lysis buffer was added to the sample at a 1 : 1 ratio. Proteinase K was added to a final concentration of 0.6 mg/mL. The viral lysate was incubated overnight at 50°C. The lysate was extracted three times with 50:49: 1 phenol:chloroform:isoamyl alcohol, once with chloroform, and the DNA precipitated in 0.4 M LiCl 2 , 95% ethanol. Tsp5091 partial digest library preparation
  • Fragments of 2-3 kb and 4-5 kb were gel-purified separately with the Gene-Clean LI kit (Bio 101, Vista, CA) and ligated into the EcoRI site ofthe PUC19 plasmid vector (Amersham Pharmacia Biotech UK Ltd., Chalfont, Buckinghamshire, England).
  • the ligation mixture was transformed into DH5- ⁇ competent cells and plated onto LB agar plates containing 50 ⁇ g/mL ampicillin and 800 ⁇ g/plate each LPTG and X-gal (Horton, P. and Nakai, K. [1997] "Better prediction of protein cellular localization sites with the k nearest neighbors classifier" Ismb. 5 : 147-152).
  • White colonies were isolated and grown overnight in 1 mL TB medium (Horton & Nakai [1997] supra) plus 50 ⁇ g/mL ampicillin.
  • Plasmid DNA was prepared using the QLAgen BioRobot 9600 and the QLAprep 96 Turbo miniprep kit. Sequencing was performed with 200-500ng of plasmid DNA as template using a 0.25X concentration of ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (#4303153; Perkin-Elmer Applied Biosystems [ABI], Foster City, CA). Cycle sequencing was performed using a PTC-200 DNA Engine (MJ Research, Watertown, MA) (25 cycles: 1 degree per second to 96 degrees; 96 for 10 seconds; 1 degree per second to 60 degrees; 60 for 4 minutes). Dye terminator removal was on Multiscreen-HV plates (Millipore) with Sephadex G-50 superfine (Sigma, St. Louis, MO) in water. The reactions were electrophoresed on an ABI 377 sequencer, and the chromatograms were edited with Analysis version 1.2.1 (ABI) and assembled as follows.
  • BLAST a new generation of protein database search programs" Nucleic Acids Res. 25:3389- 3402; Bateman, A., Birney, E., Durbin, R., Eddy, S. R., Finn, R. D., and Sonnhammer, E. L. [1999] "Pfam 3.1 : 1313 multiple alignments and profile HMMs match the majority of proteins" Nucleic Acids Res. 27:260-262). After assembling 3500 chromatograms into 6 contigs, Consed designed 43 finishing experiments. Custom oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, LA), and upon completion of the experiments, the assembly contained the entire unique region ofthe genome and one inverted terminal repeat (LTR).
  • LTR inverted terminal repeat
  • Methionine-initiated open reading frames were delineated using Vector NTI. Open reading frames that translated into proteins less than 60 amino acids were discarded from our analysis. Relevant homologies were determined by BLAST analysis (Parsons, J. D. [1995] "Miropeats: graphical DNA sequence comparisons" Comput. Appl. Biosci. 11:615-619; Ewing, B., Hillier, L., Wendl, M. C, and
  • Example 1 Gene expression in cells infected with recombinant AmEPV
  • UF5 or pTKUF5 at a 5 ⁇ g/well plasmid DNA.
  • virus infected or plasmid transfected cells were transferred into 60 mm dishes, after 24 hr, neomycin resistant colonies were selected by adding G418 at the final concentration of 200 ⁇ g/ml. G418 containing medium was changed every 3-4 days.
  • NeoR gene copy number in AmEPV derived colonies is less than those transfected with plasmids. This explanation is likely to be true as we were able to show that the AmEPV derived colonies gradually become more and more resistant to G418 and soon, some GFP positive clusters of cells were observed which become more numerous and brighter. After several changes of medium, ultimately, all cells in the well were GFP positive.
  • Example 2 Stable integration of foreign DNA sequences into mammalian cells infected with recombinant AmEPV
  • Genomic DNA was recovered from cell lines created by either infection with the virus AmEPVpTKUF5 or following transfection with a control plasmid pTR-UF5. Specifically, the recombinant AmEPVpTKUF5 was used to infect and subsequently select 293 (human kidney) cells at a multiplicity of 5 plaque forming units per cell, as described in Example 1. After growing the isolated cell lines reliably for multiple generations, DNA was isolated and digested with Hindlll before electrophoresis and blotted with a random labeled probe containing the gfp and neo genes which are contained within the LTR regions of pTR-UF5.
  • lane P contains genomic DNA from 293 cells and pTR-UF5 plasmid, showing excision of the cassette from the plasmid upon digestion.
  • a control (not shown) of 293 cells alone did not produce any endogenous cross-reacting bands.
  • the host chromosomal site in the 293 genome of integration is random, as evidenced by the different sized bands resulting from HindM digestion. Ln some cell lines, the event can be seen to have occurred more than once (multiple copies have integrated).
  • Directional integration into the long arm of chromosome 19 would be expected if the rep gene of AAV were simultaneously expressed. This experimental data proves delivery and stable integration of foreign DNA sequences by AmEPV.
  • Example 3 Growth and amplification of AmEPV
  • LD cells were maintained at 28°C in a 1:1 ratio of TC-100 medium (Gibco, Gaithersburg, MD) to EX-CELL 401 medium (JRH Biosciences, Lenexa, KN) supplemented with 10% fetal bovine serum, 50 U/ml penicillin, and 50 ⁇ g/ml streptomycin (1:1
  • TE TE-co-viral permeability
  • cells grown in 150 mm dishes are inoculated at a multiplicity of infection (m.o.i.) of 0.01 in sufficient media (5 ml) to cover the surface ofthe tissue culture vessel.
  • the cells should be no more than 70% confluent.
  • 25 ml medium is added and the infections are incubated for 4-6 days at 28°C. The infection is considered complete when most cells become occlusion body positive as seen by light microscopy, i.e. when refractile occlusion bodies can be seen.
  • infection is monitored by in situ staining of infected cells with 1 mg/ml 5-Bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside (Xgal), 4 mM potassium ferricyanide, 4 mM potassium ferrocyanide, and 2 mM MgCl 2 in phosphate buffered saline (PBS) (140 mM NaCl, 2.7 mM KC1, 10 mM Na 2 P0 4 , 1.8 mM KH 2 P0 4 , pH 7.4).
  • PBS phosphate buffered saline
  • Virus to be titered is subjected to 10-fold serial dilutions in 1 : 1 TE medium.
  • LD-652 cells are plated at 70% confluency in 6-welI dishes, each having a 34.6 mm diameter (roughly 1.4 x 10 6 cells per well). Once the cells have adhered, the medium is removed and 0.5 ml of diluted virus is added to the wells. After adsorption at 28°C for 2 hr, the inoculum is removed and 2.5 ml of overlay is added to each well.
  • the overlay is a 2:1 ratio of 1.33X TC-100 medium (containing 14% fetal calf serum) and 4% sterile low melting point agarose, equilibrated to 42°C and mixed just prior to addition to the monolayer.
  • TBS- Block (0.5% w/v blocking reagent in TBS [Boehringer Mannheim, Germany]) for 1 hr at room temperature to prevent nonspecific antibody binding.
  • the primary antibody (rabbit anti-AmEPV occlusion body antiserum) (Hall et al. [1996] supra) or secondary antibody (goat anti-rabbit conjugated to alkaline phosphatase; Fisher, Atlanta, GA) are both diluted in TBS-Block.
  • Antibody reactions and color development are performed as previously described (Harlow. E., and D. Lane [1998] Antibodies- A Laboratory Manual, E. Harlow and D. Lane, Eds., pp. 635-657. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
  • LD-652 cells (typically 10 9 cells, thirty 150 mm dishes) are infected with AmEPV at an m.o.i. of 0.01.
  • the infections (cells and medium) are harvested by scraping and centrifuged at 700 xg for 15 min. to remove cells.
  • the supernatant is centrifuged at 39,000 xg for 30 min. to pellet extracellular virus.
  • the viral pellet is resuspended in deionized water (100 ⁇ l for 40 ml of supernatant). DNAase free RNAase is added to the resuspended viral pellet at a final concentration of 50 ⁇ g/ml and incubated at 37°C for 30 min.
  • the virus sample is then heated to 50°C, and an equal volume of lysis buffer (100 mM Tris pH 8.0, 10 mM EDTA, 54% sucrose, 2% SDS, 10 mM ⁇ -mercaptoethanol) is added to the sample.
  • Proteinase K is then added to a final concentration of 0.6 mg/ml, and the viral lysate is incubated overnight at 50°C.
  • the lysate is extracted three times with 50:49: 1 phenol:chloroforrn:isoamyl alcohol and once with chloroform, and the DNA is precipitated in 0.4 M LiCl, 95% ethanol. This procedure typically yields 2 ⁇ g of genomic AmEPV DNA per 10 7 infected cells.
  • Example 7 Shuttle vector plasmid construction
  • TK thymidine kinase
  • spheroidin spheroidin
  • oligonucleotide primers was used to PCR amplify a 663-bp fragment of TK upstream flanking sequence from pMEGTK-1. These two fragments were separately inserted into pBluescript I SK(+) to produce pDUTK (Li, Y., Hall, R. L., and Moyer, R. W. [1997] "Transient, nonlethal expression of genes in vertebrate cells by recombinant entomopoxviruses" J Virol. 71:9557-9562). Foreign genes were then cloned within the TK flanks to generate shuttle vectors for the generation of recombinants (Li, Y., Hall, R.
  • oligonucleotide primers were used to amplify a 998-bp fragment of downstream spheroidin flanking sequence from pRH512. These two fragments were separately inserted into pBluescript I SK(+) to produce pDU20 (Hall, R. L., Li, Y., Feller, J. A., and Moyer, R. W. [1996] "The Amsacta moorei entomopoxvirus spheroidin gene is improperly transcribed in vertebrate poxviruses" Virology 224:427-436). Subsequent constructs were cloned within the spheroidin flanks to generate various shuttle vectors for the generation of recombinants (Hall et al. [1996] Virology 224:427-436).
  • AmEPV early promoter constructs Promoters for early poxvirus genes are active prior to viral DNA replication. We have utilized two early EPV promoters in our constructs. The first, an AmEPV early strong promoter (esp) was derived from a strongly expressed 42 kDa early protein (Li, Y., Hall, R. L., and Moyer, R. W. [1997] "Transient, nonlethal expression of genes in vertebrate cells by recombinant entomopoxviruses" J. Virol. 71:9557-9562). The second promoter was derived from the early expressed fusolin (fits) gene as described (Gauthier et al. [1995] supra).
  • esp AmEPV early strong promoter
  • the shuttle vector pTK-fus/ ⁇ cZ was constructed by PCR amplification ofthe MmEPV fusolin early promoter from pHF51 and insertion into pDUTK; lacZ was subcloned from pMC1871 (Pharmacia Biotech, Inc., Piscataway, N.J.) as described (Li, Y., Hall, R. L., and Moyer, R. W.
  • pTK-espg/ a green fluorescent protein gene (gfp) was PCR- amplified from the pTR-UF5 plasmid (Vector Core, University of Florida) (18) and cloned into pTK-esp/ ⁇ cZ replacing the esplacZ cassette as described in (Li, Y., Hall, R. L., and Moyer, R. W.
  • AmEPV late promoter constructs We have used the spheroidin (sph) promoter as an example of an AmEPV strong late promoter. This promoter has two rather unexpected properties: ( 1 ) the sph promoter appears to be insect cell specific and functions very poorly in vertebrate cells
  • pDU20/ ⁇ cZ was created by insertion of lacZ (from plasmid pMC1871, Pharmacia Biotech, Inc., Piscataway, NJ) into the BamHI site of pDU20.
  • lacZ from plasmid pMC1871, Pharmacia Biotech, Inc., Piscataway, NJ
  • the final reporter contains 1046 bp of potential spheroidin promoter sequence plus 20 bp of additional downstream spheroidin coding sequence following the TAAATG sequence before fusion to lacZ (Hall et al. [1996] supra).
  • pDU2/ cZ was constructed using the same strategy as that for pDU20/ ⁇ cZ, except that only 2 bp of spheroidin coding sequence follows the translation- starting TAAATG before fusion to lacZ (Hall et al. [1996] supra, Li, Y., R.L. Hall, S. Yuan, and R.W. Moyer [1998] "High-level expression of Amsacta moorei entomopoxvirus spheroidin depends on sequences within the gene" J. Gen. Virol. 79:613-622).
  • cowpoxvirus late ATI gene promoter to drive lacZ which functions well in both insect and vertebrate cells (Li, Y., R.L. Hall, S. Yuan, and R.W. Moyer [1998] "High-level expression of Amsacta moorei entomopoxvirus spheroidin depends on sequences within the gene" J. Gen. Virol. 79:613-622).
  • E. Construct driven by Pol II specific promoters An AmEPV construct containing reporter genes driven by Pol U rather than poxvirus promoters has also been prepared based on the plasmid pTR-UF5 (Vector Core, University of Florida) (Klein, R. L., E. M. Meyer, A. L. Peel,
  • pTR-UF5 was digested with S ⁇ /I to remove two Pstl sites then religated to form pTRUF5)S ⁇ /I. This construct was then digested with Pstl, and the fragment containing the two reporter genes was inserted into the Pstl site of pDUTK to produce pTKUF5)S ⁇ /I. This construct was then digested with Sail and the previously removed S ⁇ tl fragment was reinserted into the construct to produce pTKUF5 ( Figure 1).
  • spheroidin gene nor the thymidine kinase gene is required for propagation of AmEPV in cell culture (Palmer, C.P., D.P. Miller, S.A. Marlow, LE. Wilson, A.M. Lawrie, and
  • LD-652 cells (1.4 x 10 6 cells, 70% confluent in a 34.6 mm dish) are infected with AmEPV at an m.o.i. of 5 PFU per cell in a volume of 1 ml. Two hours post-infection, the inoculum is aspirated and 1 ml of transfection mix + DNA is added.
  • Transfection mix + DNA is prepared by separately combining 20 ⁇ l Lipofectin (Gibco, Gaithersburg, MD) and 80 ⁇ l 1:1 TE media without FBS, and 5 ⁇ g of shuttle vector plasmid
  • TK(-) cell line Cl 1.3
  • B. Selection of AmEPV recombinants For selection of recombinants inserted into the TK gene, a TK(-) cell line, Cl 1.3, was derived by serial passage of LD-652 cells in increasing concentrations of 5-bromo-2'-deoxyuridine (BudR) (10 ⁇ g/ml increasing increments of BudR at intervals of five weeks over one year).
  • BudR 5-bromo-2'-deoxyuridine
  • C11.3 cells are maintained at in 1:1 TE medium supplemented with 100 ⁇ g/ml BudR (Li, Y., Hall, R. L., and Moyer, R. W. [1997] "Transient, nonlethal expression of genes in vertebrate cells by recombinant entomopoxviruses" J. Virol.
  • AmEPV The normal host range of AmEPV is limited to Lepidoptera (butterflies), and early experiments attempting to infect vertebrate cells with AmEPV indicated no obvious deleterious effects on the cells. Given the general promiscuity of poxviruses in the binding and entering of cells and the similarity ofthe AmEPV life cycle to that of vaccinia, we had reason to believe AmEPV would infect and enter vertebrate as well as insect cells.
  • AmEPV recombinants were constructed carrying the lacZ reporter gene regulated by either of two early AmEPV promoters, the late spheroidin promoter or the ATI promoter from cowpox (TK-fus/ocZ, TK-esp/ cZ, SPH(20)t ⁇ cZ and TK-ATI/ ⁇ cZ, described above).
  • TK-fus/ocZ TK-esp/ cZ
  • SPH(20)t ⁇ cZ TK-ATI/ ⁇ cZ
  • AmEPV like other poxviruses, packages the enzymes necessary for early gene transcription within the virion. However, if vertebrate cells are co-infected with both W and AmEPV, late promoters within AmEPV are rescued and activated suggesting that vaccinia can provide factors in trans which are needed for the infection to progress and activate the late promoters. While determining the basis of host range restriction is difficult, the cytoplasmic nature of AmEPV coupled with a virus encoded transcription and replication machinery offers major advantages for vector design. We can be fairly certain that late genes are not transcribed because ofthe lack of ⁇ -galactosidase expression from late promoters in vertebrate cells and because DNA synthesis, a requirement for late mRNA synthesis does not occur. It is quite possible that incomplete uncoating ofthe virus leads to the block in gene expression.
  • uncoating of poxviruses occurs in two discrete steps. Upon entry into cells, virions are sufficiently permeabilized to allow early gene transcription from the viral core. Early proteins allow the complete uncoating ofthe core to allow transcription ofthe later classes of genes following interaction of newly synthesized DNA with intermediate and late transcription factors. The uncoating of vertebrate poxviruses has been thoroughly studied, and uncoating intermediates have been identified through differential centrifugation of cellular extracts infected with labeled virus. A viral activity specifically required for the second stage of uncoating has been identified. By analogy with vaccinia, AmEPV might be expected to encode a similar uncoating factor.
  • lymphocytic cells are more resistant to infection (Li, Y., Hall, R. L., and Moyer, R. W. [1997] "Transient, nonlethal expression of genes in vertebrate cells by recombinant entomopoxviruses" J. Virol. 71:9557-9562).
  • AmEPV can also enter cells in vivo and allow early, but not late expression of a reporter gene.
  • Example 12 The control of AmEPV induced inflammation
  • inflammation and immunogenicity to the virus and to virus-infected cells has limited transgene expression and the utility of this approach to treat chronic illnesses.
  • Inflammation is initially characterized by perivascular and peribronchiolar inflammatory cell infiltration.
  • Neutrophils and later macrophages and lymphocytes frequent the site ofthe infected area.
  • Specific cytokines can also be measured as an index ofthe inflammatory response (Ginsberg, H.S., LL. Moldawer, P.B. Sehgal, M. Redington, P.L. Kilian, R.M.
  • the early response to adenovirus infection consists of diffuse cellular infiltration of peribronchiolar and alveolar regions associated with the appearance of several classes of pro-inflammatory cytokines (Ginsberg et al. [1991] supra; Arthur etal. [1996] supra). These include TNF-a, IL-1, LL-6, and LL-8 (KC/GRO in the mouse). There is considerable experimental evidence from rodents demonstrating that these classes of cytokines, and in particular TNF-a and LL-8 (or KC/GRO), play central roles in the recruitment and activation of inflammatory cell populations in the lung.
  • vertebrate poxviruses may serve as a source of genes to provide a solution to this problem.
  • vertebrate poxvirus- encoded secreted virokines and viroceptors described including LFN-'7 ⁇ , LFN-(, TNF and LL- 1 , and chemokine receptors (Barry, M. and G. McFadden [1997] "Virus encoded cytokines and cytokine receptors" Parasitology 115:S89-100; Smith, G.L., J.A. Symons, A. Khanna, A. Vanderplasschen, and Alcami, A. [1997] "Vaccinia virus immune evasion” Immunol. Rev.
  • ITRs inverted terminal repeat sequences of AAV DNA
  • Rep 78/68 proteins The ITRs comprise two 145-nucleotide elements located at either end ofthe AAV genome. ITR sequences enclosing marker genes have been shown to allow a lower level of random genome integration when compared to the levels of specific integration observed when genes encoding the Rep 78/68 proteins are also included in constructs.

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Abstract

La présente invention concerne des vecteurs d'entomopox recombinants utilisés pour l'administration et l'expression stable d'ADN hétérologue dans des cellules de vertébrés. Plus spécifiquement, cette invention concerne un virus entomopox (poxviridae) (EPV) recombinant provenant d'amsacta moorei (AmEPV). En raison de la capacité d'EPV d'incorporer des séquences d'ADN étrangères et hétérologues, on peut utiliser les vecteurs de cette invention pour administrer des inserts d'ADN de taille supérieure à 10kb. Par conséquent, un aspect de cette invention a trait à l'utilisation des vecteurs recombinants dans l'administration et l'expression de protéines biologiques utiles dans des protocoles de thérapie génique. En outre, ladite invention concerne des nouveaux polypeptides AmEVP et les séquences de polynucléotides qui codent ces polypeptides.
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