WO2001038538A1 - Compositions de polymerase et procedes d'utilisation correspondants - Google Patents

Compositions de polymerase et procedes d'utilisation correspondants Download PDF

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Publication number
WO2001038538A1
WO2001038538A1 PCT/US2000/031909 US0031909W WO0138538A1 WO 2001038538 A1 WO2001038538 A1 WO 2001038538A1 US 0031909 W US0031909 W US 0031909W WO 0138538 A1 WO0138538 A1 WO 0138538A1
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ns5ab
seq
protein
nucleic acid
sequence
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PCT/US2000/031909
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James B. Flanegan
Sushma Ogram
Barbara J. Morasco
Linda Barone
Siu-Chi Chang Sun
James M. Groarke
Marc S. Collett
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Viropharma Incorporated
University Of Florida
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Priority to JP2001539880A priority Critical patent/JP2003514562A/ja
Priority to EP00983732A priority patent/EP1257650A4/fr
Priority to AU20447/01A priority patent/AU2044701A/en
Publication of WO2001038538A1 publication Critical patent/WO2001038538A1/fr

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/18—Togaviridae; Flaviviridae

Definitions

  • the present invention relates to the pestivirus, hepacivirus and related virus groups, and more specifically, to the RNA-directed RNA polymerase (RdRp) activities and sequences encoded by these viruses.
  • RdRp RNA-directed RNA polymerase
  • NS5AB Fused NS5A-NS5B RdRp encoding sequences and functional RdRp proteins (referred to herein as NS5AB) expressed from such sequences and compositions containing the same are disclosed. Such compositions have utility in preventative, therapeutic, diagnostic and pharmaceutical applications .
  • Pestiviruses and hepaciviruses are closely related virus groups within the Flaviviridae family. Other closely related viruses in this family include the GB virus A, GB virus A-like agents, GB virus-B and GB virus-C (also called hepatitis G virus, HGV) .
  • the pestivirus genus consists of bovine viral diarrhea virus (BVDV) , classical swine fever virus (CSFV; also called hog cholera virus) and border disease virus (BDV) of sheep. Pestivirus infections of domesticated livestock (cattle, pigs and sheep) cause significant economic losses worldwide. Human pestiviruses have not been as extensively characterized as the animal pestiviruses. However, serological surveys indicate considerable pestivirus exposure in humans.
  • the hepacivirus group (hepatitis C virus; HCV) consists of a number of closely related but genotypically distinguishable viruses that infect humans. HCV is a major cause of hepatitis globally. Most HCV infections become persistent and about 75% of cases develop chronic liver disease. Chronic HCV infection can lead to development of cirrhosis, hepatocellular carcinoma and liver failure.
  • interferon alpha IFN
  • ribavirin 1- beta-D-ribofuranosyl-lH-1, 2 , 4-triazole-3-carboxamide
  • IFN therapy for chronic hepatitis C is variable and its cure rate is low. Moreover, such therapy is often associated with considerable adverse effects. New therapies and preventatives are clearly needed for infections and diseases caused by pestiviruses, hepaciviruses and related viruses.
  • RNA viruses possess a single large open reading frame (ORF) encoding all the viral proteins necessary for virus replication. These proteins are expressed as a polyprotein that is co- and post-translationally processed by both cellular and virus-encoded proteinases to yield the mature viral proteins.
  • the viral proteins responsible for the replication of the viral genome RNA are located within approximately the carboxy-terminal two-thirds of the ORF and are termed nonstructural (NS) proteins.
  • NS nonstructural
  • the genetic organization and polyprotein processing of the nonstructural protein portion of the ORF for pestiviruses and hepaciviruses is remarkably similar.
  • the mature nonstructural (NS) proteins consist of (in sequential order from the amino-terminus of the nonstructural protein coding region to the carboxy- terminus of the ORF) :
  • the NS proteins of pestiviruses and hepaciviruses share sequence domains that are characteristic of specific protein functions.
  • the NS3 proteins of viruses in both groups possess amino acid sequence motifs characteristic of serine proteinases and of helicases (Gorbalenya et al . (1988) Nature 333:22; Bazan and Fletterick (1989) Virology 171:637-639; Gorbalenya et al . (1989) Nucleic Acid Res. 17:3889-3897).
  • the NS5B proteins of pestiviruses and hepaciviruses have the motifs characteristic of RNA-directed RNA polymerases (RdRp; (Koonin, E.V. and Dolja, V.V. (1993) Crit. Rev. Biochem. Molec. Biol. 28:375-430).
  • NS3 serine proteinase is responsible for all proteolytic processing of polyprotein precursors downstream of its position in the ORF ( iskerchen and Collett (1991) Virology 184:341-350; Bartenschlager et al. (1993) J. Virol. 67:3835-3844; Eckart et al . (1993) Biochem. Biophys. Res. Comm. 192 : 399-406; Grakoui et al . (1993) J. Virol. 67:2832-2843; Grakoui et al .
  • NS4A protein acts as a cofactor with the NS3 serine protease (Bartenschlager et al . (1994) J. Virol. 68:5045-5055; Failla et al . (1994) J. Virol. 68: 3753-3760; Lin et al . (1994) 68:8147-8157; Xu et al . (1997) J. Virol.
  • the NS3 protein of both viruses also functions as a helicase (Kim et al . (1995) Biochem. Biophys. Res. Comm. 215: 160-166; Jin and Peterson (1995) Arch. Biochem. Biophys . . 323 : 47-53 ; Warrener and Collett (1995) J. Virol. 69:1720-1726).
  • the NS5B proteins of pestiviruses and hepaciviruses have the predicted RdRp activity (Behrens et al.(1996) EMBO J. 15:12-22; Lohmann et al.(1997) J. Virol.
  • pl33 a precursor polyprotein to the mature NS5A and NS5B proteins is an "NS5AB" protein termed pl33 (Petric et al.(1992) J. Gen. Virol. 73:1867-1871). While pl33 has been shown to exist in BVDV-infected cells, its potential functional role in virus replication beyond a precursor polyprotein has not been described. Since hepaciviruses do not grow in cells in culture efficiently enough to allow the identification and study of authentic viral polyprotein precursors, the existence of a hepacivirus NS5AB protein has not heretofore been described.
  • flavivirus genomic organization and replication strategy are distinct from those of the pestiviruses and hepaciviruses.
  • the 5' end of the flaviviral genomic RNA is capped and the 5' nontranslated region (5'NTR) is short and is indicative of gene expression by ribosome scanning.
  • 5'NTR 5' nontranslated region
  • the gene expression mechanisms employed by flaviviruses are fundamentally different from those used by the pestiviruses and hepaciviruses.
  • the mature NS proteins of flaviviruses consist of (in sequential order from the amino-terminus of the nonstructural protein coding region to the carboxy- terminus of the ORF) :
  • flavivirus NS3 Sequence motifs predictive of protein function also exist in the the flavivirus ORF.
  • the flavivirus NS3 protein includes motifs which suggest that it is a trifunctional protein (Rice, in Fields Virology, 3rd edtion, pp. 931-959 (Lippincott-Raven, NY) ) .
  • flavivirus NS3 possesses a trypsin-like proteinase activity that is involved in polyprotein processing both upstream and downstream of its position in the ORF, an
  • RNA helicase activity (WO 97/27334) probably involved in RNA replication and an RNA triphosphatase activity likely involved in the formation of the 5 ' end cap structure of flavivirus RNA.
  • the flavivirus NS5 protein possesses RdRp sequence motifs and has been demonstrated to have RdRp activity (Tan et al.(1996) Virology 216:317-325).
  • the flavivirus NS5 protein also contains sequence motifs characteristic of methyltransferases . This domain of the flavivirus NS5 protein may be involved in methylation of the 5' cap structure.
  • pestivirus and hepacivirus RNAs lack a 5' cap structure.
  • the ORFs of pestiviruses and hepaciviruses including the coding regions of the NS5A and NS5B proteins, lack methyltransferase motifs (Koonin, E.V.(1993) J. Gen. Virol. 74:733-740). This is an important functional distinction and differentiating characteristic between the RdRp proteins of flaviviruses (NS5) and of the pesti- and hepaciviruses (NS5B) .
  • RNA viruses that produce their RdRp proteins via a proteolytically processed precursor polyprotein
  • RdRp activity in two well-studied cases, the immediate polyprotein precursors of mature RdRp proteins have been shown to be non-functional as RNA polymerases.
  • the functional RdRp protein termed 3Dpol, is derived from the precursor 3CDpro, which represents a fusion of the two mature viral proteins 3Cpro and 3Dpol .
  • purified 3CDpro lacked detectable activity (Van Dyke and Flanegan (1980) J. Virol. 35:732-740; Harris et al .
  • the polyprotein precursor P1234 is rapidly processed to P123 and the mature RdRp nsP4. While P123 together with nsP4 are able to synthesize viral RNA in cells, the precursor P1234 is not (Lemm, Rumenapf, Strauss, Strauss and Rice (1994) EMBO J. 13:2925-2934). Furthermore, using an engineered vaccinia virus expression system, it was shown directly that the polyprotein P34 was not able to synthesize viral RNA in cells (Lemm and Rice (1993) 67:1916-1626) . All of the foregoing suggested that any precursor protein of the NS5B RdRp of pestiviruses and hepaciviruses (herein termed NS5AB) would not be expected to function as an active polymerase.
  • NS5AB any precursor protein of the NS5B RdRp of pestiviruses and hepaciviruses
  • the present invention is based on the discovery that the NS5B RdRp precursor protein, referred to herein as NS5AB, is in fact an active RdRp enzyme.
  • NS5AB RdRp precursor protein
  • Provided herein are NS5AB encoding sequences having NS5A and NS5B joined together in their natural arrangement, NS5AB proteins and their associated activities that have utility in diagnostic, preventative, therapeutic and pharmaceutical applications.
  • the NS5AB-related molecules of the invention may also be used to advantage in assays for the identification of efficacious antiviral agents .
  • an isolated nucleic acid molecule comprises a DNA sequence representing a pestivirus NS5AB gene identified as SEQ ID NO: 1, which is present in clone DJB2-BVDV-NS5AB of the present invention.
  • An exemplary BVDV NS5AB protein has the amino acid sequence identified as SEQ ID NO: 2 encoded by the aforementioned clone.
  • nucleic acid molecules that represent nucleic acid sequences, either DNA or RNA, related to those of SEQ ID NO: 1, such as those derived from any strain, variant, serotype or genotype of a pestivirus, and the NS5AB proteins thereby encoded, are within the scope of the invention.
  • pestivirus NS5AB sequences with conservative sequence or residue substitutions of these sequences or sequences that are mutated or modified to yield variant or derivative nucleic acids and proteins, are also contemplated to be within the scope of the present invention.
  • an isolated nucleic acid molecule comprises a DNA sequence representing a hepacivirus NS5AB gene identified as SEQ ID NO : 3, which is present in clone bacHCV5AB-ll of the present invention.
  • An exemplary hepacvirus NS5AB protein has the amino acid sequence identified as SEQ ID NO : 4 encoded by the aformentioned clone.
  • an isolated nucleic acid molecule comprises a DNA sequence representing a hepacivirus NS5AB gene identified as SEQ ID NO: 5, which is present in clone bacHCV5AB-16 of the present invention.
  • An exemplary hepacvirus NS5AB protein has the amino acid sequence identified as SEQ ID NO: 6 encoded by the aformentioned clone.
  • nucleic acid molecules that represent nucleic acid sequences related to those of SEQ ID NO : 3 or SEQ ID NO: 5, such as those derived from any strain, variant, serotype or genotype of a hepacivirus, and the NS5AB proteins thereby encoded, are within the scope of the invention. Additionally, hepacivirus NS5AB sequences with conservative sequence or residue substitutions of these sequences or sequences that are mutated or modified to yield variant or derivative nucleic acids and proteins, are also contemplated to be within the scope of the present invention.
  • NS5AB proteins and polypeptides e.g., NS5AB protein and polypeptides with RdRp or other activities
  • functional NS5AB proteins and polypeptides have multiple utilities including, but not limited to, identifying agents that may modulate, either inhibit or stimulate, the activities of such functional NS5AB proteins and polypeptides .
  • variant proteins or polypeptides are contemplated to be within the scope of the present invention.
  • Such variants may or may not possess RdRp polymerase activity.
  • These variants may possess one or more changes each of which may include one or more additions, deletions, or substitutions of amino acid residues.
  • the changes will not affect, or substantially affect, the structure or useful properties of the polypeptide.
  • pestivirus and hepacivirus NS5AB variants may suitably possess functional RdRp activity, or they may be poorly functional or inactive, yet contain substantially the secondary and tertiary structure of the native polypeptide.
  • Such NS5AB molecules may be used to advantage to identify agents that specifically bind to or otherwise affect NS5AB activity.
  • NS5AB variants can be either naturally occurring (i.e., purified or isolated from a natural source) or synthetic (i.e., generated by biological expression of DNA or RNA that has been subjected to genetic engineering procedures, site-directed mutagenesis or produced by chemical synthetic techniques, all of which are well known in the art) .
  • the nucleic acid molecules of the invention are cloned and expressed in vectors.
  • vectors may be in the form of, for example, a plasmid, a replication competent or defective virus or phage vector or a replicon provided typically with an origin of replication, optionally a promoter for the expression of the polynucleotide and optionally a regulator of the promoter.
  • the vector may optionally contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector.
  • the vector may be used to advantage in in vi tro assays, for example for the production of
  • RNA or protein RNA or protein.
  • Vectors for transforming, transfecting, infecting or transducing host cells or organisms are also contemplated for use in the present invention.
  • the resulting host cells and organisms harboring or expressing pestivirus or hepacivirus nucleic acid sequences or polypeptides have utility in assays for the identification of agents that affect the activity of an NS5AB protein.
  • agents that affect the NS5AB polymerase sequences.
  • Such methods include methods of assay, as well as high throughput screening procedures that allow assessment of large numbers of agents.
  • the agents so identified may be either antagonistic or agonistic in their affect on the NS5AB sequences or proteins encoded thereby.
  • Molecules of any number of classes including but not limited to, small molecules, polymers, peptides, polypeptides, immunoglobulins or fragments thereof, oligonucleotides, antisense molecules, peptide-nucleic acid conjugates, ribozymes, polynucleotides and the like may be utilized in the methods of the present invention.
  • Antagonistic and agonistic molecules identified by practice of the invention have broad and multiple utilities.
  • Utilities for antagonists of NS5AB activity include, but are not limited to, inhibition of pestivirus and hepacivirus replication in living hosts or humans, and in in vi tro systems such as cell, tissue and organ cultures.
  • Agonists of pestivirus and hepacivirus NS5AB activity identified by practice of the invention will also have multiple utilities, both in living hosts and in in vi tro systems. For example, such agents will be useful in improving the growth, production or propagation of these viruses. Improved in vi tro propagation of pestiviruses and hepaciviruses has advantages for the production of research and diagnostic reagents as well as in vaccine production. In particular, such agents have utility in the development of animal models of HCV infection, replication or disease and for the propagation of HCV in a living host or in cell, tissue or organ culture systems .
  • kits which facilitate the use of the compositions and methods disclosed herein.
  • Exemplary kits include NS5AB nucleic acids and polypeptides of the invention, variants thereof, and alone or in association with suitable vectors.
  • One exemplary kit of the invention includes nucleic acid probes or primers suitable for the detection of the presence of NS5AB nucleic acid in a test sample.
  • an exemplary kit of the invention may include antibodies immunologically specific for pestivirus or hepacivirus NS5AB protein and other reagents to facilitate immunodetection.
  • a solid support containing immobilized NS5AB antigen may optionally be included in such a kit as a positive control.
  • kits described herein are protocols for use of the compositions of the invention for the particular application and the necessary reagents to carry out the application.
  • the reagents of a kit may vary depending on the intended application. Such reagents may include, but are not limited to buffers, solvents, media and solutions, substrates and cofactors, vectors and host cells, and detection or reporter reagents. Other accessories may optionally be included such as vials, vessels and reaction chambers.
  • hepatitis C virus shall mean any representative of a group of related viruses belonging to the hepacivirus genus of the Flaviviridae family. Also encompassed within the use of the term “hepacivirus” are the closely related viruses termed GB virus A, GB virus A-like agents, GB virus-B and GB virus-C or hepatitis G virus (HGV) .
  • BVDV bovine viral diarrhea virus
  • Nucleic acid or a “nucleic acid molecule” as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form.
  • nucleic acid molecules a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5 ' to 3 ' direction.
  • isolated nucleic acid refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated.
  • an "isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, incorporated into the genomic DNA of a procaryotic or eucaryotic cell or host organism.
  • isolated nucleic acid refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues) .
  • An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.
  • Natural allelic variants refer to nucleic acid sequences that are closely related to a particular sequence but which may possess, either naturally or by design, changes in sequence or structure.
  • closely related it is meant that at least about 60%, but often, more than 90%, of the nucleotides of the sequence match over the defined length of the nucleic acid sequence.
  • Changes or differences in nucleotide sequence between closely related nucleic acid sequences may represent nucleotide changes in the sequence that arise during the course of normal replication or duplication in nature of the particular nucleic acid sequence.
  • percent similarity when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program.
  • NS5B refers to a portion of the pestivirus or hepacivirus genome located near the 3 ' end of the viral genome that specifies the region encoding a protein.
  • NS5B refers to a portion of the pestivirus or hepacivirus genome located near the 3 ' end of the viral genome that specifies the region encoding a protein.
  • NS5B refers to a portion of the pestivirus or hepacivirus genome located near the 3 ' end of the viral genome that specifies the region encoding a protein.
  • NS5B “NS5B protein”
  • NS5B polypeptide e
  • NS5B polymerase RNA-dependent RNA polymerase
  • RdRp RNA-dependent RNA polymerase
  • the nucleic acid region encoding the NS5B protein may also be referred to as the "NS5B gene” .
  • NS5B may refer to either a nucleic acid encoding the NS5B polypeptide, to an NS5B gene or to an NS5B polypeptide, or to any portions thereof, depending on the context in which the term is used.
  • NS5B may further refer to natural allelic variants, mutants and derivatives of either NS5B nucleic acid sequences or NS5B polypeptides.
  • the NS5B nucleic acid, NS5B gene or NS5B protein referred to may be either functional or non-functional .
  • NS5AB refers to a portion of the pestivirus or hepacivirus genome located near the 3 ' end of the viral genome that specifies the region encoding together the NS5A and NS5B protein termed herein as “NS5AB” .
  • NS5A5B refers to a portion of the pestivirus or hepacivirus genome located near the 3 ' end of the viral genome that specifies the region encoding together the NS5A and NS5B protein termed herein as “NS5AB” .
  • NS5A5B The NS5AB protein is a polyprotein or precursor to the mature NS5A and NS5B proteins .
  • the NS5A and NS5B sequences are joined and aligned in their natural colinear state.
  • the nucleic acid region encoding the NS5AB protein may also be referred to as the "NS5AB gene".
  • NS5AB may refer to either a nucleic acid encoding the NS5AB polypeptide, to an NS5AB gene or to an NS5AB polypeptide, or to any portions thereof, depending on the context in which the term is used. NS5AB may further refer to natural allelic variants, mutants and derivatives of either NS5AB nucleic acid sequences or NS5AB polypeptides.
  • the NS5AB nucleic acid, NS5AB gene or NS5AB protein may be either functional or non- functional .
  • Non-cleavable derivatives of the NS5AB sequences of the invention are those sequences which have been modified such that they are no longer proteolytically cleaved by viral or other proteolytic enzymes .
  • the present invention also includes active portions, fragments, derivatives and functional or non-functional imetics of pestivirus and hepacivirus NS5AB polypeptides or proteins of the invention.
  • An "active portion" of NS5AB polypeptide means a peptide that is less than the full length NS5AB polypeptide, but which retains measurable biological activity.
  • a “fragment" or "portion” of the NS5AB polypeptide means a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, typically at least about nine to thirteen contiguous amino acids and, most preferably, at least about twenty to thirty or more contiguous amino acids. Fragments of the NS5AB polypeptide sequence, antigenic determinants, viral antigens or epitopes are useful for eliciting immune responses to a portion of the NS5AB amino acid sequence.
  • a “derivative" of the NS5AB polypeptide or a fragment thereof means a polypeptide modified by varying the amino acid sequence of the protein, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. Such derivatives of the natural amino acid sequence may involve insertion, addition, deletion, substitution or modification of one or more amino acids, and may or may not alter the essential activity of original the NS5AB polypeptide.
  • the NS5AB polypeptide or protein of the invention includes any analogue, fragment, derivative or mutant which is derived from an NS5AB polypeptide and which retains at least one property or other characteristic of the NS5AB polypeptide.
  • Different "variants" of the NS5AB polypeptide exist in nature. These variants may be alleles characterized by differences in the nucleotide sequences of the gene coding for the protein, or may involve different RNA processing or post-translational modifications. The skilled person can produce variants having single or multiple amino acid substitutions, deletions, additions or replacements.
  • variants may include inter alia : (a) variants in which one or more amino acids residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the NS5AB polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the NS5AB polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the NS5AB polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like.
  • NS5AB polypeptides of the invention include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or non-conserved positions. In another embodiment, amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues .
  • the techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to the person having ordinary skill in the art.
  • nucleic or amino acid sequence is functional for the recited assay or purpose.
  • modulating activity refers to the ability of a test substance to affect NS5AB associated activities or functions. Such activity may be agonistic or antagonistic in nature. Agonistic activity may be measured by an increase in RdRp activity as measured by increased synthesis of RNA in the presence of relevant controls. Alternatively, antagonistic activity may be measured by a decrease in RNA synthesis. Various assay methods are known to those of ordinary skill in the art to assess agents that modulate polymerase enzymatic activities .
  • membrane fraction as used herein comprises conventional lipid-containing membrane fractions or preparations derived from biological systems, as well as any combination of isolated or synthetic lipids or lipid components, liposomes, liposome formulations and micellar preparations.
  • phrases "consisting essentially of" when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID No.
  • the phrase when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.
  • a “replicon” is any genetic element, including without limitation, plasmids, cosmids, bacmids, phage or virus, capable of replication directed by replicon control sequences.
  • a replicon may be either RNA or DNA and may be single or double stranded.
  • a “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage or virus, to which another genetic sequence or element (either DNA or RNA) may be attached so as to bring about the replication of the attached sequence or element.
  • an "expression operon” refers to a nucleic acid segment that may possess transcriptional and translational control sequences, such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons) , polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
  • transcriptional and translational control sequences such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons) , polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
  • oligonucleotide refers to primers and probes of the present invention, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.
  • probe refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe.
  • a probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • the probes herein are selected to be “substantially” complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specfically.
  • the term “specifically hybridize” refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
  • primer refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of template-dependent nucleic acid synthesis.
  • suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH
  • the primer may be extended at its 3 ' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield an primer extension product.
  • the primer may vary in length depending on the particular conditions and requirement of the application.
  • the oligonucleotide primer is typically 15-25 or more nucleotides in length.
  • the primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able to anneal with the desired template strand in a manner sufficient to provide the 3 ' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template.
  • a non-complementary nucleotide sequence may be attached to the 5 ' end of an otherwise complementary primer.
  • non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
  • Amino acid residues described herein are preferred to be in the "L” isomeric form. However, residues in the "D” isomeric form may be substituted for any L-amino acid residue, provided the desired properties of the polypeptide are retained. All amino-acid residue sequences represented herein conform to the conventional left-to-right amino-terminus to carboxy-terminus orientation.
  • isolated protein or isolated and purified protein is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention.
  • this term may refer to a protein that has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in "substantially pure” form.
  • isolated is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification, addition of stabilizers, or compounding into, for example, immunogenic preparations or pharmaceutically acceptable preparations.
  • substantially pure refers to a preparation comprising at least 50-60% by weight of a given material (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-95% by weight of the given compound. Purity is measured by methods appropriate for the given compound (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
  • “Mature protein” or “mature polypeptide” refers to a polypeptide possessing the sequence of the polypeptide after any processing events that normally occur to the polypeptide during the course of its genesis, such as proteolytic processing from a polyprotein or precursor protein.
  • a “polyprotein” or “precursor” protein is a protein comprising a mature protein or proteins, which upon maturation, yields mature proteins. In designating the sequence or boundaries of a mature, polyprotein or precursor protein, the first amino of the protein sequence is designated as amino acid residue 1.
  • any amino acid residues associated with a protein not naturally found associated with that protein that precedes amino acid 1 are designated amino acid -1, -2, -3 and so on.
  • a methionine initiator codon is often utilized for purposes of efficient translation.
  • a methionine codon may be placed immediately proceeding amino acid 1 of the NS5AB protein sequence.
  • This methionine residue in the resulting polypeptide, as used herein, would be positioned at -1 relative to the NS5AB protein sequence.
  • tag refers to a chemical moiety, either a nucleotide, oligonucleotide, polynucleotide or an amino acid, peptide or protein or other chemical, that when added to another sequence, provides additional utility or confers useful properties, particularly in the detection or isolation, to that sequence.
  • a homopolymer nucleic acid sequence or a nucleic acid sequence complementary to a capture oligonucleotide may be added to a primer or probe sequence to facilitate the subsequent isolation of an extension product or hybridized product.
  • histidine residues e.g., 4 to 8 consecutive histidine residues
  • amino acid sequences, peptides, proteins or fusion partners representing epitopes or binding determinants reactive with specific antibody molecules or other molecules (e.g., flag epitope, c-myc epitope, transmembrane epitope of the influenza A virus hemaglutinin protein, protein A, cellulose binding domain, calmodulin binding protein, maltose binding protein, chitin binding domain, glutathione S-transferase, and the like) may be added to proteins to facilitate protein isolation by procedures such as affinity or immunoaffinity chromatography.
  • Chemical tag moieties include such molecules as biotin, which may be added to either nucleic acids or proteins and facilitates isolation or detection by interaction with avidin reagents, and the like. Numerous other tag moieties are known to, and can be envisioned by, the trained artisan, and are contemplated to be within the scope of this definition.
  • reporter refers to an operative genetic system in which a nucleic acid comprises a gene that encodes a product that when expressed produces a reporter signal that is a readily measurable, e.g., by biological assay, immunoassay, radioimmunoassay, or by colorimetric, fluorogenic, chemiluminescent or other methods.
  • the nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and is operatively linked to the necessary control elements for the expression of the reporter gene product.
  • the required control elements will vary according to the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional termination signals and the like .
  • transform refers to any method or means by which a nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transformation, transfection, electroporation, microin ection, PEG- fusion and the like.
  • the introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism.
  • the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid.
  • the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on or inherited to progeny cells or organisms of the recipient cell or organism.
  • the introduced nucleic acid may exist in the recipient cell or host organism only transiently.
  • a “clone” or “clonal cell population” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell or cell population that is capable of stable growth in vi tro for many generations.
  • Immune response signifies any reaction produced by an antigen, such as a viral antigen, in a host having a functioning immune system.
  • Immune responses may be either humoral in nature, that is, involve production of immunoglobulms or antibodies, or cellular in nature, involving various types of B and T lymphocytes, dendritic cells, macrophages, antigen presenting cells and the like, or both. Immune responses may also involve the production or elaboration of various effector molecules such as cytokines, lymphokines and the like. Immune responses may be measured both in in vi tro and in various cellular or animal systems. Such immune responses may be important in protecting the host from disease and may be used prophylactically and therapeutically.
  • viral antigen shall be any peptide, polypeptide or protein sequence, segment or epitope that is derived from a virus that has the potential to cause a functioning immune system of a host to react to said viral antigen.
  • antibody or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen.
  • the term includes polyclonal, monoclonal, chimeric, and bispecific antibodies.
  • antibody or antibody molecule contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunloglobulin molecule such as those portions known in the art as Fab, Fab', F(ab')2 and F(v) .
  • the term "living host” shall mean any non-human autonomous being.
  • Figure 1 depicts the nucleic acid sequence (SEQ ID NO: 1) encoding a BVDV NS5AB protein.
  • Figure 2 depicts the deduced amino acid sequence (SEQ ID NO: 2) of a BVDV NS5AB protein according to SEQ ID NO: 1.
  • FIG. 3 depicts the nucleic acid sequence (SEQ ID NO: 1
  • Figure 4 depicts the deduced amino acid sequence (SEQ ID NO: 4) of an HCV NS5AB protein according to SEQ ID NO : 3.
  • Figure 5 depicts the nucleic acid sequence (SEQ ID NO: 5) encoding an HCV NS5AB protein (clone 16) .
  • Figure 7 represents a fluorograph of an SDS gel in which radiolabeled polypeptides synthesized from a mock translation reaction (M) , or translation reactions programed by DJB2-BVDV-NS5B (5B) and DJB2-BVDV-NS5AB RNAs (5AB), were resolved.
  • M mock translation reaction
  • DJB2-BVDV-NS5AB RNAs 5AB
  • Figure 8 shows an immunoblot of an SDS gel probed with anti-BVDV NS5B antiserum in which the translation products of a mock reaction (M) , or reactions programed with DJB2-BVDV-NS5B (5B) and DJB2-BVDV-NS5AB (5AB) RNAs, were resolved.
  • Various amounts (200, 100, 50 and 10 ng) of purified NS5B were included as standards (tracks 1-4, respectively) .
  • Figure 9 shows an autoradiogram of an CH 3 HgOH agarose gel of the radiolabeled product RNA from RdRp reactions containing the translation products of a mock reaction, or reactions programed with DJB2-BVDV-NS5B (5B) and DJB2-BVDV-NS5AB (5AB) RNAs conducted with (+) or without (-) RNA template in the RdRp reaction mixture.
  • the position of the input self-priming RNA template is indicated by 'IX' and the completed duplex product by ' 2X' .
  • Figure 10 shows an immunoblot of an SDS gel probed with anti-HCV NS5B antiserum in which membrane fractions prepared from mock-infected Sf9 cells (M) or cells infected with either recombinant baculoviruses bacHCV5B-4 (5B) and bacHCV5AB-16 (5AB) have been resolved.
  • Figure 11 shows the TCA-precipitable radioactivity incorporated into reaction product RNA from RdRp reactions containing either 2 or 4 ⁇ L of membrane fractions prepared from mock-infected (mock) or bacHCV5AB-16-infected (5AB) Sf9 cells.
  • Figure 12 represents a fluorograph of an SDS gel in which radiolabeled polypeptides synthesized from a mock translation reaction (M) , or translation reactions programed by DJB2-HCV-NS5B (5B) and DJB2-HCV-NS5AB RNAs (5AB), were resolved.
  • M mock translation reaction
  • DJB2-HCV-NS5AB RNAs 5AB
  • Figure 13 shows an immunoblot of an SDS gel probed with anti-HCV NS5B antiserum in which the translation products of a mock reaction (M) , or reactions programed with DJB2-HCV-NS5B (5B) and DJB2-HCV-NS5AB (5AB) RNAs, were resolved.
  • Purified HCV NS5B produced by recombinant baculovirus bacHCV5B-4 was included as a standard (track 1) .
  • Figure 14 shows an autoradiogram of an CH 3 HgOH agarose gel in which the radiolabeled product RNA from RdRp reactions with mock, DJB2-HCV-NS5B (5B) and DJB2- HCV-NS5AB (5AB) translation products, conducted with (+) or without (-) RNA template in the RdRp reaction mixture.
  • the position of the input self-priming RNA template is indicated by 'IX' and the completed duplex product by '2X' .
  • Figure 15 shows the dose-dependent inhibition of the RdRp activities of BVDV NS5AB of Example 2 by small molecule substance 1 and of HCV NS5AB of Example 7 by a chemically distinct small molecule substance (substance 2) .
  • the present invention provides pestivirus and hepacivirus genetic material comprising NS5AB encoding nucleic acids.
  • the invention further provides pestivirus and hepacivirus recombinant NS5AB proteins expressed from these nucleic acid sequences, and further, NS5AB sequences having RdRp activity. Finally, methods of use of the NS5AB sequences and proteins are provided.
  • Expression of pestivirus and hepacivirus recombinant NS5AB gene sequences may be carried out in a variety of systems including but not limited to cell- free systems, bacterial, yeast, mammalian, insect and plant cell systems, as well as in organisms such as infected, transfected, transduced or transgenic insects, animals or plants.
  • Cell-free transcription-translation vectors are constructed which allow for the RNA transcription from plasmid DNA. Subsequent cell- free translation of such RNA transcripts are utilized for the production of pestivirus and hepacivirus NS5AB proteins.
  • recombinant baculoviruses are constructed containing NS5A5B sequences that allow for the expression of NS5AB proteins in insect cells following infection in cell culture.
  • NS5AB proteins are expressed and purified. These purified proteins possess RdRp activity that is useful in screening for antiviral agents that may be efficacious in the prevention or treatment of pestivirus and hepacivirus infections and associated diseases.
  • Pestivirus and hepacivirus NS5AB proteins so produced may be modified by particular changes in nucleotide and amino acid sequence that result in NS5AB proteins with altered functionality. Such changes may be subtle and represent conservative substitutions such as in the case of nucleotide sequences, changes in the codon sequence that do or do not alter the encoded amino acid, or for amino acid sequences, changes that result in conservative residue substitutions, additions or deletions .
  • NS5AB proteins may be expressed in a system, such as by an infectious virus genomes wherein the normal proteolytic processing of NS5AB protein to NS5A and NS5B does not occur. Prevention of this processing causes NS5AB to remain unprocessed and intact. This prevention of NS5AB cleavage may be effected by a number of methods or approaches such as by modification of amino acid residues, or cleavage site determinants in the protein that are necessary for cleavage at the NS5A-NS5B junction. Alternatively, exclusion, elimination or inactivation of the proteolytic activity responsible for this cleavage may be achieved by a variety of methods known to those of ordinary skill in the art.
  • Nucleic acid molecules encoding the pestivirus and hepacivirus NS5AB proteins of the invention may be prepared by two general methods: (1) They may be synthesized from appropriate chemical starting materials, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art .
  • nucleotide sequence information such as that provided herein for NS5AB sequences, enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis.
  • Synthetic oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices.
  • the resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC) .
  • HPLC high performance liquid chromatography
  • a 3 kilobase double-stranded molecule may be synthesized as several smaller segments of appropriate complementarity.
  • Complementary segments thus produced may be ligated such that each segment possesses appropriate cohesive termini for attachment of an adjacent segment.
  • Adjacent segments may be ligated by annealing cohesive termini in the presence of DNA ligase to construct an entire 3 kilobase double-stranded molecule.
  • a synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.
  • Nucleic acid sequences encoding NS5AB proteins may be isolated from appropriate biological sources using methods known in the art. For example, RNA isolated from BVDV grown in cell culture or from the serum of an HCV infected patient may be used as a suitable starting materials for the generation of cDNA molecules encoding pestivirus and hepacivirus NS5AB proteins, respectively.
  • nucleic acids having the appropriate level of sequence homology with the protein coding region of the DNA molecules of the present invention may be identified by using hybridization and washing conditions of appropriate stringency.
  • hybridizations may be performed, using a hybridization solution comprising, for example, 5X SSC, 5X Denhardt ' s reagent, 1.0% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide.
  • Hybridization is carried out at 37-42°C for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37 °C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes.
  • T m 81.5°C + 16.6Log [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/# duplex bp
  • the T m is 57°C.
  • the T m of a DNA duplex decreases by 1-1.5°C with every 1% decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42 °C.
  • Such a sequence would be considered substantially homologous to the sequences of the present invention.
  • clones are maintained in plasmid cloning/expression vectors, such as pBluescript plasmids (Stratagene, La Jolla, CA) or recombinant baculovirus transfer vectors such as pFastBac vectors (Gibco-BRL, Gaithersburg, MD) that are propagated in suitable E. coli host cells.
  • plasmid cloning/expression vectors such as pBluescript plasmids (Stratagene, La Jolla, CA) or recombinant baculovirus transfer vectors such as pFastBac vectors (Gibco-BRL, Gaithersburg, MD) that are propagated in suitable E. coli host cells.
  • nucleic acids of the invention may also be used as starting materials for the generation of sequence variants of the nucleic acids of the invention using any number of synthetic and molecular biologic procedures well known in the art including but not limited to site-directed mutagenesis techniques. Particular mutations may give rise to NS5AB proteins with altered characteristics such as increased enzymatic activity.
  • NS5AB protein-encoding nucleic acid molecules of the invention include cDNA, genomic DNA, RNA, and fragments thereof, which may be single- or double-stranded in nature.
  • this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule of the present invention, such as selected segments of the cDNA having substantially the sequence of any of the sequences identified in the present invention.
  • Such oligonucleotides are further useful as probes and primers for detecting or isolating additional NS5AB encoding nucleic acids .
  • a cDNA or gene may be cloned into an appropriate in vi tro transcription vector, such as pSP64 or pSP65 for in vi tro transcription, followed by cell-free translation in a suitable cell-free translation system, such as wheat germ or rabbit reticulocytes, or in other mammalian cell extracts or fractions, such as those derived from HeLa cells, for example.
  • a suitable cell-free translation system such as wheat germ or rabbit reticulocytes, or in other mammalian cell extracts or fractions, such as those derived from HeLa cells, for example.
  • Several in vi tro transcription and translation systems are commercially available, e.g., from Promega Biotech, Madison, Wisconsin or Gibco-BRL, Rockville, Maryland.
  • NS5AB proteins may be produced by expression in a suitable procaryotic or eucaryotic system.
  • a DNA molecule such as the cDNA having a sequence identified in the invention may be inserted into a plasmid vector adapted for expression in a bacterial cell, such as E. coli , or into a baculovirus vector for expression in an insect cell.
  • Such vectors comprise the regulatory elements necessary for expression of the DNA in the host cell (e.g., E. coli or insect cell), positioned in such a manner as to permit expression of the DNA in the host cell.
  • regulatory elements required for expression include, but are not limited to, promoter sequences, transcription initiation sequences, enhancer sequences and translational control sequences.
  • NS5AB proteins or derivatives thereof produced by gene expression in a recombinant cell-free, procaryotic or eucaryotic system may be prepared, enriched or purified according to any number of techniques and procedures well known in the art.
  • NS5AB proteins of the invention may be purified from appropriate sources, e.g., cell-free systems, bacterial, animal, insect or plant cultured cells or tissues, by a variety of techniques that may include cell fractionation, partitioning, reverse micelle partitioning, aqueous two-phase extraction, precipitation, chromatography, ion exchange chromatography, chelation chromatography, affinity chromatography, immunoaffinity chromatography, high pressure liquid chromatography, hydrophobic interaction chromatography, centrifugation, membrane filtration, gel filtration, immunoprecipitation, electrophoresis, isoelectric focusing, isotachophoresis and the like.
  • a commercially available expression/ secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium.
  • the recombinant proteins may be purified by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein or with nickel columns for isolation of recombinant proteins tagged with 4-8 histidine residues at their N-terminus or C- terminus .
  • affinity separation such as by immunological interaction with antibodies that bind specifically to the recombinant protein or with nickel columns for isolation of recombinant proteins tagged with 4-8 histidine residues at their N-terminus or C- terminus .
  • NS5AB proteins of the invention prepared by the aforementioned methods, may be analyzed according to standard procedures. For example, such proteins may be subjected electrophoretic analyses and to amino acid sequence analyses, as well as crystalographic analyses for structure determination according to known methods .
  • NS5AB nucleic acids and polypeptides derived from sequences of the invention have utility in numerous methods, assays and kits involving research, diagnostic, therapeutic and pharmaceutical applications, and in the development of antiviral strategies for the prevention and treatment of pestivirus and hepacivirus diseases.
  • nucleic acid sequences of the invention may be used as probes or primers for the detection, labeling, identification or isolation of related nucleic acids in biological or synthetic preparations.
  • nucleic acid sequences of the invention may be used as hybridization probes to detect the presence of pestiviruses or hepaciviruses in samples. Such hybridization probes may further be used to isolate the nucleic acids to which they are hybridized by techniques well known in the art.
  • the nucleic acid sequences of the invention may be used as primers for the detection or isolation of pestivirus, hepacivirus or related nucleic acids using techniques such as reverse transcriptase- polymerase chain reaction (RT-PCR) . Appropriate primers pairs may be further used in nested PCR applications.
  • RT-PCR reverse transcriptase- polymerase chain reaction
  • Such primers, primer pairs and probes may represent any portion of the NS5AB sequences of the invention.
  • the actual sequence of the NS5AB gene used will vary according to the specific application.
  • additional sequences may be added to the primer or probe sequence, such as homopolymer tails (tags) , sequences that represent useful restriction enzyme recognition sites, sequences encoding particular amino acid residues, initiation or termination codons or other sequences that may be useful for the particular application at hand.
  • oligonucleotides of from 10 to 80 nucleotides in length that are either the same as or complementary to the sequences of the invention are useful as hybridization probes or as primers in RT-PCR applications.
  • the entire NS5AB sequence may be employed as a capture hybridization probe, for example.
  • nucleic acid sequences of the invention may be used as primers for the generation of variants or mutants of the sequences of the invention using a variety of methodologies known in the art, including site-directed mutagenesis procedures.
  • nucleic acid sequences of the invention may be used in the construction or generation of, or incorporated into, infectious viruses, vectors or replicons. Provision or substitution of the functional NS5AB sequences of the invention for poorly functional or non-functional counterparts may serve to improve the infectious and replicative characteristics of the resulting virus or replicating unit. Such substitutions may be carried out by standard genetic engineering procedures well known in the art.
  • the resulting infectious nucleic acid would have considerable advantages over current infectious clones including, but not limited to, improved or higher level viral RNA synthesis, improved levels of infectious virus production and improved virus replication in living hosts and in in vi tro systems. Use in other systems in which the function of the pestivirus or hepacivirus
  • NS5AB protein is important, such as in complementing or trans-complementing systems, replicon systems, defective viruses, defective interfering particles and the like, would also benefit from the use of the nucleic acid sequences of the invention.
  • the nucleic acid sequences of the invention may be used in methods to elicit immune responses to NS5AB proteins.
  • the NS5AB- encoding nucleic acid sequences of the invention operationally linked to an expression operon may be introduced directly into cells, particularly into antigen presenting cells such as dendritic cells, of a living host or human possessing a functioning immune system.
  • Introduction of the sequences may utilize transfection, transformation, or transduction methods, or involve the physical uptake of particles coated with the NS5AB nucleic acid sequences, such as plasmid-coated gold particles.
  • the NS5AB sequences Once inside cells, the NS5AB sequences are expressed, processed and presented to the host's immune system.
  • Such methods are useful in the elicitation of humoral and cellular immune responses in a living host and in humans and in vaccines for pestivirus and hepacivirus infections and diseases.
  • the nucleic acid sequences of the invention may be used in cell-free systems that allow for the transcription and/or translation of NS5AB sequences.
  • NS5AB DNA operably linked with a transcriptional promoter sequence may be transcribed in vi tro to produce an NS5AB RNA molecule.
  • Such an RNA molecule may then be translated to an NS5AB polypeptide when provided with the appropriate components for the translation of RNA such as, for example, lysates derived from reticulocytes, wheat germ, HeLa cells or other cell types.
  • the translated NS5AB protein may then be used to measure its activity, such as its RdRp activity, in such cell-free systems.
  • NS5AB RdRp activity and the modulating effects of such agents on NS5AB activity, may be measured in any number of ways. For example, RNA synthesis that is directly or indirectly dependent on NS5AB polymerase activity may be quantified. In one manner, the conversion of an RNA substrate of the NS5AB RdRp to product RNA may be measured by, for example, gel electrophoresis.
  • RNA e.g., 3 H, "c, 35 S, 33 P- or "P-nucleoside triphosphate
  • the amount of a radiolabeled precursor of RNA e.g., 3 H, "c, 35 S, 33 P- or "P-nucleoside triphosphate
  • a radiolabeled precursor of RNA e.g., 3 H, "c, 35 S, 33 P- or "P-nucleoside triphosphate
  • the nucleic acid sequences of the invention may be further used in the generation of cell lines or cellular systems that express NS5AB proteins.
  • Such cell lines in which an NS5AB protein is expressed from the NS5AB genes of the invention will have utility in methods for assaying materials for antagonistic or agonistic activity toward pestiviruses or hepaciviruses.
  • assays may be established whereby intact cells expressing an NS5AB protein of the invention are contacted with agents or materials suspected of affecting the intracellular activity of the NS5AB protein, and the effect of such agents on the activity of NS5AB is measured as described above.
  • RNA substrate of an NS5AB polymerase is provided that is the antisense strand of an mRNA, the sense strand (mRNA) of which is effectively translated to produce a polypeptide capable of being detected or of producing a detectable signal (the reporter) .
  • mRNA sense strand
  • the reporter the reporter
  • an RNA molecule is provided comprising the sequence complementary to the coding sequence of luciferase (antisense strand) .
  • the activity of the NS5AB polymerase on this RNA results in the production of the sense strand of the luciferase gene, which is then translated by the cellular translational system to produce luciferase protein.
  • the luciferase protein then may be detected by antibodies to the luciferase protein or by measurement of luciferase enzymatic activity in intact cells or in cellular extracts using a luminometer or other similar device. Numerous other reporters may serve equally well in this application including but not limited to, b- galactosidase, alkaline phosphatase, fluorescent green protein and the like.
  • the cell systems that may be used in this method of the invention may be of bacterial, fungal, insect, avian, mammalian or plant origin.
  • nucleic acid sequences of the invention may be used in assays to identify agents or materials capable of interacting with or affecting the NS5AB nucleic acid sequences.
  • assays may be established in which nucleic acid sequences of the invention are provided and then contacted with agents or materials suspected of interacting with such sequences. Agents identified in such interaction assays would then have potential diagnostic utility and uses in the detection of pestiviruses or hepaciviruses in, for example, biological samples.
  • Such agents would also have potential utility in applications involving the prevention or treatment of pestivirus and hepacivirus diseases in an affected living host, including humans, and for the inhibition or enhancement of pestivirus or hepacivirus replication or propagation in living hosts and in in vi tro systems such as cell, tissue and organ cultures. Additional applications may be envisioned once the nature of the particular agent is clear.
  • Pestivirus and hepacivirus NS5AB protein compositions of the invention also have broad utility.
  • the NS5AB proteins, or peptides thereof may be used in assays for the detection of immune responses to the same.
  • protein sequences or peptides of the invention may be used in assays in which said sequences are immobilized on a matrix and used to capture antibodies directed to said sequences.
  • protein sequences or peptides of the invention may be used to detect or measure cell-mediated immune responses to the protein, such as in immune cell proliferation assays.
  • the NS5AB protein compositions of the invention also have utility in the elicitation of immune responses, such as in vaccines.
  • NS5AB proteins of the invention or peptides thereof, to a living organism with a functioning immune system will cause such organism to mount an immune response to the NS5AB sequences.
  • the NS5AB sequences may be presented to the living organism in any number of ways well known to those trained in the art and include, but are not limited to, providing free protein or peptides, formulated protein or peptides, protein- adjuvant conjugates or peptides, protein or peptides in the context of intact or disrupted cells in which NS5AB sequences are present and other such manners .
  • Immune responses so elicited may be either humoral or cellular in nature, or both.
  • NS5AB proteins of the invention may further be used to select or purify such antibodies to NS5AB.
  • an NS5AB protein may be immobilized and used to bind antibodies specific for the NS5AB protein, and thus enrich for such antibodies.
  • the NS5AB proteins and peptides of the invention may be used to produce monoclonal antibodies to the NS5AB protein using standard techniques known in the art.
  • Antibodies to the NS5AB protein, whether polyclonal or monoclonal may be further evaluated for their ability to affect the enzymatic activity of the NS5AB RdRp activity.
  • the NS5AB protein compositions of the invention have utility in assays for the detection and identification of agents capable of interacting with or affecting the NS5AB protein.
  • Assays may be established in which NS5AB polypeptide sequences of the invention are provided and then contacted with agents or materials suspected of interacting with such sequences.
  • agents or materials suspected of interacting with such sequences For example, upon provision of an NS5AB protein of the invention, or fragment or portion thereof, contacted agents may be assessed for their ability to bind specifically to the protein.
  • binding agents would then have potential diagnostic utility and uses in the detection of pestiviruses or hepacivirus in, for example, biological samples.
  • binding agents may further affect the functional activity of the NS5AB protein, such as either inhibiting or enhancing NS5AB function.
  • Agents that inhibit the function of the NS5AB protein would have potential utility in applications involving the prevention or treatment of pestivirus or hepacivirus disease in an affected living host or for the inhibition of virus replication or propagation in living hosts, including humans, in in vi tro systems such as cell, tissue and organ cultures or in biological materials.
  • Agents that enhance the function of the NS5AB protein would have potential utility in applications involving the replication, propagation or production of pestiviruses or hepaciviruses in living hosts, such as in animal models of virus replication, and in in vi tro systems such as cell, tissue and organ cultures .
  • methods of assay are provided in which the NS5AB polymerase activity furnished by an enzymatically active NS5AB protein of the invention is measured directly.
  • Agents placed in contact with said enzymatically active NS5AB polymerase may be assessed for their ability to specifically modulate this enzymatic activity.
  • Such enzymatically active polymerase may be provided in an extract or lysate of a cell in which the polypeptide was produced, in an in vi tro cell-free expression system or in an enriched or purified form.
  • NS5AB-dependent RNA synthesis typically requires certain reaction components including minimally, a buffered medium, a divalent cation, precursors of RNA (nucleoside triphosphates, NTPs) and an RNA template or substrate.
  • a primer for RNA synthesis may or may not be included, depending on the nature of the reaction conditions and whether these conditions allow for primer-independent RNA synthesis or are dependent on a primer.
  • Additional components may include monovalent cations, reducing agents, stabilizers, cofactors and inhibitors of activities unrelated to the NS5AB RdRp activity such as inhibitors of RNase, phosphatase, kinase, phosphotransferase and similar activities.
  • any number of activity detection and measurement technologies may be utilized including, but not limited to, electrophoretic, radiometric, colorimetric, fluorogenic, or chemiluminescent , any one of which may be suitable in the case of the NS5AB RdRp activity.
  • the conversion of an RNA substrate of the NS5AB RdRp to product RNA may be measured by, for example, gel electrophoresis.
  • the incorporation of a precursor of RNA into a polymer of RNA is measured, such as the incorporation of a radiolabeled NTP into trichloroacetic acid-precipitable RNA, which may then be quantified by scintillation spectrometry or phosphorimaging technologies .
  • Such precursors may alternatively be tagged with other moieties to allow their ready detection such as with biotin for detection with avidin reagents including various avidin conjugates such alkaline phosphatase and the like or with fluorescently-labeled NTPs for detection using fluorescent technologies such as fluorescence polarization.
  • NS5AB-dependent RNA synthesis may also be assessed by measuring the extension of a pre-labeled or tagged primer of RNA synthesis such as a radiolabeled or biotin-tagged oligonucleotide that is used by the polymerase to initiate RNA synthesis on a template RNA molecule.
  • Extension of the primer may be assessed by quantifying the addition of nucleoside triphosphates to the primer, by determining the length of the primer product, or by other methods known in the art.
  • the product of NS5AB RdRp activity may be detected and quantified by capture of the product RNA using hybridization techniques.
  • an oligonucleotide complementary to the product of the NS5AB RdRp reaction may be introduced during or after the reaction and hybridized to the product.
  • the extent of hybridization of the added oligonucleotide may be used as a measure of the amount of product RNA present in the mixture and may be assessed by various means known in the art .
  • RdRp activity are readily known to, or can be envisioned by, the skilled artisan and are fully contemplated here.
  • Assays involving the nucleic acid and polypeptide compositions of the invention may be formatted in any number of configurations . Particularly useful for evaluating large numbers of agents and materials are high throughput screening formats. Traditionally such assays were typically formatted in 96 well plates. However, 384, 864 and 1536 well plates may be used in such high throughput assay systems. These systems are often automated using robotics technologies to allow manipulation and processing of large numbers of samples
  • agents or materials that may be evaluated in the various assay methods of the invention for potential antagonistic or agonistic affects include but are not limited to small molecules, polymers, peptides, polypeptides, proteins, immunoglobulms or fragments thereof, oligonucleotides, antisense molecules, peptide- nucleic acid conjugates, ribozymes, polynucleotides and the like.
  • agents or materials identified using the compositions and assay methods of the invention will be broad and will include uses for the detection and isolation of pestivirus and hepacivirus nucleic acids and polypeptides, for the detection or diagnosis of pestivirus or hepacivirus infections, for the prevention and treatment of pestivirus or hepacivirus diseases in an affected living host, including humans, and for the inhibition or enhancement of pestivirus or hepacivirus replication or propagation in living hosts and in in vi tro systems such as cell, tissue and organ cultures, as well as for other uses the may be envisioned once the nature of the agent is clear.
  • kits to facilitate the use of the compositions and methods disclosed herein include kits to facilitate the use of the compositions and methods disclosed herein.
  • Exemplary kits would include NS5AB nucleic acids and polypeptides of the invention, and/or variants thereof, alone or in suitable vectors.
  • Such reagents may include, but not be limited to, buffers, solvents, media and solutions, substrates and cofactors, vectors and host cells, and detection or reporter reagents.
  • Accessory items may include vials, vessels, reaction chambers and instruction sheets .
  • NS5B and NS5AB sequences of the prototypic pestivirus were obtained by polymerase chain reaction (PCR) using plasmid pBVSD2-3 (Wiskerchen and Collett (1991) Virology 184:341-350) , which comprises a portion of a full length clone (GenBank accession number M31182), which in turn has been shown to be functional in the context of an infectious clone (Vassilev et al. (1997) J. Virol.
  • 5' primers included a Sma I restriction enzyme site, an ATG codon for translation initiation, followed by sequences corresponding to nucleotides 8705-8738 (NS5B primer) and 10197-10216 (NS5AB primer) of the published BVDV NADL sequence (Collett et al.(1988) J. Virol. 165: 191-199).
  • the N-termini of the NS5B and the NS5AB proteins was based on data reported by Xu, et al . (J. Virol. 71: 5312-5322, 1997).
  • the 3' primer was the same for both amplifications and contained sequences complementary to nucleotides
  • PCR reactions contained 100 ng of DNA template, 200 ⁇ M dNTPs, 200 pmoles of each primer, IX vent DNA polymerase buffer, 2 units of vent DNA polymerase (New England Biolabs). PCR involved 30 cycles as follows: 94°C for 90 sec, 55°C for 45 sec and 72°C for 2 min (NS5B) or 4 min (NS5AB) . After cycling, the reaction was incubated at 72°C for 10 min.
  • Plasmid DJB2 is a derivative of plasmid pT7-PVl (A) 80 , which contains a complete poliovirus type 1 genomic sequence positioned downstream of the T7 RNA polymerase promoter (Sarnow (1989) J. Virol. 63:467- 470). DJB2 consists of the 5' NTR of poliovirus, including a functional poliovirus internal ribosome entry site (IRES), fused to the 3' portion of the poliovirus 3D coding sequence, the 3' NTR and a poly(A) ao tail.
  • IRS functional poliovirus internal ribosome entry site
  • the PCR amplified NS5B and NS5AB gene fragments were ligated into DJB2 restricted with Msc I and then transformed into E. coli SURE cells (Stratagene) . Plasmids from transformants with inserts in the correct orientation were identified by restriction fragment digestion patterns and were designated DJB2-BVDV-NS5B and DJB2-BVDV-NS5AB. These translation vector constructs contained the poliovirus IRES upstream of an AUG codon followed in phase by the protein coding sequence of the inserted gene and a translation termination codon.
  • the DNA sequence of NS5AB in plasmid DJB2-BVDV-NS5AB is identified as SEQ ID NO : 1 and its deduced amino acid sequence is identified as SEQ ID NO: 2.
  • RNA transcripts were prepared from plasmids DJB2-BVDV-NS5B and DJB2-BVDV-NS5AB previously linearized by restriction with Mlu I using T7 polymerase as described (Barton et al.(1996) Methods in Enzymol . 275:35-57).
  • the RNA transcripts were phenol-chloroform- isoamyl alcohol extracted, ethanol precipitated and purified by Sephadex-G50 chromatography. Expression of the NS5B and NS5AB proteins from the
  • RNA transcripts was conducted in an HeLa S10 translation system as described by Barton et al . (1996) .
  • Reaction mixtures (50 ⁇ L final volume) contained 25 ⁇ L HeLa S10 extract, 10 ⁇ L HeLa initiation factors, 5 ⁇ L 10X nucleotide reaction mix (10 mM ATP, 2.5mM UTP, 2.5 mM GTP) , 600 mM potassium acetate, 300 mM creatine phosphate, 4 mg/mL creatine kinase, 155 mM Hepes-KOH (pH 7.4) and 50 ⁇ g/mL RNA transcript.
  • reactions contained [ 35 S] -methionine in order to radiolabel newly synthesized proteins. Reaction mixtures were incubated at 30°C for 2 h.
  • Polypeptides synthesized in translation reactions were resolved by electrophoresis in SDS-containing polyacrylamide gels.
  • the polyacrylamide gels were fluorographed and exposed to X-ray film.
  • resolved proteins were transferred from the gel to a PVDF membrane in transfer buffer (3.03 g/L Tris, 14.4 g/L glycine, 20% methanol) .
  • the membrane was blocked in 5% dry milk in TBS-T (20 mM Tris, 137 mM NaCl, 0.1% Tween 20) for 1 h at room temperature and was then rinsed several times with TBS-T.
  • Rabbit antibody specific to the BVDV NS5B protein Collett et al .
  • RNA transcripts derived from DJB2-BVDV-NS5B and DJB2-BVDV-NS5AB in the presence of [ 35 S] -methionine resulted in the production of radiolabeled 75 kDal and 130 kDal polypeptides, respectively (Fig. 7, tracks 2 and 3, respectively) . That these polypeptides represented NS5B sequence- containing protein was demonstrated by their immunoreactivity with antiserum specific for NS5B sequences (Fig. 8) .
  • the 75 kDal protein produced from DJB2-BVDV-NS5B RNA (track 7) co-migrated with purified NS5B produced from a recombinant baculovirus (tracks 1- 4) .
  • the 130 kDal protein was the only polypeptide recognized specifically by the anti-NS5B antiserum and was the size expected for the NS5AB protein (track 6) . Importantly, a protein the size of the NS5B was not detected in the 5AB RNA translation reaction.
  • DJB2-BVDV-NS5AB RNA produces the expected 130 kDal NS5AB protein, and further, that the NS5AB protein is not processed to NS5B in this expression system.
  • Plasmid SAO-2a is a derivative of plasmid DJB2-BVDV-NS5B in which nucleotides 6234-7205 of the poliovirus sequence have been excised.
  • RNA transcripts from SA0-2a are approximately 3 kb in length and possess a CGCG sequence at their 3' terminus. The presence of this CGCG sequence has previously been shown to allow for self-priming of RNA synthesis to occur with poliovirus RdRp and results in production of a 2X-sized (6 kb) product (Barton et al . , 1996) . RdRp reactions were incubated at 30°C for 60 min. and radiolabeled product RNAs were analyzed by CH 3 HgOH-l% agarose gel electrophoresis as described (Barton et al . , 1996).
  • a 2X-sized (6 kb) radiolabeled RNA product was detected when the NS5B translation product was used in the polymerase assay (Fig. 9, track 2) .
  • the 2x-sized product was also found when the NS5AB translation product was evaluated (Fig. 9, track 6) .
  • the 2x-sized product was not detected in the mock translation reaction (Fig. 9, track 4) .
  • the RdRp activity observed with the NS5AB protein was not due to processing of NS5AB to NS5B since NS5B was not detected in the ' immunoblot analysis of the NS5AB translation products (Example 1) .
  • the detergent NP-40 was added to the translation products prior to their addition to the polymerase assays. This resulted in about a two-fold increase in the amount of radiolabeled product RNA in both the NS5B and NS5AB reactions.
  • NS5AB sequences may be cloned into any number of surrogate expression systems that are routinely used by the trained artisian and may include, but are not limited to, cell-free systems, bacterial, yeast, mammalian cell, insect cell and plant cell systems.
  • the HCV NS5AB sequences may be obtained from the serum of an infected individual. RNA isolated from the serum of an HCV-infected patient was used to amplify the HCV NS5AB gene using an RT-nested PCR protocol . An aliquot of this RNA was added to the 50 ⁇ L reaction mixture of the Titan One Tube RT-PCR System
  • the reaction mixture consisted of IX RT-PCR buffer containing 1.5mM MgCl 2 , 200 ⁇ M each dNTP, 5 mM dithiothreitol, 10 units RNasin (Promega), 300 nM of forward reverse primers, and 1 ⁇ L enzyme mix (AMV and Expand High Fidelity Enzyme mix) .
  • the RT-PCR cycle was performed as follows: Step 1, 50°C 50 minutes; Step 2, 94°C 45 seconds; Step 3, 94°C 45 seconds; Step 4, 55°C 45 seconds; Step 5, 72°C 150 seconds; Step 6, repeat steps 3-5 for 14 cycles; Step 7, 94°C 45 seconds; Step 8, 55°C 45 seconds; Step 9, 72°C 150 seconds plus 5 seconds each additional cycle; Step 10, repeat steps 7-9 for 24 cycles.
  • a portion of this first round reaction was added to the nested PCR reaction using Expand High Fidelity PCR System (Boehringer-Mannheim) .
  • the reaction mix consisted of IX PCR buffer containing 1.5 mM MgCl 2 , 200 ⁇ M each dNTP, 300 nM forward and reverse primers, and 1 ⁇ L enzyme mix.
  • the forward primer included an Rsr II restriction enzyme site, an ATG codon for translation initiation, followed by the first 18 nucleotides corresponding to the first 6 codons of the NS5A protein.
  • the reverse primer contained sequences complementary to the 3' end of the NS5B gene including the stop codon, followed by a Not I restriction enzyme site.
  • the nested PCR cycle was performed as follows: Step 1, 94°C 30 seconds; Step 2, 94°C 45 seconds; Step 3, 52°C 45 seconds; Step 4, 72°C 150 seconds; Step 5, repeat steps 2-4 for 9 cycles; Step 6, 94°C 45 seconds; Step 7, 60°C 45 seconds; Step 8, 72°C 150 seconds plus 5 seconds each additional cycle; Step 9, repeat steps 6-8 for 14 cycles.
  • the resulting product was purified on a 1% agarose gel using Qiagen's QIAquick Gel Extraction Kit, digested with Rsr II and Not I restriction enzymes and cloned into pFASTBAC (Gibco-BRL) previously digested with Rsr II and Not I.
  • the ligation mixture was transformed into E. coli DH5 cells.
  • One of the transformed bacterial colonies yielded plasmid bacHCV5AB-ll .
  • a second colony yielded plasmid bacHCV5AB-16.
  • the DNA sequence and deduced amino acid sequence of the NS5AB gene in plasmids bacHCV5AB-ll and bacHCV5AB-16 are identified as SEQ ID NO : 3 (Fig. 3), SEQ ID NO:4
  • Plasmid bacHCV5AB-16 was used to generate a recombinant baculovirus by the transposition method of the Bac-to-Bac system (Gibco-BRL) . After transformation of the bacHCV5AB-16 plasmid DNA into E. coli DHlOBac cells, several colonies containing bacmid DNA were transfected into Sf9 insect cells according to the protocol supplied by the manufacturer. Recombinant baculoviruses were isolated and amplified. NS5AB protein expression in recombinant baculovirus-infected cells was verified by Western immunoblot analysis with antisera specific to either the HCV NS5A or NS5B sequences.
  • a lysate of Sf9 insect cells infected with the bacHCV5AB-16 virus was prepared. An aliquot of the lysate was electrophoresed on an SDS-containing polyacrylamide gel, the resolved proteins in the gel were transferred to a PVDF membrane as described in Example 1, and the 126 kilodalton HCV NS5AB protein was detected using either HCV NS5A- or NS5B-specific antiserum.
  • Sf9 cells were either mock-infected or infected with recombinant baculoviruses bacHCV5B-4 or bacHCV5AB-16. After 60 hours, cells were pelleted, washed and resuspended in 1/50 the culture volume in hypotonic buffer (10 mM Tris-HCl, pH 7.5 , 10 mM NaCl) supplemented with protease inhibitors. After 30 minutes on ice, the cells were disrupted by 20 strokes with a dounce homogenizer and sonicated briefly. Cellular debris and nuclei were removed by centrifugation at 900 x g for 5 minutes.
  • hypotonic buffer 10 mM Tris-HCl, pH 7.5 , 10 mM NaCl
  • the membrane-containing supernatant was pelleted by centrifugation at 15,000 x g for 30 minutes. This pellet was resuspended in 1/300 the culture volume in hypotonic buffer by gentle douncing and then mixed with a 67% sucrose solution prepared in hypotonic buffer to achieve a final sucrose concentration of 60%. This solution was layered onto 67% sucrose solution in a ultracentrifuge tube and was then overlaid with 50% sucrose solution followed by 10% sucrose solution. This discontinuous sucrose gradient was subjected to centrifugation at 90,000 x g for 16 hours. The membrane band located at the 10%-50% sucrose interface was recovered. As shown in the immunoblot depicted in Fig.
  • the RdRp activity of the membrane-associated NS5AB protein was assessed in a standard RdRp reaction (Example 2) .
  • RdRp activity was measured by quantification of trichloroacetic acid (TCA) - precipitable radioactivity incorporated into reaction product RNA after 60 min of incubation.
  • TCA trichloroacetic acid
  • the baculovirus-produced NS5AB protein (5AB) possessed RdRp activity. Only background activity was seen in the mock-infected baculovirus material (mock) .
  • the NS5AB gene in plasmid bacHCV5AB-ll was used as a template to PCR amplify both the NS5AB and the NS5B genes.
  • the 5' primers for NS5AB and NS5B included the Sma I restriction enzyme site, an ATG codon for translation initiation followed by sequences corresponding to the amino terminus of NS5A and NS5B, respectively.
  • the 3' primer which included a stop codon for the HCV polyprotein coding region followed by the Not I restriction enzyme site, was used to amplify both the HCV NS5AB and NS5B genes.
  • PCR was performed essentially as described in Example 1. The PCR amplified fragments were ligated into plasmid DJB2 and transformed into E. coli as described in Example 1. Plasmid inserts from bacterial clones were verified correct by restriction digestion patterns and sequencing, and were designated DJB2-HCV-NS5B and DJB2- HCV-NS5AB.
  • RNA transcripts were prepared from plasmids DJB2- HCV-NS5B and DJB2-HCV-NS5AB and were used to express the NS5B and NS5AB proteins, all as described in Example 1.
  • Figure 12 shows the polypeptides translated from RNA transcripts derived from DJB2-HCV-NS5B and
  • DJB2-HCV-NS5AB in the presence of [ 35 S] -methionine .
  • the radiolabeled NS5B and NS5AB polypeptides are the only proteins produced, respectively (Fig. 12, tracks 2 and 3, respectively). That these polypeptides represented HCV NS5B sequence-containing proteins was confirmed by their immunoreactivity with antiserum specific for HCV NS5B sequences (Fig. 13).
  • the immunoreactive protein produced from DJB2-HCV-NS5B RNA (track 4) co-migrated with purified HCV NS5B produced from a recombinant baculovirus (track 1) and protein produced from the DJB2-HCV-NS5AB RNA translation was recognized specifically by the anti-NS5B antiserum (track 2).
  • a protein the size of the NS5B was not detected in the NS5AB RNA translation reaction.
  • DJB2-HCV-NS5AB RNA produces the expected NS5AB protein, and further, that the NS5AB protein is intact and is not processed to NS5B in this expression system.
  • Example 6 To determine if the polypeptides of Example 6 possessed RdRp activity, aliquots of each of the translation reactions of Example 6 were evaluated in RNA polymerase reactions as described in Example 2. RdRp reactions were incubated at 30°C for 60 min. and radiolabeled product RNAs were analyzed by CH 3 HgOH-l% agarose gel electrophoresis as described in Example 2. Radiolabeled RNA product of 2X size was detected when either the NS5B or the NS5AB translation product was used in the polymerase assay. These results demonstrate that, not only does the HCV NS5B protein possess RdRp activity, but, unexpectedly, so does the HCV NS5AB protein.
  • the RdRp activity of viral proteins may represent an important target for therapeutic intervention or prevention of infections and diseases caused by RNA viruses.
  • Use of the NS5AB activity of the present invention to discover agents capable of modulating, interfering with or inhibiting this activity represents a novel, previously unanticipated means of discovering and developing antiviral agents.
  • the transcription-translation products of Examples 2 and 7 may be used to test and evaluate potential biological and chemical compounds for inhibitory activity against the RdRp.
  • the NS5AB- containing materials of the above examples were incubated in standard RdRp reaction mixtures supplemented with increasing concentrations of particular chemical substances suspected of inhibiting the NS5AB RdRp activity.
  • the RdRp activity was quantified by measurement of radioactivity incorporated into TCA-precipitable product. As is shown in Fig.
  • an assay for polymerase activity that is capable of high volume screening, in other words, a high-throughput assay, is desirable.
  • assay methodologies well known to the trained artisan that allow the efficient screening of large numbers of samples [see, for example, Cole, JL, in Meth Enzymology 275;310-328 (1996)], any one of which may be suitable in the case of the NS5AB RdRp activity.
  • These methodologies may utilize any number of activity detection and measurement technologies including, but not limited to, radiometric, colorimetric, fluorogenic, or chemiluminescent signals.
  • a high throughput assay of the RdRp activity of the functional NS5AB proteins of the invention is provided to enable the screening of large numbers of chemicals or other potential inhibitors for activity against the enzyme.
  • the assay is formatted in 96-well microplates and measures polymerase activity on an RNA template-primer by the incorporation of radiolabeled NTP into TCA-precipitable RNA product. Radioactivity may be quantified by either direct scintillation spectrometry or phosphorimaging technology.
  • the present invention demonstrates that protein molecules derived from the colinear expression - of the sequences of the NS5A gene and the NS5B gene of pestiviruses, hepaciviruses and related viruses in their natural configuration are functional polypeptides, termed NS5AB, possessing RNA-dependent RNA polymerase activity.
  • NS5AB functional polypeptides
  • This novel composition and activity provides a target for identification of test substances useful in diagnostic, preventative, therapeutic and pharmaceutical applications .

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Abstract

Cette invention se rapporte à des compositions et à des procédés pour protéines d'ARN-polymérase dirigées sur l'ARN de nature virale.
PCT/US2000/031909 1999-11-23 2000-11-21 Compositions de polymerase et procedes d'utilisation correspondants WO2001038538A1 (fr)

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