AU768177B2 - Hepatitis C virus NS5B compositions and methods of use thereof - Google Patents

Description

WO 99/51781 PCT/US99/07404 Hepatitis C Virus NS5B Compositions and Methods of Use Thereof Field of the Invention The present invention relates to hepatitis C virus (HCV), and more specifically, to the HCV NS5B gene and the encoded protein. In particular, the invention relates to novel compositions comprising HCV sequences, functional HCV NS5B sequences, functionally improved compositions of HCV NS5B sequences, and to the use of such NS5B sequences in research, diagnostic, therapeutic and pharmaceutical applications.

Cross Reference to Related Applications This application claims priority under 35 U.S.C.

§119(e) to US Provisional Application 60/080,509 filed April 2, 1998 and US Provisional Application 60/090,356 filed June 23, 1998, the disclosures of each being incorporated by reference as though set forth herein in full.

Background of the Invention Hepatitis C virus (HCV) is a major cause of hepatitis globally. The World Health Organization estimates that 150 million people worldwide are presently infected with the virus. Most infections become persistent and about 60% of cases develop into chronic liver disease. Chronic HCV infection can lead to development of cirrhosis, hepatocellular carcinoma and liver failure.

For treatment of hepatitis due to HCV, interferon WO 99/51781 PCT/US99/07404 alpha (IFN) is currently approved in the U.S. IFN treatment is associated with improved serum enzyme response in 20-40% of patients. The remainder are nonresponsive to IFN treatment. For responders, a sustained improvement of aminotransferase levels is seen in only 10-20% of patients; the majority of patients relapse upon cessation of IFN treatment. While IFN represents the first treatment of chronic hepatitis C, its effectiveness is variable, its cure rate is low, and associated adverse effects are considerable.

Vaccines under development for HCV generally consist of recombinant versions of the putative viral structural proteins El, E2), or genes encoding these. It is believed that virus neutralizing antibodies do exist, can be elicited, and may be able to inhibit or prevent HCV infection. However, to date, no vaccine has been demonstrated safe and effective for HCV. Indeed, given the inherent genetic diversity of HCV, with virus isolates exhibiting immunologically distinct envelope proteins that are not neutralized by pre-existing antibodies, vaccine development will be a formidable task.

HCV can not yet be efficiently propagated in vitro.

This deficiency has compromised a clear understanding of several aspects of HCV replication. While numerous HCV isolates have been molecularly cloned from infected patients and have been sequenced, much remains to be learned regarding the required sequence features and elements which facilitate efficient replication. From study of these sequences in a variety of surrogate gene expression systems, knowledge of the molecular biology WO 99/51781 PCT/US99/07404 of HCV has expanded greatly in recent years. These advancements, which have been facilitated by existing knowledge and understanding of the molecular biology of the related pestiviruses and flaviviruses, have yielded important insights into the virus-specified components essential for virus replication that may be suitable targets for antiviral drug discovery strategies.

As mentioned above, HCV is a genetically heterogeneous virus. It exists in nature, and in infected individuals, as a "quasispecies", which means that within the virus population from any particular source, the viral genetic material is present as a collection of closely related, but non-identical sequences. This genetic heterogeneity of HCV sequences may be a consequence of natural errors introduced during the normal replication process of HCV, of selection pressures placed on the virus by the host, or others factors. It is believed that the quasispecies nature of HCV may contribute to the virus's ability to avoid elimination by the host's defense systems and establish persistent infections.

The quasispecies nature of HCV has made it difficult to define the sequence of "functional" genes and genomes, particularly in the absence of a controlled system for the efficient replication of the virus.

However, functionality of segments of HCV sequences has been studied in a variety of surrogate expression systems. Several gene products have been shown to be functional in in vitro assays in that they exhibit enzymological activities that are predicted to exist based on the presence of particular amino acid sequence WO 99/51781 PCT/US99/07404 motifs in these gene products. For example, the NS3 protein possesses serine proteinase, nucleoside triphosphatase and helicase activities and the protein possesses RNA-dependent RNA polymerase (RdRp) activity.

The recent demonstration of an infectious molecular clone of HCV [Kolykhalov et al., Science 277:570-574 (1997); Yanagi et al., Proc. Natl. Acad. Sci. USA 94:8738-8743 (1997)] allows definition of a complete functional HCV genome and consequently of a sequence for a set of functional HCV gene products from this particular strain of HCV obtained from one particular source. It is noteworthy that Kolykhalov et al.

indicate that the difficulties in obtaining a functional genome included: 1) the highly variable quasispecies nature of HCV in infected patients; 2) the required in vitro amplification (cDNA synthesis and PCR amplification) of the sequences due to the small quantities of viral RNA in clinical samples; and 3) the necessity of molecular cloning (in E. coli). Each of these aspects allows for the possibility and presentation of defective (poorly functional or nonfunctional) HCV sequences or for errors in transcribing or amplifying HCV sequences. Kolykhalov et al. indicated that RNAs produced from 34 full genome molecular clones failed to exhibit infectivity in chimpanzees. Nucleotide sequence analyses showed numerous sequence changes among the 6 full length clones that were completely sequenced. Only after a "consensus sequence" clone was generated, was positive infectivity demonstrable. Yanagi et al. also indicated the high WO 99/51781 PCT/US99/07404 degree of sequence variability among individual molecular clones obtained from a single source material and the non-infectious nature of RNA transcripts from full-length molecular clones. Yanagi et al. concluded that a large proportion of HCV genomes are defective.

In fact, recent work indicates a bias toward the preferential selection of defective HCV genomes during molecular cloning procedures [Forns, et al., Proc Natl Acad Sci USA 94:13909-13914 (1997)]. Of 25 random clones derived from a control plasmid by Forns et al., only 8% were functional in polyprotein synthesis.

Thus, it is clear from these examples that mere knowledge of a sequence derived from HCV is insufficient to conclude that such a sequence encodes useful viral genes or functional viral gene products.

Regarding the NS5B gene, while many partial and complete gene sequences have been entered into various databases, only a very few of these sequences have been demonstrated to be functional based on in vitro assays of RdRp activity.

The present invention provides novel and functional, as well as functionally improved, hepatitis C virus NS5B sequences for use in research, diagnostic, therapeutic and pharmaceutical applications, and for use in assays for the identification of efficacious antiviral agents.

SUMMARY OF THE INVENTION The present invention provides novel HCV nucleotide sequences from which recombinant HCV proteins WO 99/51781 PCT[US99/07404 having demonstrable RdRp activity may be derived.

Sequence modifications that result in improved functional activity are also provided. Such functional recombinant HCV NS5B proteins have utility in research, diagnostic, therapeutic and pharmaceutical applications.

In particular, the recombinant HCV NS5B proteins of the invention have utility in antiviral drug discovery strategies.

In a preferred embodiment of the invention, an isolated nucleic acid molecule is provided that comprises a DNA sequence identified as SEQ ID NO: 1, which is present in clone 4 of the present invention.

An exemplary HCV NS5B protein has the amino acid sequence identified as SEQ ID NO: 2 encoded by clone 4.

Additional nucleic acid molecules that represent nucleic acid sequences related to those of SEQ ID NO: 1, including but not limited to those identified by SEQ ID NO: 3, 4, 5, 6 and 7 encoding, respectively, the amino acid sequences identified by SEQ ID NO: 8, 9, 10, 11 and 12 of the HCV NS5B are also contemplated to be within the scope of the present invention. It is further contemplated that conservative sequence or residue substitutions of these sequences are also within the scope of the invention.

In an additional embodiment of the invention, functionally improved hepatitis C polymerase nucleic acid and amino acid sequences are provided. Exemplary sequences include those nucleic acid sequences that encode amino acid sequences related to those identified WO 99/51781 PCT/US99/07404 by SEQ ID NO: 2 in which particular codons have been substituted to encode particular amino acid changes in the sequence. Examples of useful changes include but are not limited to changes at amino acid positions number 75, number 177 and number 543 of the mature protein.

In another embodiment of the invention, functionally improved hepatitis C polymerase nucleic acid and amino acid sequences are provided in which particular codons are substituted to encode particular amino acids not normally found in known sequences of For example, changes including but not limited to residue changes at amino acid positions number 1 and number 2 of the mature NS5B protein are useful according to this invention.

As described above, nucleic acids encoding variant proteins or polypeptides are contemplated to be within the scope of the present invention. Sequence ID NO: 13 provides an example of such a variant sequence. Such variants may or may not possess HCV 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. Preferably, the changes will not affect, or substantially affect, the structure or useful properties of the polypeptide.

Thus, HCV NS5B variants may suitably possess functional activity such as those described above, or they may be poorly functional or inactive, yet contain substantially the secondary and tertiary structure of the native polypeptide. Such NS5B molecules may be used to advantage to identify agents that specifically bind WO 99/51781 PCT/US99/07404 to or otherwise affect the HCV NS5B activity. HCV variants can be either naturally occurring purified or isolated from a natural source) or synthetic generated by biological expression of DNA that has been subjected to site-directed mutagenesis or produced by chemical synthetic techniques well known in the art).

The nucleic acid molecules of the invention may be cloned and expressed in vectors. Such 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 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 in vitro, for example for the production of RNA or protein. The vector may be further used to transform, transfect, infect or transduce a host cell or an organism. The present invention further contemplates the use of host cells and organisms harboring or expressing the HCV nucleic acid sequences or polypeptides of the invention for the identification of agents that affect the activity of the HCV NS5B protein.

In yet another embodiment of the invention, methods are provided for the identification of agents that affect the HCV NS5B polymerase sequences. Such methods include high throughput screening procedures that allow assessment of large numbers of agents. The agents WO 99/51781 PCT/US99/07404 identified by use of the HCV NS5B nucleic acids and polypeptides of the invention, variants thereof, or methods of the invention, may be either antagonistic or agonistic in their affect on the NS5B sequences. These agents may include 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. It is specifically contemplated that both antagonistic and agonistic molecules identified by practice of the invention have broad and multiple utilities. Such utilities for antagonists of HCV NS5B activity include, but are not limited to, uses for the inhibition of HCV replication in humans, in other living hosts and in in vitro systems such as cell, tissue and organ cultures. Agonists of HCV NS5B activity identified by practice of the invention will also have multiple utilities, both in living hosts and in in vitro systems. For example, such agents will be useful 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.

According to another aspect of the invention, kits are provided to facilitate the use of the compositions and methods disclosed herein. Exemplary kits would include HCV NS5B nucleic acids and polypeptides of the invention, variants thereof, alone or in association with suitable vectors. Also included would be protocols for use of the compositions of the invention for the WO 99/51781 PCT/US99/07404 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 also be included such as vials, vessels and reaction chambers.

The following definitions are provided to aid in understanding the subject matter regarded as the invention.

As used herein, "hepatitis C virus" or "HCV" shall mean any representative of a diverse group of related viruses classified within the hepacivirus genus of the Flaviviridae family.

"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. In discussing 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. With reference to nucleic acids of the invention, the term "isolated nucleic acid" is sometimes used. This term, when applied to DNA, 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. For example, an "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or WO 99/51781 PCT/US99/07404 virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.

When applied to RNA, the term "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 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", "mutants" and "derivatives" of particular sequences of nucleic acids 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. By closely related, it is meant that at least about 75%, but often, more than 90%, of the nucleotides of the sequence match over the defined length of the nucleic acid sequence referred to using a specific SEQ ID NO. 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. Other changes may be specifically designed and introduced into the sequence for specific purposes, such as to change. an amino acid codon or sequence in a regulatory region of the nucleic acid. Such specific changes may be made in vitro using WO 99/51781 PCT/US99/07404 a variety of mutagenesis techniques or produced in a host organism placed under particular selection conditions that induce or select for the changes. Such sequence variants generated specifically may be referred to as "mutants" or "derivatives" of the original sequence.

The terms "percent similarity", "percent identity" and "percent homology" when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program.

The term "NS5B" refers to a portion of the HCV genome located near the 3' end of the viral genome that specifies the region encoding a protein, termed the protein", "NS5B polypeptide", "NS5B polymerase" or combinations of these terms which are used interchangeably herein. NS5B in its natural state, functions as an RNA-dependent RNA polymerase (RdRp).

The nucleic acid region encoding the NS5B protein may also be referred to as the "NS5B gene". Thus, the term "NS5B" may refer to either a nucleic acid encoding the polypeptide, to an NS5B gene or to an 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 polypeptides. The NS5B nucleic acid, NS5B gene or protein referred to may be either functional or non-functional.

The present invention also includes active portions, fragments, derivatives and functional or non-functional mimetics of HCV NS5B polypeptides or WO 99/51781 PCT/US99/07404 proteins of the invention. An "active portion" of HCV polypeptide means a peptide that is less than the full length HCV NS5B polypeptide, but which retains measurable biological activity.

A "fragment" or "portion" of the HCV 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 HCV NS5B polypeptide sequence, antigenic determinants, viral antigens or epitopes are useful for eliciting immune responses to a portion of the HCV amino acid sequence.

A "derivative" of the HCV NS5B 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 or substitution of one or more amino acids, and may or may not alter the essential activity of original the HCV NS5B polypeptide. As mentioned above, the HCV NS5B polypeptide or protein of the invention includes any analogue, fragment, derivative or mutant which is derived from a HCV polypeptide and which retains at least one property or other characteristic of the HCV NS5B polypeptide.

Different "variants" of the HCV NS5B polypeptide exist in nature. These variants may be alleles characterized WO 99/51781 PCT/US99/07404 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. These variants may include inter alia: variants in which one or more amino acids residues are substituted with conservative or non-conservative amino acids, variants in which one or more amino acids are added to the HCV NS5B polypeptide, (c) variants in which one or more amino acids include a substituent group, and variants in which the HCV 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 HCV NS5B polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like. Other HCV NS5B 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.

To the extent such allelic variations, analogues, fragments, derivatives, mutants, and modifications, WO 99/51781 PCT/US99/07404 including alternative nucleic acid processing forms and alternative post-translational modification forms result in derivatives of the HCV NS5B polypeptide that retain any of the biological properties of the HCV polypeptide, they are included within the scope of this invention.

The term "functional" as used herein implies that the nucleic or amino acid sequence is functional for the recited assay or purpose.

The phrase "consisting essentially of" when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID No:. For example, 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, for example, a plasmid, cosmid, bacmid, phage or virus, that is capable of replication largely under its own control. 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 ATG or AUG codons), polyadenylation signals, terminators, and the WO 99/51781 PCT/US99/07404 like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.

The term "oligonucleotide," as used herein 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.

The term "probe" as used herein 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 WO 99/51781 PCT/US99/07404 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"). In particular, 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.

The term "primer" as used herein 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. When presented with an appropriate nucleic acid template, suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable WO 99/51781 PCT/US99/07404 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. For example, in diagnostic applications, 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 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. For example, a non-complementary nucleotide sequence may be attached to the 5' end of an otherwise complementary primer. Alternatively, 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 isomeric form. However, residues in the isomeric form may be substituted for any L-amino acid residue, provided the desired properties of the polypeptide are retained.

WO 99/51781 PCT/US99/07404 All amino-acid residue sequences represented herein conform to the conventional left-to-right amino-terminus to carboxy-terminus orientation.

The term "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.

Alternatively, 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.

The term "substantially pure" refers to a preparation comprising at least 50-60% by weight of a given material 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 chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like) "Mature protein" or "mature polypeptide" shall mean 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 WO 99/51781 PCT/US99/07404 protoelytic processing from a polyprotein precursor. In designating the sequence or boundaries of a mature protein, the first amino of the mature protein sequence is designated as amino acid residue 1. In the case of the mature NS5B protein, its normal biogenesis entails its proteolytic cleavage from a precursor polyprotein.

Thus, the first amino acid residue of the mature protein represents a serine residue for all known HCV sequences. As used herein, any amino acid residues associated with a mature protein not naturally found associated with that protein that precedes amino acid 1 are designated amino acid -3 and so on. For recombinant expression systems, a methionine initiator codon is often utilized for purposes of efficient translation. Thus, in the case of NS5B, a methionine codon may be place immediately proceeding the serine codon. This methionine residue in the resulting polypeptide, as used herein, would be positioned at -1 relative to the mature NS5B protein sequence.

The term "tag," "tag sequence" or "protein 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. Thus, for example, 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. In the case of protein tags, WO 99/51781 PCT/US99/07404 histidine residues 4 to 8 consecutive histidine residues) may be added to either the amino- or carboxy-terminus of a protein to facilitate protein isolation by chelating metal chromatography.

Alternatively, amino acid sequences, peptides, proteins or fusion partners representing epitopes or binding determinants reactive with specific antibody molecules or other molecules 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.

As used herein, the terms "reporter," "reporter system", "reporter gene," or "reporter gene product" shall mean 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, by biological assay, immunoassay, radioimmunoassay, or by colorimetric, fluorogenic, chemiluminescent or other methods. The nucleic acid may be either RNA or DNA, linear or WO 99/51781 PCT/US99/07404 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.

The terms "transform", "transfect", "transduce", shall refer 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, transfection, electroporation, microinjection, PEGfusion and the like.

The introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism. In bacterial, yeast, plant and mammalian cells, for example, the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid. Alternatively, 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. In other manners, the introduced nucleic acid may exist in the recipient cell or host organism only transiently.

A "clone" or "clonal cell population" is a WO 99/51781 PCT/US99/07404 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 vitro for many generations.

An "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 immunoglobulins 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 vitro 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.

A "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.

An "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. As used herein, antibody or antibody molecule contemplates both an intact WO 99/51781 PCT/US99/07404 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).

As used herein, the term "living host" shall mean any non-human autonomous being.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows an alignment of the amino acid sequences of several NS5B proteins of the invention.

The approximate positions of conserved sequence motifs present in RdRp enzymes identified by Koonin [J Gen Virol. (1991) 72:2197-206] (Roman numerals) and Poch et al. [EMBO J. (1989) 8:3867-74] (letters) are indicated above the clone 4 sequence.

Figure 2 shows the purification of the HCV protein. Sf9 cells infected with a recombinant baculovirus expressing the HCV NS5B gene were harvested and lysed. Cleared lysate was subjected to a series of protein purification steps. Samples for each step of the purification procedure were electrophoresed on an SDS-containing polyacrylamide gel, which was then silver-stained. Lane 1: cleared cell lysate; lane 2, flow-through material from DEAE column; lane 3, material from heparin column; lane 4, material from Cibracron blue column. M molecular mass standards; numbers in kilodaltons.

Figure 3 is an alignment of the clone 4 sequence of the invention with the genotype la consensus WO 99/51781 PCT/US99/07404 sequence derived from an infectious clone (GenBank Accession Number AF009606) and a genotype la sequence (PCT WO 97/12033) previously shown to have RdRp activity. The dashed lines indicate amino acid residue identity.

Figure 4 shows an alignment of the clone 4 sequence of the invention with a genotype Ib "consensus" amino acid sequence derived from sequences of HCV sequences obtained from the GenBank database and with genotype lb NS5B sequences previously shown to be functional (GenBank Accession Numbers M58335 and Z97730). The dashed lines indicate amino acid residue identity.

Figure 5 shows a protein sequence alignment of the HCV genotype lb NS5B gene reported by Yanagi et al.

[Virology (1998) 244:161-172; GenBank Accession Number AF054247] and clone 4 of the present invention. The dashed lines indicate amino acid residue identity.

Figure 6 shows results of a high throughput assay for HCV NS5B RdRp activity.

Figure 7 shows the sequence of SEQ ID NO: 1.

Figure 8 shows the sequence of SEQ ID NO: 3.

Figure 9 shows the sequence of SEQ ID NO: 4.

Figure 10 shows the sequence of SEQ ID NO: WO 99/51781 PCT/US99/07404 Figure 11 shows the sequence of SEQ ID NO: 6.

Figure 12 shows the sequence of SEQ ID NO: 7.

Figure 13 shows the amino acid sequence of clone 20(V-1M), SEQ ID NO: 13.

DETAILED DESCRIPTION OF THE INVENTION HCV is the major causative agent of transfusionassociated and sporadic non-A, non-B hepatitis. A high number of HCV-infected patients develop chronic hepatitis that eventually leads to cirrhosis and often progresses to hepatocellular carcinoma. There is an urgent need for new effective treatments of this disease.

HCV is an enveloped virus with a stranded linear RNA genome of approximately 9.4 kilobases. This nucleic acid encodes a large polyprotein that is processed by viral and cellular proteases into at least 9 different viral polypeptides.

In one of its aspects, the present invention provides HCV genetic material derived from an HCVinfected patient. The invention further provides HCV recombinant NS5B proteins expressed from these sequences and sequences that have RdRp activity.

Expression of recombinant HCV NS5B gene sequences may be carried out in a variety of systems including but not limited to bacterial, yeast, mammalian, insect and plant cell systems, as well as in organisms such as infected, transfected, transduced or transgenic insects, animals or plants. In one embodiment of the invention, WO 99/51781 PCT/US99/07404 recombinant baculoviruses were constructed to express HCV NS5B gene sequences in insect cells following infection in culture.

RNA extracted from the serum of a patient infected with HCV was subjected to a reverse transcriptase-nested polymerase chain reaction procedure using primer oligonucleotides designed to amplify the NS5B gene sequences. Primers for the nested PCR reaction allowed direct cloning of the NS5B gene into a baculovirus expression transfer vector, providing an initiator methonine codon immediately preceeding the first amino acid of the authentic NS5B coding sequence. The gene from several transfer vector clones was sequenced and used to generate recombinant baculoviruses.

Infection of Sf9 cells with these viruses, followed by Western immunoblotting with HCV NS5B sequence-specific antiserum, showed clear production of the 68 kilodalton protein.

In another aspect of the invention, NS5B sequences that are expressed, purified and evaluated for RdRp activity may possess varying levels of RdRp activity, from highly active to little or no activity, and may thus represent functional, poorly functional or non-functional sequences, respectively. Such sequences would also be assessed for activity in accordance with the present invention.

In a further aspect of the invention, HCV proteins may be modified by particular changes in nucleotide and amino acid sequence that result in RdRp enzymes with altered functionality. Such changes may be subtle and represent conservative substitutions such as WO 99/51781 PCT/US99/07404 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.

In yet a further aspect of the invention, 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 HCV disease.

Based on the discoveries described herein, it is demonstrated that simple knowledge of, or mere inspection of, a nucleotide or amino acid sequence of an gene or protein is insufficient to enable one to deduce or predict that the particular NS5B sequence represents a sequence that has enzymatic activity or functional utility.

Preparation of HCV NS5B Nucleic Acid Molecules and HCV Proteins and Uses Thereof in Assay Methods and Kits A. Nucleic Acid Molecules Nucleic acid molecules encoding the HCV proteins of the invention may be prepared by two general methods: They may be synthesized from appropriate chemical starting materials, or they may be isolated from biological sources. Both methods utilize protocols well known in the art.

The availability of nucleotide sequence WO 99/51781 PCT/US99/07404 information, such as that provided herein for HCV 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). Long, double-stranded polynucleotides, such as a DNA molecule of the present invention, must be synthesized in stages due to the size limitations inherent in current oligonucleotide synthetic methods. Thus, for example, 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 HCV NS5B proteins may be isolated from appropriate biological sources using methods known in the art. For example, RNA isolated from the serum of an HCV infected patient may be used as a suitable starting material for the generation of cDNA molecules encoding HCV NS5B proteins.

In accordance with the present invention, nucleic acids having the appropriate level of sequence homology WO 99/51781 PCT/US99/07404 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. For example, hybridizations may be performed, using a hybridization solution comprising, for example, 5X SSC, 5X Denhardt's reagent, 1.0% SDS, 100 pg/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide.

Hybridization is carried out at 37-42 0 C for at least six hours. Following hybridization, filters are washed as follows: 5 minutes at room temperature in 2X SSC and 1% SDS; 15 minutes at room temperature in 2X SSC and 0.1% SDS; 30 minutes-i hour at 37 0 C in IX SSC and 1% SDS; 2 hours at 42-65 0 C in 1X SSC and 1% SDS, changing the solution every 30 minutes.

One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology is as follows (Sambrook et al., 1989): Tm 81.5 0 C 16.6Log 0.41(% G+C) 0.63 formamide) 600/#bp in duplex As an illustration of the above formula, using [0.368] and 50% formamide, with GC content of 42% and an average probe size of 200 bases, the Tm is 57 0 C. The T, of a DNA duplex decreases by 1-1.5 0 C with every 1% decrease in homology. Thus, targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42 0 C. Such a sequence would be considered substantially homologous to the sequences of the present invention.

WO 99/51781 PCT/US99/07404 Nucleic acids of the invention may be maintained as DNA in any convenient cloning vector. In one embodiment, 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.

The 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 HCV NS5B proteins with altered characteristics such as increased enzymatic activity.

HCV NS5B 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. Thus, 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 HCV encoding nucleic acids.

WO 99/51781 PCT/US99/07404 B. Proteins HCV NS5B proteins of the present invention may be prepared in a variety of ways, according to any number of known methods. The protein may be purified from appropriate sources, cultured cells, tissues or organs, by a variety of techniques that may include partitioning and precipitation procedures, affinity purification methods, conventional chromatography procedures, high performance chromatography techniques and the like.

The availability of nucleic acids molecules encoding HCV NS5B protein enables production of the protein using in vitro expression methods known in the art. For example, a cDNA or gene may be cloned into an appropriate in vitro transcription vector, such as pSP64 or pSP65 for in vitro RNA synthesis, followed by cell-free translation of the RNA in a suitable cell-free translation system, such as extracts of wheat germ, rabbit reticulocytes or HeLa cells. In vitro transcription and translation systems are commercially available Promega Biotech, Madison, WI; Gibco-BRL, Gaithersburg, MD) Alternatively, according to a preferred embodiment of the invention, larger quantities of HCV NS5B protein may be produced by expression in suitable prokaryotic or eukaryotic systems such as bacterial, fungal, mammalian or plant systems. For example, part or all of a DNA molecule, such as a cDNA 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 WO 99/51781 PCT/US99/07404 elements necessary for expression of the DNA in the host cell E. coli or insect cell), positioned in such a manner as to permit expression of the DNA in the host cell. Such regulatory elements required for expression may include promoter sequences, transcriptional initiation and termination sequences, enhancer sequences, translational control sequences and the like.

The HCV NS5B proteins or derivatives thereof produced by gene expression in a recombinant prokaryotic or eukaryotic system may be purified according to methods known in the art. In one embodiment, 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. If expression/secretion vectors are not used, an alternative approach involves purifying the recombinant protein from extracts of expressing cells, tissues or organs by standard protein purification techniques or by affinity separation techniques, such as by immunological interaction with antibodies that bind specifically to the recombinant protein or by nickel columns for isolation of recombinant proteins tagged with 5-8 histidine residues at their N-terminus or C-terminus.

Such methods are commonly used by skilled practitioners.

The HCV NS5B proteins of the invention, prepared by the aforementioned methods, may be analyzed according to standard procedures. For example, such proteins may be subjected to electrophoretic analyses and to amino acid sequence analyses, as well as to crystallographic analyses for structure determination according to known WO 99/51781 PCT/US99/07404 methods. Such analyses provide useful information regarding the functionality of the NS5B protein and on means to affect that functionality, such as in the design of molecules that may inhibit the function of the NS5B protein.

C. Assay Methods and Kits The HCV NS5B sequences of the invention may be used in a variety of ways having utility in research, diagnostic, therapeutic and pharmaceutical applications.

Representative methods of use for the compositions of the invention are described below.

In one aspect, the nucleic acid sequences of the invention, and sequences complementary to these, may be used as probes or primers for the detection, labeling, identification or isolation of related nucleic acids in biological or synthetic preparations. For example, nucleic acid sequences of the invention may be used as hybridization probes to detect the presence of HCV 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.

Additionally, the nucleic acid sequences of the invention may be used as primers for the detection or isolation of HCV 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. Such primers, primer pairs and probes may represent any WO 99/51781 PCT/US99/07404 portion of the NS5B sequences of the invention. The actual sequence of the NS5B gene used will vary according to the specific application. Moreover, additional sequences may be added to the HCV 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. Typically, 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. Alternatively, the entire NS5B sequence may be employed as a capture hybridization probe, for example.

Several examples of useful oligonucleotide primers and probes of the invention comprise sequences set forth in Table 1, sequences complementary to these and potions of these sequences.

Table 1 Oligonucleotides useful as probes and primers 5'-TCAATGTCCTACACATGGAC-3' SEQ ID NO: 14 5'-CTACACATGG-3' SEQ ID NO: 5'-CTCTGATTACACCATGCGCTGCGGAGGAGAGCAAGCTGCCC-3' SEQ ID NO: 16 5'-AATGCGCTGAGCAACTCTTTGCTGCGCC-3' SEQ ID NO: 17 '-CCATAACATGGTCTATGCCACAACATCCCGCAGCGCAAGCCAGCGGC-3' SEQ ID NO: 18 -GAAGAAGGTAACTTTGACAGG-3' SEQ ID NO: 19 '-CAAGTCCTGGATGACCACTACCG-3' SEQ ID NO: 5'-GACGTGCTCAAGGA-3' SEQ ID NO: 21 5'-ATGAAGGCGAAGGCGTCC-3' SEQ ID NO: 22 5'-GGAAGAAGCCTGTAAG-3' SEQ ID NO: 23 -GAACCTATCCAGCAAGGCCGTTAA-3' SEQ ID NO: 24 WO 99/51781 5'-ACCAGAGAAAGGAGGCCGC-3' 5'-ACCCAGACTTGGGG-3' '-TCTCCACCCTTCCTCAGGCT-3' 5'-CGAGTTCCTGGTGAATGCC-3' 5'-TGCCCTATGGGCTTCGCATATGAC-3' 5'-TTTCGACTCAACGGTCACCGAGAAT-3' 5'-GTTGAGGAGTCAATT-3' 5'-TTGGCCCCCGAAGCCAGACA-3' 5'-AAGGTCGCTTACAGAGC-3' 5'-ATCGGGGGTCCCCTGAC-3' 5'-TAACTCAAAAGGGCAGAG-3' 5'-ATGTTACTTGAAGGCCTCT-3' 5'-GATGCTTGTGTGCGGAGACGACCTC-3' 5'-GGTCGCGCACGATGCATCTGGCAAAAGGGTA-3' 5'-CACCACCCCTCTTGCGCGG-3' 5'-CTCCATCCTTCTAGCTCAGGAGCAA-3' '-AGTTACTGTCCCAGGGGGGG-3' 5'-TCCGGCTGCGTCCCAGT-3' 5'-CCGACCCCGCTGGTTCATGTGGTGCC-3' 5'-CTACCTGCTCCCGAACCGA-3' 5'-CTACCTGCTCCCCAACCGA-3' PCT/US99/07404 SEQ ID NO: SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 28 SEQ ID NO: 29 SEQ ID NO: SEQ ID NO: 31 SEQ ID NO: 32 SEQ ID NO: 33 SEQ ID NO: 34 SEQ ID NO: SEQ ID NO: 36 SEQ ID NO: 37 SEQ ID NO: 38 SEQ ID NO: 39 SEQ ID NO: SEQ ID NO: 41 SEQ ID NO: 42 SEQ ID NO: 43 SEQ ID NO: 44 SEQ ID NO: Additionally, the 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.

In another aspect, the 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 functionally superior NS5B sequences of the invention for poorly functional or non-functional counterparts will serve to improve the infectious and replicative WO 99/51781 PCT/US99/07404 characteristics of the resulting virus or replicating unit. For example, Rice et al. (PCT WO 98/39031) provide an infectious nucleic acid of HCV. The polymerase encoded by this infectious clone is shown in the present invention to be substantially inferior in its RdRp activity to those of the present invention.

Therefore, substitution of the NS5B encoding gene of Rice et al. with those of the present invention would be expected to result in improved virus replication due to the improved characteristics of the NS5B gene and encoded protein of the present invention. 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 of HCV, 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 vitro systems.

Use in other systems in which the function of the HCV polymerase 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.

In another embodiment, the nucleic acid sequences of the invention may be used in methods to elicit immune responses to the HCV NS5B protein. For example, the nucleic acid sequences of the invention operationally linked to an expression operon may introduced directly into cells, particularly into WO 99/51781 PCT/US99/07404 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 NS5B nucleic acid sequences, such as plasmid-coated gold particles. Once inside cells, the NS5B 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 HCV.

The nucleic acid sequences of the invention may be further used in the generation of cell lines or cellular systems that express the HCV NS5B protein. Such cell lines in which a functional NS5B protein is expressed from the NS5B genes of the invention will have utility in methods for assaying materials for antagonistic or agonistic activity toward HCV. For example, assays may be established whereby intact cells expressing an HCV NS5B protein of the invention are contacted with agents or materials suspected of affecting the intracellular activity of the HCV NS5B protein, and the affect of such agents on the HCV NS5B activity is measured. The affect of such agents on the HCV NS5B activity may be measured in any number of ways. For example, RNA synthesis that is directly or indirectly dependent on the HCV polymerase activity may be quantified. In one manner, the amount of a radiolabeled precursor of RNA 3

H-

uridine) that is incorporated into trichloroacetic acidprecipitable RNA that is dependent on NS5B activity may be measured.

WO 99/51781 PCT/US99/07404 Alternatively, such cell systems may utilize a reporter system in which the production of the reporter signal is dependent on RNA synthesis by the HCV polymerase. In one embodiment, a RNA substrate of the HCV NS5B 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). For example, an RNA molecule is provided comprising the sequence complementary to the coding sequence of luciferase (antisense strand). The activity of the HCV NS5B 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, pgalactosidase, alkaline phosphatase, fluorescent green protein and the like. Furthermore, the cell systems that may be used in this method of the invention may be of bacterial, fungal, insect, avian, mammalian or plant origin.

Further, the nucleic acid sequences of the invention may be used in assays to identify agents or materials capable of interacting with or affecting the HCV NS5B nucleic acid sequences. For example, assays WO 99/51781 PCT/US99/07404 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 HCV in, for example, biological samples. Such agents would also have potential utility in applications involving the prevention or treatment of HCV disease in an affected living host, including humans, and for the inhibition or enhancement of HCV replication or propagation in living hosts and in in vitro systems such as cell, tissue and organ cultures.

Additional applications may be envisioned once the nature of the particular agent is clear.

The HCV NS5B protein compositions of the invention also have broad utility. In diagnostic applications, for example, the NS5B proteins, or peptides thereof, may be used in assays for the detection of immune responses to the same. For example, 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.

Additionally, 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 HCV NS5B protein compositions of the invention also have potential utility in the elicitation of immune WO 99/51781 PCT/US99/07404 responses, such as in vaccines. For example, provision of the NS5B 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 NS5B sequences. The NS5B sequences may be presented to the living organism in any number of assays well known to those trained in the art and include, but are not limited to, providing free protein or peptides, formulated protein or peptides, adjuvanted protein or peptides, protein or peptides in the context of intact or disrupted cells in which NS5B sequences are present and other such manners. Immune responses so elicited may be either humoral or cellular in nature, or both. Such immune responses may be important in protecting living hosts from HCV disease and may also provide or serve as a source of useful immunological reagents such as antibodies, that may further have therapeutic or diagnostic utility. The NS5B proteins of the invention, or peptide portions thereof, may further be used to select or purify such antibodies to For example, NS5B protein may be immobilized and used to bind antibodies specific to the NS5B protein, and thus enrich for such antibodies. Furthermore, the proteins and peptides of the invention may be used to produce monoclonal antibodies to the NS5B protein using standard techniques known in the art. Antibodies to the protein, whether polyclonal or monoclonal, may be further evaluated for their ability to affect the WO 99/51781 WO 99/178 1PCT/US99/07404 enzymatic activity of the NS5B RdRp activity.

Several examples of portions of the NS5B protein sequences useful in the above applications comprise sequences or portions thereof, provided in Table 2.

Table 2 Peptides Useful in Methods of the Invention SMSYTWTGALITPCAAEESKLP INALSNSLLRHHNMVYATTSRSASQRQK KVTFDRLQVLDDHYRDVLKEMKAKASTVKA SEQ ID NO: 46 SVEEACKLTPPHSARSKFGYGAKVRNLSSKAVN{IHSVWKDLLEDT SEQ ID NO: 47 ETPIDTTIMAKNEVFCVQPEKGGRKPARLIVYPDLGVRVC SEQ ID NO: 48

SSYGFQYSPGQRVEFLVNAWKSKKCPMGFAYDTRCFDSTVTENDIRVEESIY

QCCDLAPEARQAIRSLTERLYIGGPLTNSKGQSCGYRRcRASGVLTTScG SEQ ID NO: 49

CTMLVCGDDLVVICESAGTQEDAASLRVFTEAMTRYSAPPGDPPQPEYDLELITSC

SEQ ID NO: SVAHDASGKRVYYLTRDPTTPLARAAWETARHTPVNS SEQ ID NO: 51 CLRKILGVPPLRVWRHRARSVRAKLLSQGG SEQ ID NO: 52 FVAGYSGGDIYHSLSRARPRWFMWC SEQ ID NO: 53 The protein compositions of the invention have utility in assays for the detection and identification of agents capable of interacting with or affecting the HCV NS5B protein. Assays may be established in which HCV NS5B polypeptide sequences of the invention are provided and then contacted with agents or materials suspected of interacting with such sequences. For example, upon provision of the HCV NS5B3 protein of the inventi on, or fragment or portion thereof, contacted WO 99/51781 PCT/US99/07404 agents may be assessed for their ability to bind specifically to the protein. Such binding agents would then have potential diagnostic utility and uses in the detection of HCV in, for example, biological samples.

Such binding agents may further affect the functional activity of the HCV protein, such as either inhibiting or enhancing the HCV NS5B function. Agents that inhibit the function of the NS5B protein would have potential utility in applications involving the prevention or treatment of HCV disease in an affected living host or for the inhibition of HCV replication or propagation in living hosts, including humans, in in vitro systems such as cell, tissue and organ cultures or in biological materials. Agents that enhance the function of the protein would have potential utility in applications involving the replication, propagation or production of HCV in living hosts, such as in animal models of HCV replication, and in in vitro systems such as cell, tissue and organ cultures.

In another embodiment, methods of assay are provided in which the HCV NS5B polymerase activity furnished by an enzymatically active NS5B protein of the invention is measured directly. Agents placed in contact with said enzymatically active NS5B polymerase may be assessed for their ability to specifically affect 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 WO 99/51781 PCT/US99/07404 vitro cell-free expression system or in an enriched or purified form.

There are numerous means by which the enzymatic activity of the HCV NS5B protein provided in an extract, cell-free system or enriched form may be assessed, and these are well known in the art. dependent RNA synthesis typically requires certain reaction components including minimally a buffered medium, a divalent cation, precursors of RNA (nucleoside triphosphates, NTPs), an RNA template and a primer for RNA synthesis on that template. Additional components may include monovalent cations, reducing agents, stabilizers, cofactors and inhibitors of activities unrelated to the NS5B RdRp activity such as inhibitors of RNase, phosphatase, kinase, phosphotransferase and similar activities.

Measurement of NS5B-dependent RNA synthesis may be assessed in numerous manners. In one example, 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 WO 99/51781 PCT/US99/07404 detection using fluorescent technologies such as fluorescence polarization.

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.

Alternatively, the product of NS5B RdRp activity may be detected and quantified by capture of the product RNA using hybridization techniques. For example, an oligonucleotide complementary to the product of the 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.

Other means -of detection of the products of 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 WO 99/51781 PCT/US99/07404 such high throughput assay systems. These systems are often automated using robotics technologies to allow manipulation and processing of large numbers of samples.

The 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, immunoglobulins or fragments thereof, oligonucleotides, antisense molecules, peptidenucleic acid conjugates, ribozymes, polynucleotides and the like. The potential utility of 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 HCV nucleic acids and polypeptides, for the detection or diagnosis of HCV, for the prevention and treatment of HCV disease in an affected living host, including humans, and for the inhibition or enhancement of HCV replication or propagation in living hosts and in in vitro 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.

Another feature of the invention includes kits to facilitate the use of the compositions and methods disclosed herein. Exemplary kits would include HCV nucleic acids and polypeptides of the invention, and/or variants thereof, alone or in suitable vectors. Also included would be protocols for use of the compositions of the invention for the particular application and the necessary reagents to carry out the application. Such reagents may include, but not be limited to, buffers, WO 99/51781 PCT/US99/07404 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.

The following examples are provided to describe the invention in further detail. These examples, which set forth the preferred mode presently contemplated for carrying out the invention, are intended to illustrate and not to limit the invention.

EXAMPLE 1 Cloning and Expression of HCV NS5B Genes RNA isolated from the serum of an HCV-infected patient was used to amplify the HCV NS5B gene using an RT-nested PCR protocol. Using the following first round primers: 5'-TGA GGA TGT CGT CTG CTG CTC AAT GTC C-3' and 5'-GGG ATG GCC TAT TGG CCT GGA GT-3', the RT reaction was performed at 50 0 C for 50 minutes followed by a 1 minute 94 0 C denaturation, followed by 40 cycles of first round PCR, 15 cycles of 94 0 C for 30 seconds, 0 C for 30 seconds 68 0 C for 2 minutes and 25 cycles of 94 0 C for 30 seconds, 50 0 C for 30 seconds, 68 0 C for 2 minutes.

A portion of first round PCR reaction mixture was then used in the second round PCR reaction in which the following nested primers were incorporated: 5'-AAC AGA TCT GAA TTC TTA TAA ATA TGT CAA TGT CCT ACA CAT GGA C-3' and 5'-TGC TCT AGA GCG GCC GCT CAT CAT CGG TTG GGG AGC AGG TAG-3', which included EcoRI and NotI restriction WO 99/51781 PCT/US99/07404 sites, respectively (underlined) for subsequent cloning purposes. The second round PCR involved an initial denaturation at 94 0 C for 1 minute, followed by 10 cycles of 94 0 C for 45 seconds, 50 0 C for 30 seconds, 68 0 C for 2 minutes and 20 cycles of 94 0 C for 30 seconds, 50 0 C for seconds, 68 0 C for 2 minutes.

The resultant PCR product was purified, digested with EcoRI and NotI and ligated to pFastBac plasmid (Gibco-BRL) previously digested with EcoRI and NotI.

The ligation mixture was transformed into DH5 E. coli cells. Plasmids from bacterial colonies containing the HCV NS5B gene were used to generate a recombinant baculovirus by the transposition method of the Bac-to-Bac system (Gibco-BRL). After transformation of the HCV NS5B gene-containing pFastBac plasmid DNA into E. coli DH1OBac cells, several colonies containing bacmid DNA were transfected into Sf9 insect cells according to the protocol supplied by the manufacturer.

protein expression in recombinant baculovirus-infected cells was verified by Western immunoblot analysis with antiserum specific to the HCV sequences.

Six independently isolated clones (clones 4, 14, 21, 11, 16 and 20) of the HCV NS5B gene from a single RNA preparation derived from one patient were sequenced.

The nucleotide sequences of these clones are provided by SEQ ID NO: 1, 3, 4, 5, 6 and 7, respectively. All six sequences were derived from a genotype lb virus and are closely related, but not identical, to one another.

Each of the sequences set forth above is unique and novel sequence, not presently listed in the GenBank WO 99/51781 PCT/US99/07404 database. The deduced amino acid sequences of nucleotide sequences identified by SEQ ID NO: 1, 3, 4, 6 and 7 are provided by SEQ ID NO: 2, 8, 9, 10, 11 and 12, respectively. These NS5B amino acid sequences are aligned relative to clone 4 (SEQ ID NO: 2) in Figure 1. As revealed in this alignment, the sequence of the protein of clones 14, 21, 11, 16 and 20 differs from that of clone 4 by 2, 5, 3, 4 and 3 amino acids, respectively.

EXAMPLE 2 Purification of HCV NS5B Proteins HCV NS5B proteins may be obtained in purified form from recombinant protein-expressing cell systems by any number of procedures known to the trained artisan, several of which are exemplified in Current Protocols in Molecular Biology, Frederick M. Ausubel et al. eds., John Wiley Sons, 1995 which is incorporated by reference herein.

In the case of the present example, baculovirus clone 4-infected insect cells were disrupted in lysis buffer (50% glycerol, 20 mM Tris-HCl, pH 7.5, 10 mM dithiothreitol (DTT), 0.5 M NaCI, 1 mM EDTA, 2% Triton X-100) supplemented with a Complete Protease Inhibitor tablet (Boehringer Mannheim). MgC 2 1 was then added to a final concentration of 10 mM, followed by 10 units DNase I (RQ-1, Promega). After 30 minutes on ice, the lysate WO 99/51781 PCT/US99/07404 was clarified by centrifugation at 35,000 rpm for minutes at 4 0

C.

The clarified lysate was diluted to a final NaCI concentration of 0.3 M with elution buffer glycerol, 20 mM Tris-HCl, pH 7.5, 10 mM DTT, 1 mM EDTA, Triton X-100), and incubated with DEAE Sepharose equilibrated in elution buffer containing 0.3 M NaCl at 4°C. The mixture was then poured into a column, and flow-through material was collected. Flow-through material was diluted to a final NaCi concentration of 0.2 M with elution buffer and loaded onto a heparin Sepharose column equilibrated in elution buffer containing 0.2 M NaCl. Bound proteins were eluted with a linear gradient of NaCl (200 mM to 1 M NaCl in elution buffer) while fractions were collected.

fractions were pooled and loaded onto a Cibacron Blue column equilibrated in elution buffer containing 0.4 M NaCi. Bound proteins were eluted with a linear gradient of NaCi (400 mM to 4M NaCl in elution buffer) while fractions were collected.

fractions were pooled and dialyzed against 50% glycerol, 10 mM Tris-HCl, pH 7.2, 50 mM NaCl, 1 mM EDTA, 0.01% Triton X-100 and stored at -20 0

C.

Typical purification results are shown in Figure 2.

EXAMPLE 3 Enzymatic Activity of Purified HCV NS5B Proteins There are numerous methodologies for the measurement of RNA-dependent RNA polymerase activity WO 99/51781 PCT/US99/07404 that are well known to one of ordinary skill in the art.

One approach for measuring the HCV NS5B RdRp activity uses a purified recombinant NS5B protein in an in vitro RdRp assay. For example, Behrens et al. [EMBO J. 15:12-22 (1996)] describe the baculovirus expression, purification and enzymatic activity of the HCV NS5B RdRp derived from the BK strain of HCV. In another example, PCT WO 97/12033 [PCT/US96/15571] discloses the bacterial expression, purification and enzymatic activity of the HCV NS5B RdRp derived from an HCV sample obtained from the Centers for Disease Control and several truncated and modified versions of this sequence. Another example is Lohmann et al. [J Virol 71:8416-8428 (1997)], in which the NS5B gene derived from a chronically infected patient was expressed with recombinant baculoviruses in insect cells, purified and enzymatically evaluated. Yet another example is Yuan et al. [Biochem Biophys Res Comm 232:231-235 (1997)], in which the bacterial expression, purification and enzymatic activity of the HCV NS5B RdRp derived from a patient with chronic sporadic hepatitis is described.

Purified NS5B protein prepared according to Example 2 was incubated in a 10 pL standard reaction mixture consisting of 20 mM HEPES, pH 7.5, 3 mM MgCl 2 1 mM dithiothreitol, 400U/mL RNasin (Gibco/BRL), 0.5 mM each of UTP, ATP and CTP, 0.1 M 32 P]GTP and 0.03 4g pOF1213 RNA at 30 0 C for 60 minutes. The reaction is terminated by the addition of cold trichloroacetic acid (TCA) and sodium pyrophosphate. The TCA-precipitable WO 99/51781 PCT/US99/07404 radioactivity was then quantified to determine the extent of RdRp activity.

In addition to the NS5B proteins derived from clones 4, 14, 21, 11, 16 and 20 of the invention, the NS5B gene of the sequence derived from the genotype la consensus sequence infectious clone as described by Kolykhalov et al. (GenBank Accession Number AF009606) was expressed and purified in the same manner as the proteins of the invention. Table 3 presents a direct comparison of the enzymatic activities of the proteins of the invention and the consensus sequence protein of Kolykhalov.

Table 3 Comparison of RdRp Activities of Various HCV Proteins specific amino acid changes Protein activity* relative to clone 4 clone 4 (SEQ ID NO: 2) 9.3 clone 14 (SEQ ID NO: 8) 1.7 2 clone 21 (SEQ ID NO: 9) 1.9 clone 11 (SEQ ID NO: 10) 0.2 3 clone 16 (SEQ ID NO: 11) 0.1 4 clone 20 (SEQ ID NO: 12) <0.1 3 AF009606 0.9 71 pmole NTP incorporated per gg NS5B protein per 60 minutes under standard reaction conditions.

Surprisingly, a considerable range of enzymatic activities was observed among these distinct proteins. The NS5B protein derived from clone 4 possessed the greatest activity in this comparison, while the activities of clone 14 and 21 were moderate, those of clone 11 and 16 were minimal and that of clone was undetectable. The activity of the genotype la consensus NS5B protein of Kolykhalov was substantially less than that clone 4 of the invention. Lohmann et al.

WO 99/51781 PCT/US99/07404 reported a specific activity of their genotype lb protein on a heteropolymeric RNA template to be 1.7 pmol/,g/120 minutes, similar to the activity reported here for the Kolykhalov enzyme, but also significantly below that of the clone 4 enzyme.

This example demonstrates a range of functional activities among cloned and expressed NS5B sequences, from very active to inactive. In this analysis of 6 proteins of the invention, there are three instances (clones 11, 16 and 20) of NS5B proteins that are poorly functional or non-functional and three instances (clones 4, 14 and 21) of NS5B proteins that are functional.

It is further revealed here that surprisingly very few changes in amino acid sequence can be sufficient to dramatically alter the enzymatic activity of the protein. While sequence changes within known or predicted active sites or conserved sequence motifs of an enzyme might be expected to effect an enzyme's activity, most amino acid changes among the 6 sequences of this comparison lie outside consensus motifs of RdRp enzyme identified by Koonin Gen.

Virol. 72:2197-2206 (1991)] and Poch et al. [EMBO J.

8:3867-3874 (1989)]. The two exceptions to this are as follows. Clone 16 possesses a single residue change within motif III and VI, and clone 21 possesses a single conservative amino acid change within motif VI (Figure This notwithstanding, the effect of these amino acid changes, and of the others among the NS5B sequences of this example, on the RdRp activity of the protein is surprising and could not have been WO 99/51781 PCT/US99/07404 anticipated from prior art.

The data presented in this example also demonstrate that the NS5B RdRp activity derived from clone 4 has dramatically superior activity over that of the derived from the infectious clone identified by GenBank Accession Number AF009606. This result indicates that the mere generation of a consensus sequence of an protein does not necessarily provide an NS5B protein with optimal functionality.

EXAMPLE 4 HCV NS5B Sequence Alignments The NS5B sequences of the invention are distinct from NS5B sequences previously characterized as functional. This is demonstrated by the sequence alignments presented in Figures 3, 4 and The sequence disclosed by Kolykhalov et al.

(GenBank Accession Number AF009606) represents a consensus sequence of known NS5B sequences derived from genotype la HCV. Figure 3 shows an alignment of the amino acid sequence of clone 4 (SEQ ID NO: 2) of the invention with that of this genotype la consensus sequence. There are 71 amino acid differences between these two sequences (excluding the initiator methionine residue in clone This demonstrates that the clone 4 sequence of the invention is distinct from the genotype la consensus sequence. Alignment of this genotype la consensus sequence with all other sequences of the invention similarly demonstrates the distinct WO 99/51781 PCT/US99/07404 nature of the sequences of the invention. Also included in the alignment in Figure 3 is the genotype la sequence disclosed in PCT WO 97/12033, the NS5B protein from which was demonstrated to possess RdRp activity.

Inspection of this alignment reveals 72 amino acid differences between the sequences, again demonstrating the unique nature of the sequences of the invention.

The sequences of the invention are also distinct from a genotype lb consensus sequence. A genotype lb consensus sequence was generated based on alignment of 29 genotype lb NS5B sequences found in the GenBank database. Comparison of this consensus sequence with that of the clone 4 (SEQ ID NO: 2) of the invention shows that these sequences are distinct (Figure 4) After the initiator methionine (at position the clone 4 sequence differs from this genotype Ib consensus sequence by 13 amino acids. Alignment of this genotype Ib consensus sequence with all other sequences of the invention similarly demonstrates the distinct nature of the sequences of the invention. Also included in the alignment in Figure 4 are the genotype lb sequences disclosed by Behrens et al. (GenBank Accession Number M58335) and by Lohmann et al. (GenBank Accession Number Z97730). The NS5B proteins encoded by these sequences have been previously demonstrated to possess RdRp activity. Clone 4 is again distinct in that it possesses 17 amino acid changes relative to M58335 and 12 changes relative to Z97730.

The sequences of the invention are further distinct from a genotype lb sequence derived from an infectious clone [Yanagi et al. Virology 244:161-172 (1998); WO 99/51781 PCT/US99/07404 GenBank Accession Number AF054247]. As shown in alignment presented in Figure 5, the clone 4 sequence (SEQ ID NO: 2) differs by 25 amino acid residues, excluding the methionine residue at position D1 in clone 4.

These alignments reveal the unique nature of sequences of the invention relative to these genotype lb sequences.

Therefore, based on comparisons with both genotype la and genotype lb consensus sequences, the sequences of the invention do not represent a consensus sequence. Thus, based on the consensus sequence method for deducing sequence functionality, it would not be apparent that clone 4 is a functional RdRp or a functionally superior RdRp. Moreover, based on comparisons with NS5B sequences previously demonstrated to have functional RdRp activity, the sequence of clone 4 and of the other sequences of the invention are distinct from these RdRp sequences, and again, would not a priori be assumed to possess RdRp activity, and further not suspected of possessing superior RdRp activity.

EXAMPLE HCV NS5B Proteins with Improved Activity Based on sequence comparisons among NS5B sequences of the invention, a number of modified NS5B sequences were constructed in which particular amino acids were changed. These modified sequences were generated by WO 99/51781 PCT/US99/07404 standard site-specific mutagenesis procedures [Picard et al. Nucleic Acid Res. 22:2587-2591 (1994)]. Modified genes were engineered in the baculovirus expression system as described in Example 1, purified according to Example 2 and evaluated for RdRp activity according to Example 4.

The sequence of clone 20 differs from that of clone 4 at three amino acid residues. The presence of a valine residue at position -1 of the clone 20 protein instead of an initiator methionine suggests that the NS5B protein produced from clone 20 is initiated at the methionine at position 2, and further, that protein produced from this methionine residue is inactive. The V change in clone 20 is likely an artifact of molecular cloning procedures (mistaken sequence in a primer oligonucleotide). The V residue position -1 in clone 20 was changed to M, yielding clone 20(V-1M), identified by SEQ ID NO: 13, which now differs from the sequence of clone 20 by one amino acid (at residue and from clone 4 by two amino acids (at residues 177 and 543). The RdRp activity of the purified NS5B protein derived from clone 20(V-1M) was dramatically increased over that of clone 20 and significantly improved over that of clone 4 (Table 4).

Additional residue changes among the NS5B sequences of the invention can lead to useful and functional proteins. For example, when the alanine residue at position 75 in clone 4 is changed to valine the residue found at this position in clone 14, the resulting clone, clone (A75V), shows considerable RdRp activity (Table In another example, the asparagine WO 99/51781 PCT/US99/07404 residue in clone 4 at position 177 was changed to aspartic acid the residue found at this position in all other clones of the invention, yielding clone 4(N177D). This clone showed very good RdRp activity (Table In a third example, when the serine at position 543 in clone 4 is changed to proline as is found in clone 20, yielding clone 4(S543P), a functional RdRp is generated (Table 4).

Table 4 RdRp Activities of Various HCV NS5B Proteins Protein# specific activity clone 20 (SEQ ID NO: 12) 0.1 clone 20(V-1M) (SEQ ID NO: 13) 23.5 clone 4 (SEQ ID NO: 2) 9.3 clone 4(A75V) (SEQ ID NO: 54) clone 4(N177D) (SEQ ID NO: 55) 7.2 clone 4(S543P) (SEQ ID NO: 56) pmole NTP incorporated per 1tg NS5B protein per 60 minutes under standard reaction conditions.

All of the changes in table 4 may also be made to the other encoding amino acid sequences set forth herein i.e. SEQ ID NOS: 8, 9, 10, 11, 12, 13. and thus are contemplated to be within the scope of the invention.

This example demonstrates that amino acid residue changes may be made in the sequence of the NS5B protein and that such changes may maintain the functional activity of the resulting protein. Moreover, the amino acid substitutions may be either conservative or nonconservative in nature.

Additional changes of the amino acid residues among the NS5B sequences of the invention in various combinations are contemplated to be within the scope of the invention.

WO 99/51781 PCT/US99/07404 EXAMPLE 6 Novel HCV NS5B Proteins with Improved Activity Another strategy for investigating functional sequences involves the introduction of unique amino acid substitutions at positions where amino acid residues are conserved in known NS5B sequences. For example, all known NS5B sequences have as the amino terminal amino acid (position 1) a serine residue. Several modified versions of clone 4 were constructed in which this serine was substituted with either alanine [clone 4(S1A)], glycine [clone 4(S1G)] or threonine [clone 4(SIT)]. Surprisingly, all of the NS5B proteins derived from these novel sequences exhibited substantial RdRp activity. However, substitution at this position with tyrosine [clone 4(S1Y)] yielded an inactive protein (Table Similarly, all known NS5B sequences have a methionine residue at position 2. Modified versions of clone 4 were constructed in which this methionine was substituted with either alanine [clone 4(M2A)], leucine [clone 4(M2L)] or. threonine [clone 4(M2T)].

Surprisingly, all of these novel NS5B proteins exhibited substantial RdRp activity.

In a final example, addition of an amino acid residue prior to the first residue of the mature protein (serine at position as in clone 4(MAS), in which an alanine residue is inserted before the serine residue, yields a substantially functional NS5B RdRp (Table WO 99/51781 PCT/US99/07404 These findings, in which amino acid substitutions are made at residues for which there is no naturally occurring variation and that result in the generation of functional, as well as dramatically improved functional, NS5B proteins, were totally unanticipated.

Moreover, these data demonstrate that the nature of amino acid residue changes capable of giving rise to functional variants of NS5B is not limited to conservative amino acid substitutions. For example, while a substitution of threonine for serine at position 1 can be viewed as a conservative amino acid substitution, the substitution with glycine represents a dramatic change in the chemical nature of the amino acid side-chain. Similarly, substitution of the methionine residue at position 2 with leucine represents a conservative change. However, replacement with threonine, a clear non-conservative change, yields a functional and functionally improved NS5B protein.

Based on these discoveries, it is therefore fully contemplated here that additional changes of the amino acid residues among the NS5B sequences of the invention, both conservative and non-conservative in nature, at various residue positions and in various combinations will yield useful NS5B compositions and are therefore within the scope of the invention.

WO 99/51781 PCT/US99/07404 Table RdRp Activities of Various HCV NS5B Proteins Protein# specific activity clone 4 (SEQ ID NO: 2) 9.3 clone 4(S1G) (SEQ ID NO: 57) 23.0 clone 4(S1A) (SEQ ID NO: 58) clone 4(S1T) (SEQ ID NO: 59) 3.8 clone 4(S1Y) (SEQ ID NO: 60) <0.1 clone 4(M2A) (SEQ ID NO: 61) 14.0 clone 4(M2L) (SEQ ID NO: 62) 13.0 clone 4(M2T) (SEQ ID NO: 64) 22.3 clone 4(MAS) (SEQ ID NO: 65) 15.0 pmole NTP incorporated per jg NS5B protein per 60 minutes under standard reaction conditions.

while changes to SEQ ID N02 are exemplified herein, the recited alterations may also be made to the other NS5B amino acid sequences, i.e. SEQ ID NOS: 8, 9, 10, 11, 12, 13. Thus, sequences so altered are also contemplated to be within the scope of the invention While Examples 5 and 6 demonstrate that various changes in the amino acid sequence of SEQ ID NO: 2 may be made to advantage, it is fully contemplated that the residue changes exemplified here will also have similar utility, either singly or in various combinations, in other NS5B sequences, including sequences of the invention 8, 9, 10, 11 and 13, those of Tables 4 and any natural allelic variants, mutants and derivatives of these and other NS5B sequences. That is to say, the changes described in Examples 5 and 6, when introduced into any NS5B sequence, will likely provide a functional or functionally improved NS5B, and are fully contemplated to be within the scope of the present invention.

WO 99/51781 PCT/US99/07404 Several examples of nucleic acid sequences encoding variant NS5B protein sequences useful in the practice of the invention comprise sequences provided in Table 6.

Table 6 Nucleic acid sequences encoding variant NS5B Proteins* Parental SEQ ID Codon Position/Change SEQ ID NO: 1 A codon encoding alanine at position 75 to any codon encoding valine .0 SEQ ID NO: 1 A codon encoding asparagine at position 177 to any codon encoding aspartic acid SEQ ID NO: 1 A codon encoding serine at position 543 to any codon encoding proline SEQ ID NO: 1 A codon encoding serine at position 1 to any codon encoding glycine SEQ ID NO: 1 A codon encoding serine at position 1 to any codon encoding alanine SEQ ID NO: 1 A codon encoding serine at position 1 to any codon encoding threonine SEQ ID NO: 1 A codon encoding serine at position 1 to any codon encoding tyrosine SEQ ID NO: 1 A codon encoding methionine at position 2 to any codon encoding alanine SEQ ID NO: 1 A codon encoding methionine at position 2 to any codon encoding leucine SEQ ID NO: 1 A codon encoding methionine at position 2 to any codon encoding threonine SEQ ID NO: 1 Insertion of any codon encoding alanine at -1 While SEQ ID NO: 1 is exemplified in this table, the alterations recited may also be introduced into other NS5B encoding nucleic acid sequences set forth herein, SEQ ID NOS: 3, 4, 6 and 7. Such altered nucleic acid sequences are also contemplated for use in the present invention.

The examples of Table 6 demonstrate various beneficial changes in the nucleic acid sequence of SEQ ID NO: 1. In view of these results, it is expected that the changes exemplified in SEQ ID NO:1 will also have similar utility, either singly or in various combinations, in other NS5B sequences, including sequences of the invention 3, 4, 5, 6 and WO 99/51781 PCT/US99/07404 any natural allelic variants, mutants and derivatives of these and other NS5B sequences. That is to say, the changes described in this Table 6, when introduced into any NS5B sequence, will likely provide a functional or functionally improved NS5B, and are thus included in the scope of the present invention.

EXAMPLE 7 Utility of HCV NS5B Protein for Discovery of Antiviral Compositions The discovery of novel inhibitors of viral polymerases and related proteins often times requires the screening of large numbers of chemical compounds or mixtures of chemical compounds. Thus, an assay for polymerase activity that is capable of high volume screening, in other words, a high throughput assay, is desirable. There are a variety of 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)], and may utilize any number of activity detection and measurement technologies including, but not limited to, radiometric, colorimetric, fluorogenic, or chemiluminescent, any one of which may be suitable in the case of the HCV NS5B RdRp activity.

In one approach, a high throughput assay of the RdRp activity of the functional HCV NS5B proteins of the invention is provided to enable the screening of large numbers of chemicals or other potential inhibitors for WO 99/51781 PCT/US99/07404 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 trichloroacetic acid (TCA)-precipitable RNA product. Radioactivity may be quantified by either direct scintillation spectrometry or phosphorimaging technology. A phosphorimage of assay results obtained with the NS5B protein of Example 4 [clone 4] for one screening plate is presented in Fig.

6. The first and last (12) columns of the plate contain activity and background controls and a titration of a reference inhibitor compound. Thus, wells Al, Bl, A12 and B12 show the activity of the enzyme in the absence of any test compound (100% of the expected activity). In columns 1 and 12, wells C through F, a compound that was discovered to inhibit the HCV RdRp activity by use of the methods of the invention is included in decreasing concentrations. Wells HI and H12 lack the HCV enzyme and illustrate the background (0% RdRp activity) in the assay. In this example, the remaining 80 wells contain a collection of small organic compounds that were tested for their ability to affect the RdRp activity of the HCV NS5B protein. Thus, it can be seen that the material in wells G2, D10 and represent potent inhibitors of the HCV RdRp activity.

This example demonstrates that HCV NS5B proteins may be used to advantage to identify and assess agents or materials that may affect the HCV RdRp and HCV replication.

In summary, the disclosure of the present invention demonstrates that simple knowledge of an HCV WO 99/51781 PCT/US99/07404 sequence is insufficient to allow one to conclude, or even reliably predict that such sequence is a functional sequence or has functional utility. Additionally, an HCV NS5B consensus sequence is not the only, and not necessarily the optimal, functional NS5B protein encoding sequence. The present invention provides novel sequences with unanticipated functionality.

Additionally, the present invention demonstrates that particular sequence changes in HCV NS5B can lead to unanticipated and significantly improved functionality.

In light of the foregoing, the NS5B sequences of the invention represent novel sequences with demonstrated functionality and utility and unique improvements over prior art. Finally, the HCV NS5B sequences of the invention have broad utility in research, diagnostic, therapeutic and pharmaceutical applications.

While certain embodiments of the present invention have been described and/or exemplified above, various other embodiments will be apparent to those skilled in the art from the foregoing disclosure. The present invention is, therefore, not limited to the particular embodiments described and/or exemplified, but is capable of considerable modification without departure from the scope of the appended claims.

EDITORIAL NOTE APPLICATION NUMBER 38610/99 The following Sequence Listing pages 1/20-20/20 are part of the description. The claims pages follow on pages 66-81.

WO 99/51781 1 SEQUENCE LISTING <110> Viropharma, Incorporated <120> Hepatitis C Virus NS5B Compositions and Methods of Use Thereof <130> HEPPOL <150> 60/080,509 <151> 1998-04-02 <150> 60/090,356 <151> 1998-06-23 <160> 53 <170> FastSEQ for Windows Version PCT/US99/07404 <210> 1 <211> 1779 <212> DNA <213> Hepatitis C Virus <400> atgtcaatgt aagctgccca acaacatccc ctggatgacc gctaaacttc aaatttggct cactccgtgt gcaaaaaatg atcgtgtacc tccacccttc cgggtcgagt gacacccgct taccaatgtt ctttatatcg cgtgcgagcg tctgcagcct ctcgtcgtta acggaggcta ttggagctaa agggtatact gctagacaca tgggcaagga gaaaaagccc cctcagatca ggtgaaatca acttgtggca ccggctgcgt atatatcaca tctgtagggg cctacacatg tcaatgcgct gcagcgcaag actaccggga tatccgtgga atggggcgaa ggaaggactt aggttttctg cagacttggg ctcaggctgt tcctggtgaa gtttcgactc gtgacttggc ggggt cc cct gcgtactgac gtcgagctgc tctgtgaaag tgactaggta taacatcatg acctcacccg ctccagttaa tgattctgat tagattgtga ttcaacgact atagggtggc ~ggcaaag agtacctctt cccagttgga gcctgtctcg taggcatcta gacaggcgct gagcaactct ccagcggcag cgtgctcaag agaagcctgt ggacgtccgg gctggaagac cgttcaacca ggttcgcgtg gatgggctcc tgcctggaag aacggtcacc ccccgaagcc gactaactca gactagctgc aaagctccag cgcgggaacc ctctgcccc ctcctccaac tgaccccacc ctcctggcta gactcacttc gatctacggg ccatggtctt ttcatgcctc cgtni-r,-nr caactgggca cttatccggc tgcccgaccc cctgctcccg ctgattacac ttgctgcgcc: aagaaggtaa gagatgaagg aagctgacgc aacctatcca actgagacac gagaaaggag tgcgagaaaa tcatacggat tcaaagaaat gagaatgaca agacaggcca aaagggcaga ggtaataccc gactgcacga caagaggacg cctggggac c gtgtcggtcg acccctcttg ggc aac at ca ttctccatcc gcccactact agcgcgtttt.

aggaagcttg gtaaggacca tggttcgttg cgctggttca aaccgatga catgcgctgc accataacat cttttgacag cgaaggcgtc ccccacattc gcaaggccgt caattgacac gccgcaagcc tggccctcta tccagtactc gc cctatggg tccgtgttga taaggtcgct gctgcggtta tcacatgtta tgcttgtgtg cggcgagcct cgccccaacc cgcacgatgc cgCgggctgc tcatgtatgc ttctagctca ccattgagcc cactccacag gggtaccacc cccaggggg agcttaaact ctggttacag tgtggtgcct ggaggagagc ggtctatgcc gctgcaagtc cacagtcaag agccagatcc taaccacatc caccatcatg agctcgccta caacgtggtc tcctggacag cttcgcatat ggagtcaatt tacagagcgg tcgccggtgc cttgaaggcc cggagacgac acgagtcttc agaatacgac atctggcaaa gtgggagaca gcccactctg ggagcaactt acttgaccta ttactctcca cttgcgagtc aacfc~tqcc cactccaatt cgggggagac actcctactt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1779 <210> 2 <211> 592 <212> PRT WO 99/51781 PCT/US99/07404 2 Met 1 Ala Arg Arg Tyr Ala Ser Ser Glu Val 145 Ile Tyr Gly Trp Phe 225 Tyr Leu Gin Ser Arg 305 Leu Leu Asp Ser Leu 385 Ala Ala Ile <213> H <400> 2 Ser Met S Glu Glu S 2 His His A Gin Lys L Arg Asp V Lys Leu L Ala Arg S 1 Ser Lys A 115 Asp Thr G 130 Phe Cys V Val Tyr P Asn Val V 1 Phe Gin T 195 Lys Ser L 210 Asp Ser T Gin Cys C Thr Glu A 2 Ser Cys G 275 Cys Gly A 290 Ala Ala L Val Val I Arg Val P 3 Pro Pro C 355 Asn Val S 370 Thr Arg I Arg His 1 Pro Thr I 4 Leu Leu I 435 er er 0 sn ys al eu er 00 la lu al ro al 80 yr *ys hr ;ys rg 60 ly sn 'ys :le >he 40 ;1n er Asp rhr Leu 420 Ala Tyr 5 Lys Met Val Leu Ser Lys Val Thr Gin Asp 165 Ser Ser Lys Val Asp 245 Leu Tyr Thr Leu Cys 325 Thr Pro Val Pro Pro 405 Trp Gin Thr Leu Val Thr Lys 70 Val Phe Asn Pro Pro 150 Leu Thr Pro Cys Thr 230 Leu Tyr Arg Leu Gin 310 Glu Glu Glu Ala Thr 390 Val Ala Glu Trp Pro Tyr Phe 55 Glu Glu Gly His Ile 135 Glu Gly Leu Gly Pro 215 Glu Ala Ile Arg Thr 295 Asp Ser Ala Tyr His 375 Thr Asn Arg Gin Thr Ile Ala 40 Asp Met Glu Tyr Ile 120 Asp Lys Val Pro Gin 200 Met Asn Pro Gly Cys 280 Cys Cys Ala Met Asp 360 Asp Pro Ser Met Leu 440 epatitis C Virus Gly Asn 25 Thr Arg Lys Ala Gly 105 His Thr Gly Arg Gin 185 Arg Gly Asp Glu Gly 265 Arg Tyr Thr Gly Thr 345 Leu Ala Leu Trp Ile 425 Glu Ala 10 Ala Thr Leu Ala Cys 90 Ala Ser Thr Gly Val 170 Ala Val Phe Ile Ala 250 Pro Ala Leu Met Thr 330 Arg Glu Ser Ala Leu 410 Leu Lys Leu Leu Ser Gin Lys 75 Lys Lys Val Ile Arg 155 Cys Val Glu Ala Arg 235 Arg Leu Ser Lys Leu 315 Gin Tyr Leu Gly Arg 395 Gly Met Ala Ile Ser Arg Val Ala Leu Asp Trp Met 140 Lys Glu Met Phe Tyr 220 Val Gin Thr Gly Ala 300 Val Glu Ser Ile Lys 380 Ala Asn Thr Leu Thr Asn Ser Leu Ser Thr Val Lys 125 Ala Pro Lys Gly Leu 205 Asp Glu Ala Asn Val 285 Ser Cys Asp Ala Thr 365 Arg Ala Ile His SAsp 445 Pro Ser Ala Asp Thr Pro.

Arg 110 Asp Lys Ala Met Ser 190 Val Thr Glu Ile Ser 270 Leu Ala Gly Ala Pro 350 Ser Val Trp Ile Phe 430 Cys Cys Leu Ser Asp Val Pro Asn Leu Asn Arg Ala 175 Ser Asn Arg Ser Arg 255 Lys Thr Ala Asp Ala 335 Pro Cys Tyr Glu Met 415 Phe Glu Ala Leu Gin His Lys His Leu Leu Glu Leu 160 Leu Tyr Ala Cys Ile 240 Ser Gly Thr Cys Asp 320 Ser Gly Ser STyr SThr 400 STyr Ser Ile Tyr Gly 450 Ala His Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gin Ile Ile 455 460 WO 99/51781 WO 9951781PCT/US99/07404 Gin 465 Gly Arg Leu His Gly Leu 470 Val 3 Ser Ala Phe Se Ala Ser Cys Le ~r Leu 475 ~u Arg His Ser Tyr Lys Leu Gly Ser Pro 480 Val Pro 495 Glu Ile Asn 490 Arg Pro Leu Arg Leu Ser Gin 515 Trp Ala Val Val 500 Gly Arg His Arg Ala 505 Thr Ser Val Arg Gly Arg Ala Cys Gly Lys Tyr 525 Pro Ala Lys Leu 510 Leu Phe Asn Ala Ala Ser Arg Thr Lys Leu Thr Pro Ile 540 Gin 545 Ile Asp Leu Ser Gly 550 Ser Phe Val Ala Tyr His Ser Leu 565 Ser Arg Ala Arg Pro 570 Ile Giy Tyr Ser 555 Arg Trp Phe Tyr Leu Leu Gly Gly Met Trp 575 Asp 560 Cys Leu Leu Leu Leu 580 Val Gly Val Pro Asn Arg 590 <210> 3 <211> 1779 <212> DNA <213> Hepatitis C Virus <400> 3 atgtcaatgt aagctgccca acaacatccc ctggatgacc gctaaacttc aaatttggct cactccgtgt gcaaaaaatg atcgtgtacc tccacccttc cgggtcgagt gacacccgct taccaatgtt ctttatatcg cgtgcgagcg tctgcagcct ctcgtcgtta acggaggcta ttggagctaa agggtatact gctagacaca tgggcaagga gaaaaagccc cctcagatca ggtgaaatca tggagacatc acttgtggca ccggctgcgt atatatcaca tctgtagggg cctacacatg tcaatgcgct gcagcgcaag actaccggga tatccgtgga atggggcgaa ggaaggactt aggttttctg cagacttggg ctcaggctgt tcctggtgaa gtttcgactc gtgacttggc ggggtcccct gcgt ac tgac gtcgagctgc tctgtgaaag tgactaggta taacatcatg acctcacccg ctccagttaa tgattctgat tagattgtga ttcaacgact atagggtggc gggccagaag agtacctctt cccagttgga gcctgtctcg taggcatcta gacaggcgct gagcaactct ccagcggcag cgtgctcaag agaagcctgt ggacgtccgg gc tggaagac cgttcaacca ggttcgcgtg gatgggctcc tgcctggaag aacggtcacc ccccgaagcc gactaactca gactagctgc aaagctccag cgcgggaacc ctctgccccc ctcctccaac tgaccccacc ctcctggcta gactcacttc gatctacggg ccatggtctt ttcatgcctc cgtccgcgct caactgggca cttatccggc tgcccgaccc cctgctcccc ctgattacac ttgctgcgcc aagaaggtaa gagatgaagg aagctgacgc aacctatcca actgagacac gagaaaggag tgcgagaaaa tcatacggat tcaaagaaat gagaatgaca agacaggcca aaagggcaga ggtaataCc gactgcacga caagaggacg cctggggacc gtgtcggt cg acccctcttg ggcaacatca ttctccatcc gcccactact agcgcgtttt aggaagcttg aagttactgt gtaaggacca tggttcgttg cgctggttca aaccgatga catgcgctgc accataacat cttttgacag cgaaggtgtc ccccacattc gcaaggccgt caattgacac gccgcaagcc tggccctcta tccagtactc gccctatggg tccgtgttga taaggtcgct gctgcggt ta tcacatgtta tgcttgtgtg cggcgagcct cgccccaacc cgcacgatgc cgcgggctgC tcatgtatgc ttctagctca ccattgagcc cactccacag gggtaccacc cccagggggg agcttaaact ctggttacag tgtggtgcct ggaggagagc ggtctatgcc gctgcaagtc cacagtcaag agccagatcc taaccacatc caccatcatg agctcgccta cgacgtggtc tcctggacag cttcgcatat ggagtcaatt tacagagcgg tcgccggtgc cttgaaggcc cggagacgac acgagtcttc agaatacgac atctggcaaa gtgggagaca gcccactctg ggagcaactt acttgaccta ttactctcca cttgcgagtc gagggctgcc cactccaatt cgggggagac actcctactt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1779 <210> 4 <211> 1779 <212> DNA <213> Hepatitis C Virus <400> 4 atgtcaatgt cctacacatg gacaggcgct ctgattacac catgcgctgc ggaggagagc WO 99/51781 WO 99/1 781PCTIUS99/07404 4 aagctgccca acaacatccc ctggatgacc gctaaacttc aaatttggct cactccgtgt gcaaaaaatg atcgtgtacc tccacccttc cgggtcgagt gacacccgct taccaatgtt ctttatatcg cgtgcgagcg tctgcagcct ctcatcgtta acggaggcta ttggagctaa agggtatact gctagacaca tgggcaagga gaaaaagCc cctcagatca ggtgaaatca tggagacatc acttgtggca ccggctgcgt atataccaca tctgtagggg tcaatgcgct gcagcgcaag act a ccggga tatccgtgga atggggcgaa ggaaggactt aggttttctg cagacttggg ctcaggctgt tcctggtgaa gtttcgactc gtgacttggc ggggtcccct gcgtactgac gtcgagctgc tctgtgaaag tgactaggta taacatcatg acctcacccg ctccagttaa tgattctgat tagattgtga ttcaacgact atagggtggc gggccagaag agtacctctt cccagttgga gcctgtCtcg taggcatcta gagcaactct ccagcggcag cgtgctcaag agaagcctgt ggacgtccgg gctggaagac cgttcaacca ggttcgcgtg gatgggctcc tgcctggaag gacggtcacc ccccgaagcc gactaactca gactagctgc aaagctccag cgcgggaacc ctctgccccc ctcctccaac tgaccccacc ctcctggcta gactcacttc gatctacggg ccatggtctt ttcatgcctc cgtccgcgct caactgggca cttatccggc tgcccgaccc cctgctcccc ttgctgcgcc aagaaggtaa gagatgaagg aagctgacgc aacctatcca.

actgagacac gagaaaggag tgcgagaaaa tcatacggat tcaaagaaat gagaatgaca agacaggcca.

aaagggcaga.

ggtaataccc gactgcacga caagaggacg cctggggacc gtgtcggtcg acccctcttg ggcaacatca ttctccatcc gcccactact agcgcgtttt aggaagcttg aagttactgt gtaaggacca.

tggttcgttg tgctggttca.

aaccgatga accataacat cttttgacag cgaaggcgtc ccccacattc gcaaggccgt caattgacac gccgcaagcc tggccctcta tccagtactc gccctatggg tccgtgttga taaggtcgct gctgcggtca tcacatgtta tgcttgtgtg cggcgagcct cgccccaacc cgcacgatgc cgcgggctgc tcatgtatgc ttctagctca ccattgagcc cactccacag gg t a ccac c cccagggggg agcttaaact ctggttacag tgtggtgcct ggtctatgcc gctgcaagtc cacagtcaag agccagatcc taaccacatc caccatcatg agctcgccta cgacgtgatc tcctggacag cttcgcatat ggagtcaatt t acagagcgg tcgccggtgc cttgaaggcc cggagacgac acgagtcttc agaatacgac atctggcaaa gtgggagaca gcccactctg ggagcaactt acttgaccta ttactctcca cttgcgagtc gagggctgcc cactccaatt cgggggagac actcctactt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1779 <210> <211> 1779 <212> DNA <213> Hepatitis C virus <400> atgtcaatgt cctacacatg aagctgccca tcaatgcgct acaacatccc gcagcgcaag ctggatgacc actaccggga gctaaacttc tatccgtgga aaatttggct atggggcgaa cactccgtgt ggaaggactt gcaaaaaatg aggttttctg atcgtgtacc cagacttggg tccacccttc ctcaggctgt cgggtcgagt tcctggtgaa gacacccgct gtttcgactc taccaatgtt gtgacttggc ctttatatcg ggggtcccct cgtgcgagcg gcgtactgac tctgcagcct gtcgagctgc ctcgtcgtta tctgtgaaag acggaggcta tgactaggta ttggagctaa taacatcatg agggtatact acctcacccg gctagaCaca ctccagttaa tgggcaagga tgattctgat gaaaaagccc tagattgtga cctcagatca. ttcaacgact ggtgaaatca atagggtggc gacaggcgct gagcaactct ccagcggcag cgtgctcaag agaagcctgt ggacgtccgg gctggaagac cgttcaacca ggttcgcgtg gatgggctcc tgcctggaag aacggtcacc ccccgaagcc gactaactca gactagctgc aaagctccag cgcgggaacc ctctgccc ctcctccaac tgaccccac ctcctggcta gac tc actt c gatctacggg ccatggtctt ttcatgcctc ctgattacac ttgctgcgcc aagaaggtaa gagatgaagg aagctgacgc aacctatcca.

actgagacac gagaaaggag tgcgagaaaa tcatacggat ccaaagaaat gagaatgaca.

agacaggcca aaagggcaga.

ggtaatacc c gactgcacga.

caagaggacg cctggggacc gtgtcggtcg acccctcttg ggcaacacca ttctccatcc gcccactact agcgcgtttt aggaagcttg catgcgctgc accataacat cttttgacag cgaaggcgtc ccccacattc gcaaggccgt caattgacac gccgcaagcc tggccctcta tccagtactc gccctatggg tccgtgttga taaggtcgct gctgcggtta tcacatgtta tgcttgtgtg cggcgagcct cgccccaacc cgcacgatgc cgcgggctgC tcatgtatgc ttctagctca ccattgagcc cactccacag gggtaccacc ggaggagagc ggtctatgcc gctgcaagtc cacagtcaag agccagatcc taaccacatc caccatcatg agctcgccta cgacgtggtc ccctggacag cttcgcatat ggagtcaatt tacagagcgg tcgccggtgc cttgaaggcc cggagacgac acgagtcttc agaatacgac atctggcaaa gtgggagaca gcccactctg ggagcaactt acttgaccta ttactctcca cttgcgagtc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 WO 99/51781 WO 99/178 1PCT/US99/07404 tggaqacatc gggccagaag acttgtggca agtacctctt ccggctgcgt cccagttgga atatatcaca gcctgtctcg tctgtagggg taggcatcta cgtccgcgct caactgggca cttatccggc tgcccgaccc cctgctcccc aagttactgt cccagggggg gagggctgcc gtaaggacca agcttaaact cactccaatt tggttcgttg ctggttacag cgggggagac cgctggttca tgtggtgcct actcctactt aaccgatga 1560 1620 1680 1740 1779 <210> 6 <211> 1779 <212> DNA <213> Hepatitis C Virus <400> 6 atgtcaatgt aagctgccca acaacatccc ctggatgacc gc caaac tt c aaatttggct cactccgtgt gcaaaaaatg atcgtgtacc tccacccttc cgggtcgagt gacacccgct taccaatgtt ctttatatcg cgtgcgagcg tctgcagcct ttcgtcgtta acggaggcta ttggagctaa agggtatact gdtagacaca tgggcaagga gaaaaagccc cctcagatca ggtgaaatca tggagacatc acttgtggca ccggctgcgt atatatcaca tctgtagggg cctacacatg tcaatgcgct gcagcgcaag actaccggga tatccgtgga atggggcgaa ggaaggactt aggtcttctg cagacttggg ctcaggctgt tcctggtgaa gtttcgactc gtgacttggc ggggtcccct gcgtactgac gtcgagctgc tctgtgaaag tgactaggta taacatcatg acctcacccg ctccagttaa tgattctgat tagattgtga ttcaacgact atagggtggc gggccagaag agtacctctt cccagttgga gcctgtCtcg taggcatcta gagaggcgct gagcaactct ccagcggcag cgtgctcaag agaagcctgt ggacgtccgg gctggaagac cgttcaacca ggttcgcgtg gatgggctcc tgcctggaag aacggtcacc ccccgaagcc gactaactca gactagctgc aaagctccag cgcgggaacc ctctgccccc ctcctccaac tgaccccacc ctcctggcta gactcacttc gatctacggg ccatggtctt ttcatgcctc cgtccgcgCt c aa ct ggg ca cttatccggc tgcccgaccc cctgctcccc ctgattacac catgcgctgc ggaggagagc ttgctgcgcc aagaaggtaa gagatgaagg aagctgacgc aacctatcca actgagacac gagaaaggag tgcgagaaaa tcatacggat tcaaagaaat gagaatgaca agacaggcca aaagggcaga.

ggt aa tacc c gactgcacga caagaggacg cctggggacc gtgtcggtcg acccctcttg ggcaacatca.

ttctccatcc gcccactact agcgcgtttt aggaagcttg aagttactgt gtaaggacca.

tggttcgttg cgctggttca.

aaccgatga accataacat cttttgacag cgaaggcgtc ccccacattc gcaaggccgt caattgacac gccgcaagcc tggccctcta tccagcactc gccctatggg tccgtgttga taaggtcgct gctgcggtta tcacatgtta tgcttgtgtg cggcgagcct cgccccaacc cgcacgatgc cgcgggctgc tcatgtatgc ttctagctca ccattgagcc cactccacag gggtaccaCC cccagggggg agcttaaact ctggttacag tgtggtgcct ggtctatgc gctgcaagtc cacagtcaag agccagatcc taaccacatc caccatcatg agctcgccta cgacgtggtc tcctggacag cttcgcatat ggagtcaatt tacagagcgg tcgccggtgc cttgaaggcc cggagacgac acgagtcttc agaatacgac atctggcaaa gtgggagaca gcccactctg ggagcaactt acttgaccta ttactctcca cttgcgagtc gagggctgc cactccaatt cgggggagac actcctactt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1779 <210> <211> <212> <213> 7 1779

DNA

Hepatitis C Virus <400> 7 gtgtcaatgt cctacacatg aagctgccca tcaatgcgct acaacatccc gcagcgcaag ctggatgacc actaccggga gctaaaCttc tatccgtgga aaatttggct atggggcgaa cactccgtgt ggaaggactt gcaaaaaatg aggttttctg atcgtgtacc cagacttggg tccacccttc ctcaggctgt cgggtcgagt tcctggtgaa gacactcgct gtttcgactc gacaggcgct gagcaactct ccagcggcag cgtgctcaag agaagcctgt ggacgtccgg gc tggaagac cgttcaacca ggttcgcgtg gatgggctcc tgcctggaag aacggtcacc ctgattacac ttgctgcgcc aagaaggtaa gagatgaagg aagctgacgc aacctatcca.

actgagacac gagaaaggag tgcgagaaaa.

tcatacggat tcaaagaaat gagaatgaca catgcgctgc accataacat cttttgacag cgaaggcgtc ccccacattc gcaaggccgt caattgacac gccgcaagcc tggccctcta tccagtactc gccctatggg tccgtgttga ggaggagagc ggtctatgcc gctgcaagtc cacagtcaag agccagatc taaccacatc caccatcatg agctcgccta cgacgtggtc tcctggacag cttcgcatat ggagtcaatt 120 180 240 300 360 420 480 540 600 660 720 WO 99/51781 WO 99/178 1PCTIU S99/07404 6 taccaatgtt ctttatatcg cgtgcgagcg tctgcagcct ctcgtcgtta acggaggcta ttggagctaa agggtatact gctagacaca tgggcaagga gaaaaagcc cctcagatca ggtgaaatca tggagacatc act tgtggc a ccggctgcgc atatatcaca tctgtagggg gtgacttggc ggggtcccct gcgtactgac gtcgagctgc tctgtgaaag tgactaggta taacatcatg acctcacccg ctccagttaa tgattctgat tagattgtga ttcaacgact atagggtggc gggccagaag agtacctctt cccagttgga gcctgtctcg taggcatcta ccccgaagcc gactaactca gactagctgc aaagctccag cgcgggaacc ctctgccccc ctcctccaac tgaccccacc ctcctggcta gactcacttc gatctacggg ccatggtctt ttcatgcctc cgtccgcgct caactgggca cttatccggc tgcccgaccc cctgctcccc agacagg cca aaagggcaga ggtaataccc gactgcacga caagaggacg cctggggacc gtgtcggtcg acccctcttg ggcaacatca ttctccatcc gcccactact agcgcgtttt aggaagcttg aagttactgt gtaaggacca tggttcgttg cgctggttca aaccgatga taaggtcgct gctgcggtta tcacatgtta tgcttgtgtg cggcgagcct cgccccaacc cgcacgatgc cgcgggctgc tcatgtatgc ttctagctca ccattgagcc cactccacag gggtaccacc cccagggggg agcttaaact ctggttacag tgtggtgcct tacagagcgg tcgccggtgc cttgaaggcc cggagacgac acgagtcttc agaatacgac atctggcaaa gtgggagaca gcccactctg ggagcaactt acttgaccta ttactctcca cttgcgagtc gagggctgcc cactccaatt cgggggagac actcctactt 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1779 <210> 8 <211> 592 <212> PRT <213> Hepatitis C Virus <400> 8 Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Met 1 Ala Cys Ala 10 Ala Giu Glu Ser Leu Pro Ile Asn 25 Thr Leu Ser Asn Arg His His Arg Gin Lys Asn Met Val Tyr Thr Ser Arg Ser Leu Ser Leu Leu Ala Ser Gin Asp Asp His Lys Val Thr Tyr Arg Phe 55 Glu Arg Leu Gin Val Val Asp Val Leu Ala Lys Val Met Lys Ala Ser Thr Val Lys Leu Leu Giu Giu Ala Leu Thr Pro Pro His Ser Ala Arg Ser Ser Lys 115 Glu Asp Thr Ser 100 Al a Phe Gly Tyr Gly 105 His Lys Asp Val Val Asn His Ile 120 Asp Ser Val Trp Lys 125 Ala Arg Asn Leu 110 Asp Leu Leu Lys Asn Giu Glu Thr Pro Thr Thr Ilie 130 Val Phe Cys Val Gin Lys Gly Giy Pro Ala Arg Leu 160 Val Tyr Pro Asp 165 Ser Gly Val Arg Val 170 Al a Giu Lys Tyr Asp Val Gly Phe Gin 195 Trp Lys Ser Val 180 Tyr Thr Leu Pro Gin 185 Arg Val Met Gly Met Ala Leu 175 Ser Ser Tyr 190 Val Asn Ala Thr Arg Cys Ser Pro Gly Val Giu Phe Leu 205 Asp Lys Lys Cys 210 Phe Asp Pro 215 Glu Gly Phe Ala Tyr 220 Val Ser Thr Val 225 Tyr Thr 230 Leu Asn Asp Ile Arg 235 Arg Giu Giu Ser Ile 240 Gin Cys Cys Ala Pro Giu Al a 250 Pro Gin Ala Ile Arg Ser 255 Leu Thr Giu Gin Ser Cys 275 Arg 260 Gly Tyr Ile Gly Gly 265 Arg Leu Thr Asn Ser Lys Gly 270 Leu Thr Thr Tyr Arg Arg Cys 280 Ala Ser Gly WO 99/51781 WO 99/178 1PCT/US99/07404 7 Ser Arg 305 Leu Leu Asp Ser Leu 385 Ala Ala Ile Tyr Gin 465 Gly Pro Leu Trp Gin 545 Ile Leu Met 1 Ala Arg Arg Tyr Ala Ser Ser Cys 290 Al a Val Arg Pro Asn 370 Thr Arg Pro Leu Gly 450 Arg Glu Leu Ser Ala 530 Leu Tyr Leu Gly Ala Val Val Pro 355 Val1 Arg His Thr Leu 435 Al a Leu Ile Arg Gin 515 Val Asp His Leu Asn Lys Ile Phe 340 Gin Ser Asp Thr Leu 420 Al a His His Asn Val1 500 Gly Arg Leu Ser Leu 580 Thr Leu Cys 325 Thr Pro Val Pro Pro 405 Trp Gin Tyr Gly Arg 485 Trp Gly Thr Ser Leu 565 Ser Leu Gin, 310 Giu Glu Glu Ala Thr 390 Val Al a Glu Ser Leu 470 Val Arg Arg Lys Gly 550 Ser Val Thr 295 Asp Ser Ala Tyr His 375 Thr Asn Arg Gin Ile 455 Ser Ala His Ala Leu 535 Trp Arg Gly Cys Cys Al a Met Asp 360 Asp Pro Ser Met Leu 440 Glu Ala Ser Arg Ala 520 Lys Phe Ala Val Tyr Thr Gly Thr 345 Leu Al a Leu Trp Ile 425 Glu Pro Phe Cys Ala 505 Thr Leu Val Arg Gly Leu Met Thr 330 Arg Glu Ser Al a Leu 410 Leu Lys Leu Ser Leu 490 Arg Cys Thr Ala Pro 570 Ile Lys Leu 315 Gin Tyr Leu Gly Arg 395 Gly Met Al a Asp Leu 475 Arg Ser Gly Pro Gly 555 Arg Tyr Al a 300 Val Glu Ser Ilie Lys 380 Ala Asn Thr Leu Leu 460 His Lys Val1 Lys Ile 540 Tyr Trp Leu Ser Cys Asp Ala Thr 365 Arg Ala Ile His Asp 445 Pro Ser Leu Arg Tyr 525 Pro Ser Phe Leu Ala Ala Gly Asp Ala Ala 335 Pro Pro 350 Ser Cys Val Tyr Trp Giu Ile Met 415 Phe Phe 430 Cys Glu Gin Ile Tyr Ser Gly Val 495 Ala Lys 510 Leu Phe Ala Ala Gly Gly Met Trp 575 Pro Asn 590 Cys Asp 320 Ser Gly Ser Tyr Thr 400 Tyr Ser Ile Ile Pro 480 Pro Leu Asn Ser Asp 560 Cys Arg <210> 9 <211> 5 <212> P: <213> H <400> 9 Ser Met S Glu Glu S 2 His His A Gin Lys L Arg Asp V Lys Leu L Ala Arg S 1 Ser Lys A 115 92

RT

epatitis C Virus er er 0 sn ys al eu er 00 l1a Tyr Lys Met Val Leu Ser Lys Val1 Thr Leu Val Thr Lys 70 Val Phe Asn Trp Pro Tyr Phe 55 Glu Glu Gly His Thr Ile Al a Asp Met Glu Tyr Ile 120 Gly Asn 25 Thr Arg Lys Ala Gly 105 His Al a 10 Ala Thr Leu Ala Cys 90 Ala Ser Leu Leu Ser Gin Lys 75 Lys Lys Val Ile Ser Arg Val Val1 Leu Asp Trp Pro Ser Ala Asp Thr Pro Arg 110 Asp Cys Leu Ser Asp Val Pro Asn Leu Al a Leu Gin His Lys His Leu Leu WO 99/51781 PCT/US99/07404 8 Glu Asp Thr Glu Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Asn Glu 130 135 140 Val Phe Cys Val Gin Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu 145 150 155 160 Ile Val Tyr Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu 165 170 175 Tyr Asp Val Ile Ser Thr Leu Pro Gin Ala Val Met Gly Ser Ser Tyr 180 185 190 Gly Phe Gin Tyr Ser Pro Gly Gin Arg Val Glu Phe Leu Val Asn Ala 195 200 205 Trp Lys Ser Lys Lys Cys Pro Met Gly Phe Ala Tyr Asp Thr Arg Cys 210 215 220 Phe Asp Ser Thr Val Thr Glu Asn Asp Ile Arg Val Glu Glu Ser Ile 225 230 235 240 Tyr Gin Cys Cys Asp Leu Ala Pro Glu Ala Arg Gin Ala Ile Arg Ser 245 250 255 Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn Ser Lys Gly 260 265 270 Gin Ser Cys Gly His Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr 275 280 285 Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala Ser Ala Ala Cys 290 295 300 Arg Ala Ala Lys Leu Gin Asp Cys Thr Met Leu Val Cys Gly Asp Asp 305 310 315 320 Leu Ile Val Ile Cys Glu Ser Ala Gly Thr Gin Glu Asp Ala Ala Ser 325 330 335 Leu Arg Val Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly 340 345 350 Asp Pro Pro Gin Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser 355 360 365 Ser Asn Val Ser Val Ala His Asp Ala Ser Gly Lys Arg Val Tyr Tyr 370 375 380 Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr 385 390 395 400 Ala Arg His Thr Pro Val Asn Ser Trp Leu Gly Asn Ile Ile Met Tyr 405 410 415 Ala Pro Thr Leu Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser 420 425 430 Ile Leu Leu Ala Gin Glu Gin Leu Glu Lys Ala Leu Asp Cys Glu Ile 435 440 445 Tyr Gly Ala His Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gin Ile Ile 450 455 460 Gin Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro 465 470 475 480 Gly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val Pro 485 490 495 Pro Leu Arg Val Trp Arg His Arg Ala Arg Ser Val Arg Ala Lys Leu 500 505 510 Leu Ser Gin Gly Gly Arg Ala Ala Thr Cys Gly Lys Tyr Leu Phe Asn 515 520 525 Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Pro Ala Ala Ser 530 535 540 Gin Leu Asp Leu Ser Gly Trp Phe Val Ala Gly Tyr Ser Gly Gly Asp 545 550 555 560 Ile Tyr His Ser Leu Ser Arg Ala Arg Pro Cys Trp Phe Met Trp Cys 565 570 575 Leu Leu Leu Leu Ser Val Gly Val Gly Ile Tyr Leu Leu Pro Asn Arg 580 585 590 <210> <211> 592 WO 99/51781 PCT/US99/07404 9 <212> PRT <213> Hepatitis C Virus <400> Met Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys Ala 1 5 10 Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu Ser Asn Ser Leu Leu 25 Arg His His Asn Met Val Tyr Ala Thr Thr Ser Arg Ser Ala Ser Gin 40 Arg Gln Lys Lys Val Thr Phe Asp Arg Leu Gin Val Leu Asp Asp His 55 Tyr Arg Asp Val Leu Lys Glu Met Lys Ala Lys Val Ser Thr Val Lys 70 75 Ala Lys Leu Leu Ser Val Glu Glu Ala Cys Lys Leu Thr Pro Pro His 90 Ser Ala Arg Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg Asn Leu 100 105 110 Ser Ser Lys Ala Val Asn His Ile His Ser Val Trp Lys Asp Leu Leu 115 120 125 Glu Asp Thr Glu Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Asn Glu 130 135 140 Val Phe Cys Val Gin Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu 145 150 155 160 Ile Val Tyr Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu 165 170 175 Tyr Asp Val Val Ser Thr Leu Pro Gin Ala Val Met Gly Ser Ser Tyr 180 185 190 Gly Phe Gin Tyr Ser Pro Gly Gin Arg Val Glu Phe Leu Val Asn Ala 195 200 205 Trp Lys Pro Lys Lys Cys Pro Met Gly Phe Ala Tyr Asp Thr Arg Cys 210 215 220 Phe Asp Ser Thr Val Thr Glu Asn Asp Ile Arg Val Glu Glu Ser Ile 225 230 235 240 Tyr Gin Cys Cys Asp Leu Ala Pro Glu Ala Arg Gin Ala Ile Arg Ser 245 250 255 Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn Ser Lys Gly 260 265 270 Gin Ser Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr 275 280 285 Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala Ser Ala Ala Cys 290 295 300 Arg Ala Ala Lys Leu Gin Asp Cys Thr Met Leu Val Cys Gly Asp Asp 305 310 315 320 Leu Val Val Ile Cys Glu Ser Ala Gly Thr Gin Glu Asp Ala Ala Ser 325 330 335 Leu Arg Val Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly 340 345 350 Asp Pro Pro Gin Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser 355 360 365 Ser Asn Val Ser Val Ala His Asp Ala Ser Gly Lys Arg Val Tyr Tyr 370 375 380 Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr 385 390 395 400 Ala Arg His Thr Pro Val Asn Ser Trp Leu Gly Asn Thr Ile Met Tyr 405 410 415 Ala Pro Thr Leu Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser 420 425 430 Ile Leu Leu Ala Gin Glu Gin Leu Glu Lys Ala Leu Asp Cys Glu Ile 435 440 445 Tyr Gly Ala His -Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gin Ile Ile WO 99/51781 PCT/US99/07404 450 455 460 Gin Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro 465 470 475 480 Gly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val Pro 485 490 495 Pro Leu Arg Val Trp Arg His Arg Ala Arg Ser Val Arg Ala Lys Leu 500 505 510 Leu Ser Gin Gly Gly Arg Ala Ala Thr Cys Gly Lys Tyr Leu Phe Asn 515 520 525 Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Pro Ala Ala Ser 530 535 540 Gin Leu Asp Leu Ser Gly Trp Phe Val Ala Gly Tyr Ser Gly Gly Asp 545 550 555 560 Ile Tyr His Ser Leu Ser Arg Ala Arg Pro Arg Trp Phe Met Trp Cys 565 570 575 Leu Leu Leu Leu Ser Val Gly Val Gly Ile Tyr Leu Leu Pro Asn Arg 580 585 590 <210> 11 <211> 592 <212> PRT <213> Hepatitis C Virus <400> 11 Met Ser Met Ser Tyr Thr Arg Thr Gly Ala Leu Ile Thr Pro Cys Ala 1 5 10 Ala Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu Ser Asn Ser Leu Leu 25 Arg His His Asn Met Val Tyr Ala Thr Thr Ser Arg Ser Ala Ser Gin 40 Arg Gin Lys Lys Val Thr Phe Asp Arg Leu Gin Val Leu Asp Asp His 55 Tyr Arg Asp Val Leu Lys Glu Met Lys Ala Lys Val Ser Thr Val Lys 70 75 Ala Lys Leu Leu Ser Val Glu Glu Ala Cys Lys Leu Thr Pro Pro His 90 Ser Ala Arg Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg Asn Leu 100 105 110 Ser Ser Lys Ala Val Asn His Ile His Ser Val Trp Lys Asp Leu Leu 115 120 125 Glu Asp Thr Glu Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Asn Glu 130 135 140 Val Phe Cys Val Gin Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu 145 150 155 160 Ile Val Tyr Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu 165 170 175 Tyr Asp Val Val Ser Thr Leu Pro Gin Ala Val Met Gly Ser Ser Tyr 180 185 190 Gly Phe Gin His Ser Pro Gly Gin Arg Val Glu Phe Leu Val Asn Ala 195 200 205 Trp Lys Ser Lys Lys Cys Pro Met Gly Phe Ala Tyr Asp Thr Arg Cys 210 215 220 Phe Asp Ser Thr Val Thr Glu Asn Asp Ile Arg Val Glu Glu Ser Ile 225 230 235 240 Tyr Gin Cys Cys Asp Leu Ala Pro Glu Ala Arg Gin Ala Ile Arg Ser 245 250 255 Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn Ser Lys Gly 260 265 270 Gin Ser Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr 275 280 285 Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala Ser Ala Ala Cys WO 99/51781 WO 9951781PCTIUS99/07404 11 Arg 305 Phe Leu Asp Ser Leu 385 Al a Ala Ile Tyr Gin 465 Gly Pro Leu Trp, Gin 545 Ile Leu 290 Ala Ala Val Val Arg Val Pro Pro 355 Asn Vai 370 Thr Arg Arg His Pro Thr Leu Leu 435 Gly Ala 450 Arg Leu Giu Ile Leu Arg Ser Gin 515 Ala Vai 530 Leu Asp Tyr His Leu Leu 295 Lys Leu Gin Asp Cys Ile Phe 340 Gin Ser Asp Thr Leu 420 Ala His His Asn Vai 500 Giy Arg Leu Ser Leu 580 Cys 325 Thr Pro Val Pro Pro 405 Trp Gin Tyr Gly Arg 485 Trp Gly Thr Ser Leu 565 Ser 310 Glu Giu Giu Ala Thr 390 Val1 Ala Giu Ser Leu 470 Val Arg Arg Lys Gly 550 Ser Val Ser Al a Tyr His 375 Thr Asn Arg Gin Ile 455 Ser Al a His Ala Leu 535 Trp Arg Gly Ala Met Asp 360 Asp Pro Ser Met Leu 440 Glu Ala Ser Arg Al a 520 Lys Phe Ala Val Thr Gly Thr 345 Leu Ala Leu Trp Ile 425 Glu Pro Phe Cys Ala 505 Thr Leu Val Arg Gly 585 Met Thr 330 Arg Giu Ser Ala Leu 410 Leu Lys Leu Ser Leu 490 Arg Cys Thr Ala Pro 570 Ile Leu 315 Gin T'yr Leu Gly Arg 395 Gly Met Al a Asp Leu 475 Arg Ser Gly Pro Gly 555 Arg Tyr 300 Val Giu Ser Ilie Lys 380 Al a Asn Thr Leu Leu 460 His Lys Val1 Lys Ile 540 Tyr Trp Leu Cys Asp Al a Thr 365 Arg Ala Ile His Asp 445 Pro Ser Leu Arg Tyr 525 Pro Ser Phe Leu Gly Ala Pro 350 Ser Val Trp Ile Phe 430 Cys Gin Tyr Giy Ala 510 Leu Ala Gly Met Pro 590 Asp Asp 320 Ala Ser 335 Pro Gly Cys Ser Tyr Tyr Giu Thr 400 Met Tyr 415 Phe Ser Glu Ile Ile Ile Ser Pro 480 Val Pro 495 Lys Leu Phe Asn Ala Ser Gly Asp 560 Trp Cys 575 Asn Arg Val Al a Arg Arg Tyr Ala Ser Ser <210> <211> <212> <213> <400> Ser Met Giu Giu His His Gin Lys Arg Asp Lys Leu Ala Arg Ser Lys 12 Ser Tyr Ser Lys Asn Met Lys Vai Val Leu Leu Ser Ser Lys 100 Ala Val Thr Leu Val1 Thr Lys 70 Val1 Phe Asn Trp Pro Tyr Phe 55 Glu Glu Gly His Thr Ile Al a Asp Met Giu Tyr Ile Gly Asn 25 Thr Arg Lys Al a Gly 105 His 12 592

PRT

Hepatitis C Virus Ala 10 Ala Thr Leu Ala Cys 90 Ala Ser Leu Leu Ser Gin Lys 75 Lys Lys Val Ile Ser Arg Val Val Leu Asp Trp Pro Ser Al a Asp Thr Pro Arg 110 Asp Cys Leu Ser Asp Vai Pro Asn Leu Ala Leu Gin His Lys His Leu Leu 115 120 125 Giu Asp Thr Glu -Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Asn GiU WO 99/51781 PCT/US99/07404 12 130 135 140 Val Phe Cys Val Gin Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu 145 150 155 160 Ile Val Tyr Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu 165 170 175 Tyr Asp Val Val Ser Thr Leu Pro Gin Ala Val Met Gly Ser Ser Tyr 180 185 190 Gly Phe Gin Tyr Ser Pro Gly Gin Arg Val Glu Phe Leu Val Asn Ala 195 200 205 Trp Lys Ser Lys Lys Cys Pro Met Gly Phe Ala Tyr Asp Thr Arg Cys 210 215 220 Phe Asp Ser Thr Val Thr Glu Asn Asp Ile Arg Val Glu Glu Ser Ile 225 230 235 240 Tyr Gin Cys Cys Asp Leu Ala Pro Glu Ala Arg Gin Ala Ile Arg Ser 245 250 255 Leu Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn Ser Lys Gly 260 265 270 Gin Ser Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr 275 280 285 Ser Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala Ser Ala Ala Cys 290 295 300 Arg Ala Ala Lys Leu Gin Asp Cys Thr Met Leu Val Cys Gly Asp Asp 305 310 315 320 Leu Val Val Ile Cys Glu Ser Ala Gly Thr Gin Glu Asp Ala Ala Ser 325 330 335 Leu Arg Val Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly 340 345 350 Asp Pro Pro Gin Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser 355 360 365 Ser Asn Val Ser Val Ala His Asp Ala Ser Gly Lys Arg Val Tyr Tyr 370 375 380 Leu Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr 385 390 395 400 Ala Arg His Thr Pro Val Asn Ser Trp Leu Gly Asn Ile Ile Met Tyr 405 410 415 Ala Pro Thr Leu Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser 420 425 430 Ile Leu Leu Ala Gin Glu Gin Leu Glu Lys Ala Leu Asp Cys Glu Ile 435 440 445 Tyr Gly Ala His Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gin Ile Ile 450 455 460 Gin Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro 465 470 475 480 Gly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val Pro 485 490 495 Pro Leu Arg Val Trp Arg His Arg Ala Arg Ser Val Arg Ala Lys Leu 500 505 510 Leu Ser Gin Gly Gly Arg Ala Ala Thr Cys Gly Lys Tyr Leu Phe Asn 515 520 525 Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Pro Ala Ala Pro 530 535 540 Gin Leu Asp Leu Ser Gly Trp Phe Val Ala Gly Tyr Ser Gly Gly Asp 545 550 555 560 Ile Tyr His Ser Leu Ser Arg Ala Arg Pro Arg Trp Phe Met Trp Cys 565 570 575 Leu Leu Leu Leu Ser Val Gly Val Gly Ile Tyr Leu Leu Pro Asn Arg 580 585 590 <210> 13 <211> 592 <212> PRT WO 99/51781 PCT/US99/07404 13 Met 1 Ala Arg Arg Tyr Ala Ser Ser Glu Val 145 Ile Tyr Gly Trp Phe 225 Tyr Leu Gin Ser Arg 305 Leu Leu Asp Ser Leu 385 Ala Ala Ile <213> <400> Ser Met Glu Glu His His Gin Lys Arg Asp Lys Leu Ala Arg Ser Lys 115 Asp Thr 130 Phe Cys Val Tyr Asp Val Phe Gin 195 Lys Ser 210 Asp Ser Gin Cys Thr Glu Ser Cys 275 Cys Gly 290 Ala Ala Val Val Arg Val Pro Pro 355 Asn Val 370 Thr Arg Arg His Pro Thr Leu Leu 13 Ser Ser Asn Lys Val Leu Ser 100 Ala Glu Val Pro Val 180 Tyr Lys Thr Cys Arg 260 Gly Asn Lys Ile Phe 340 Gin Ser Asp Thr Leu 420 Ala Tyr 5 Lys Met Val Leu Ser Lys Val Thr Gin Asp 165 Ser Ser Lys Val Asp 245 Leu Tyr Thr Leu Cys 325 Thr Pro Val Pro Pro 405 Trp Gin Thr Leu Val Thr Lys 70 Val Phe Asn Pro Pro 150 Leu Thr Pro Cys Thr 230 Leu Tyr Arg Leu Gin 310 Glu Glu Glu Ala Thr 390 Val Ala Glu Trp Pro Tyr Phe 55 Glu Glu Gly His Ile 135 Glu Gly Leu Gly Pro 215 Glu Ala Ile Arg Thr 295 Asp Ser Ala Tyr His 375 Thr Asn Arg SGin Ile Thr lie Ala 40 Asp Met Glu Tyr Ile 120 Asp Lys Val Pro Gin 200 Met Asn Pro Gly Cys 280 Cys Cys Ala Met Asp 360 Asp Pro Ser Met Leu 440 Gli Gly Asn 25 Thr Arg Lys Ala Gly 105 His Thr Gly Arg Gin 185 Arg Gly Asp Glu Gly 265 Arg Tyr Thr Gly Thr 345 Leu SAla Leu STrp Ile 425 i Glu Pro Ala Leu 10 Ala Leu Thr Ser Leu Gn Ala Lys 75 Cys Lys 90 Ala Lys Ser Val Thr Ile Gly Arg 155 Val Cys 170 Ala Val Val Glu Phe Ala Ile Arg 235 Ala Arg 250 Pro Leu Ala Ser Leu Lys Met Leu 315 Thr Gin 330 Arg Tyr Glu Leu Ser Gly Ala Arg 395 SLeu Gly 410 SLeu Met SLys Ala Leu Asp Iie Ser Arg Val Val Leu Asp Trp Met 140 Lys Glu Met Phe Tyr 220 Val Gin Thr Gly Ala 300 Val Glu Ser Ile Lys 380 Ala Asn Thr Leu Leu Thr I Asn Ser 1 Leu 2 Ser Thr Val Lys 125 Ala Pro Lys Gly Leu 205 Asp Glu Ala Asn Val 285 Ser Cys Asp Ala Thr 365 Arg Ala Ile His SAsp 445 Pro Pro Ser Ala Asp rhr Pro Arg 110 Asp Lys Ala Met Ser 190 Val Thr Glu Ile Ser 270 Leu Ala Gly Ala Pro 350 Ser Val Trp Ile Phe 430 Cys Gin Cys Leu Ser Asp Val Pro Asn Leu Asn Arg Ala 175 Ser Asn Arg Ser Arg 255 Lys Thr Ala Asp Ala 335 Pro Cys Tyr Glu Met 415 Phe Glu Ile Hepatitis C Virus Ala Leu Gin His Lys His Leu Leu Glu Leu 160 Leu Tyr Ala Cys Ile 240 Ser Gly Thr Cys Asp 320 Ser Gly Ser STyr Thr 400 Tyr Ser Ile Ile 435 Tyr Gly 450 Ala His Tyr Ser 455 460 WO 99/51781 PCT/US99/07404 14 Gin Arg Leu His Gly Leu Ser Ala Phe Ser Leu His Ser Tyr Ser Pro 465 470 475 480 Gly Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val Pro 485 490 495 Pro Leu Arg Val Trp Arg His Arg Ala Arg Ser Val Arg Ala Lys Leu 500 505 510 Leu Ser Gin Gly Gly Arg Ala Ala Thr Cys Gly Lys Tyr Leu Phe Asn 515 520 525 Trp Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Pro Ala Ala Pro 530 535 540 Gin Leu Asp Leu Ser Gly Trp Phe Val Ala Gly Tyr Ser Gly Gly Asp 545 550 555 560 Ile Tyr His Ser Leu Ser Arg Ala Arg Pro Arg Trp Phe Met Trp Cys 565 570 575 Leu Leu Leu Leu Ser Val Gly Val Gly Ile Tyr Leu Leu Pro Asn Arg 580 585 590 <210> 14 <211> <212> DNA <213> Hepatitis C Virus <400> 14 tcaatgtcct acacatggac <210> <211> <212> DNA <213> Hepatitis C Virus <400> ctacacatgg <210> 16 <211> 41 <212> DNA <213> Hepatitis C Virus <400> 16 ctctgattac accatgcgct gcggaggaga gcaagctgcc c 41 <210> 17 <211> 28 <212> DNA <213> Hepatitis C Virus <400> 17 aatgcgctga gcaactcttt gctgcgcc 28 <210> 18 <211> 47 <212> DNA <213> Hepatitis C Virus <400> 18 ccataacatg gtctatgcca caacatcccg cagcgcaagc cagcggc 47 <210> 19 <211> 22 <212> DNA <213> Hepatitis C Virus WO 99/51781 PCT/US99/07404 <400> 19 gaagaaggta acttttgaca gg 22 <210> <211> 23 <212> DNA <213> Hepatitis C Virus <400> caagtcctgg atgaccacta ccg 23 <210> 21 <211> 14 <212> DNA <213> Hepatitis C Virus <400> 21 gacgtgctca agga 14 <210> 22 <211> 18 <212> DNA <213> Hepatitis C Virus <400> 22 atgaaggcga aggcgtcc 18 <210> 23 <211> 16 <212> DNA <213> Hepatitis C Virus <400> 23 ggaagaagcc tgtaag 16 <210> 24 <211> 24 <212> DNA <213> Hepatitis C Virus <400> 24 gaacctatcc agcaaggccg ttaa 24 <210> <211> 19 <212> DNA <213> Hepatitis C Virus <400> accagagaaa ggaggccgc 19 <210> 26 <211> 14 <212> DNA <213> Hepatitis C Virus <400> 26 acccagactt gggg 14 <210> 27 <211> WO 99/51781 PCT/US99/07404 16 <212> DNA <213> Hepatitis C Virus <400> 27 tctccaccct tcctcaggct <210> 28 <211> 19 <212> DNA <213> Hepatitis C Virus <400> 28 cgagttcctg gtgaatgcc 19 <210> 29 <211> 24 <212> DNA <213> Hepatitis C Virus <400> 29 tgccctatgg gcttcgcata tgac 24 <210> <211> <212> DNA <213> Hepatitis C Virus <400> tttcgactca acggtcaccg agaat <210> 31 <211> <212> DNA <213> Hepatitis C Virus <400> 31 gttgaggagt caatt <210> 32 <211> <212> DNA <213> Hepatitis C Virus <400> 32 ttggcccccg aagccagaca <210> 33 <211> 17 <212> DNA <213> Hepatitis C Virus <400> 33 aaggtcgctt acagagc 17 <210> 34 <211> 17 <212> DNA <213> Hepatitis C Virus <400> 34 atcgggggtc ccctgac 17 \If\ On/ 1 Q PCT/US99/07404 v 77.. 0 1 17 <210> <211> 18 <212> DNA <213> Hepatitis C Virus <400> taactcaaaa gggcagag 18 <210> 36 <211> 19 <212> DNA <213> Hepatitis C Virus <400> 36 atgttacttg aaggcctct 19 <210> 37 <211> <212> DNA <213> Hepatitis C Virus <400> 37 gatgcttgtg tgcggagacg acctc <210> 38 <211> 31 <212> DNA <213> Hepatitis C Virus <400> 38 ggtcgcgcac gatgcatctg gcaaaagggt a 31 <210> 39 <211> 19 <212> DNA <213> Hepatitis C Virus <400> 39 caccacccct cttgcgcgg 19 <210> <211> <212> DNA <213> Hepatitis C Virus <400> ctccatcctt ctagctcagg agcaa <210> 41 <211> <212> DNA <213> Hepatitis C Virus <400> 41 agttactgtc ccaggggggg <210> 42 <211> 17 <212> DNA <213> Hepatitis C Virus WO 99/51781 WO 9951781PCT/US99/07404 18 20 <400> 42 tccggctgcg tcccagt <210> 43 <211> 26 <212> DNA <213> Hepatitis C Virus <400> 43 ccgaccccgc tggttcatgt ggtgcc <210> 44 <211> 19 <212> DNA <213> Hepatitis C Virus <400> 44 ctacctgctc ccgaaccga <210> <211> 19 <212> DNA <213> Hepatitis C Virus <400> ctacctgctc cccaaccga <210> 46 <211> <212> PRT <213> cHepatitis C Virus <400> 46 Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu 1 5 10 Glu Glu Ser Lys Leu Pro Ile Asn Ala Leu 25 His His Asn Met Val Tyr Ala Thr Thr Ser 40 Gin Lys Lys Val Thr Phe Asp Arg Leu Gin 55 Arg Asp Val Leu Lys Giu Met Lys Ala Lys 70 Ile Thr Pro Cys Ala Ser Asn Ser Leu Leu Arg Ser Ala Ser Gin Val Leu Asp Asp His Ala Ser Thr Val Lys 75 Ala Arg Arg Tyr Ala <210> <211> <212> <213> <400> Val Glu Phe Gly Asn His <210> <211> <212> <213> 47 47

PRT

cHepatitis C Virus 47 Glu Ala Cys Lys Leu Thr Pro Pro His Ser Ala Arg Ser 10 Tyr Gly Ala Lys Asp Val Arg Asn Leu Ser Ser Lys Ala 25 Ile His Ser Val Trp Lys Asp Leu Leu Glu Asp Thr 40 48

PRT

cHepatitis C Virus Gli 1 Va Pr WO 99/5178 <400> u Thr Pro 1 Gin Pro o Asp Leu <210> <211> <212> <213> <400> r Ser Tyr 1 Asn Ala r Arg Cys u Ser Ile e Arg Ser r Lys Gly u Thr Thr 1 19 48 Ile Asp Thr Thr Ile Met Ala Lys Asn Giu Val Phe Cys 5 10 Giu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile Val Tyr 25 Gly Val Arg Val Cys PCT/US99/07404 Se: 1 Va Th Gi 11 Se Le 49 102

PRT

cHepatitis C virus 49 Gly Phe Gin Tyr Ser 5 Trp Lys Ser Lys Lys Phe Asp Ser Thr Val 40 Tyr Gin Cys Cys Asp 55 Leu Thr Glu Arg Leu 70 Gin Ser Cys Giy Tyr Ser Cys Gly 100 56

PRT

cHepatitis C virus Leu Val Cys Gly Asp 5 Gin Glu Asp Ala Ala Tyr Ser Ala Pro Pro 40 Leu Ile Thr Ser Cys Pro Cys 25 Thr Leu Tyr Arg Gly 10 Pro Giu Ala Ile Arg 90 Arg Gly Asp Giu Gly Arg Val Giu Phe Ala Ile Arg Ala Arg Pro Leu Ala Ser Phe Leu Tyr Asp Val Glu Gin Ala Thr Asn Gly Val Cys 1 Ala Met Asp Ser 1 Asp Thr <210> <211> <212> <213> <400> Thr Met Gly Thr Thr Arg Leu Giu <210> <211> <212> <213> <400> Val Ala Pro Thr Pro Val <210> <211> <212> <213> Asp Leu Val Val Ile Cys Giu Ser 10 Ser Leu Arg Val Phe Thr Glu Ala 25 Gly Asp Pro Pro Gin Pro Glu Tyr 51 37

PRT

cHepatitis C Virus 51 His Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu Thr Arg 5 10 Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala Arg His 25 Asn Ser 52 29

PRT

cHepatitis C Virus WO099/51781 20 20 PCTIUS99/07404 <400> 52 Cys Leu Arg Lys Leu Gly Val Pro Pro Leu Arg Val Trp Arg His Arg 1 5 10 Ala Arg Ser Val Arg Ala Lys Leu Leu Ser Gin Gly Gly <210> 53 <211> <212> PRT <213> Hepatitis C Virus <400> 53 Phe Val Ala Gly Tyr Ser Gly Gly Asp Ile Tyr His Ser Leu Ser Arg 1 5 10 Ala Arg Pro Arg Trp Phe Met Trp, Cys

Claims (52)

10-10-03;03: 16PM;PIZZEYS ;61 7 32218077 8/ 59 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. An isolated nucleic acid molecule encoding a hepatitis C virus (HQV) NSSB protein, said nucleic acid molecule having a sequence selected from the group consisting of SEQ ID NOs: 1, 3, and 4. 2. The isolated nucleic acid molecule of claim 1, which comprises the sequence of SEQ ID NO. 1. 3. The isolated nucleic acid molecule of claim 1, which comprises the sequence of SEQ ID NO: 3. 4. The isolated nucleic acid molecule of claim 1, which comprises the sequence of SEQ ID NO: 4. An isolated nucleic acid molecule encoding a HCV protein, said NSSB protein having a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 63, and 64. o ooo o o f 6. wherein The said isolated nucleic acid molecule of claim NS5B protein comprises the sequence of SEQ ID NO: isolated nucleic acid molecule of claim NS5B protein comprises the sequence of SEQ ID NO: 7. wherein 8. 8. wherein The said The said isolated nucleic acid molecule of claim NS5B protein comprises the sequence of SEQ ID NO: COMS ID No: SMBI-00448164 Received by IP Australia: Time 15:26 Date 2003-10-10 S10-10-03;03:1 6PM PIZZEYS ;61 7 32218077 9/ 59 9. An isolated HCV NS5B protein having a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 63, and 64. The isolated HCV NS5B protein of claim 9, which comprises the sequence of SEQ ID NO: 2.

11. The isolated HCV NS5B protein of claim 9, which comprises the sequence of SEQ ID NO: 8.

12. The isolated HCV NSSB protein of claim 9, which o comprises the sequence of SEQ ID NO: 9. 15 13. The nucleic acid molecule of claim 5, further comprising a tag sequence.

14. The nucleic acid molecule of claim 13, said tag sequence being selected from the group consisting of homopolymeric nucleic acid sequences, polyhistidine, 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 biotin.

15. The HCV NS5B protein of claim 9, further comprising a protein tag sequence. 67 COMS ID No: SMBI-00448164 Received by IP Australia: Time 15:26 Date 2003-10-10 10-10-03;03:16PM;PIZZEYS :61 7 32218077 I0/ 59

16. The HCV NS5B protein of claim 15, wherein said protein tag sequence is selected from the group consisting of said tag sequence being selected from the group consisting of homopolymeric nucleic acid sequences, polyhistidine, 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 biotin.

17. A vector comprising a nucleic acid sequence encoding a HCV NS5B protein, said protein consisting essentially of an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 15 54, 55, 56, 57, 58, 59, 61, 62, 64, and

18. A method for assaying a test compound, for a modulating activity against hepatitis C virus comprising: i) providing an enzymatically active HCV NS5B protein comprising a sequence S: selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and ii) contacting said protein with a test d 25 compound suspected of modulating hepatitis C virus activity; and iii) measuring modulation of said HCV NS5B activity by said test compound.

19. The method of claim 18, wherein said HCV NS5B protein further comprises a tag sequence. 68 COMS ID No: SMBI-00448164 Received by IP Australia: Time 15:26 Date 2003-10-10 S10-10-03:03:11PM:PlZZEYS :s1 7 32218077 HI/ 59 A method for assaying a compound, for antagonistic activity against hepatitis C virus comprising: i) providing an enzymatically active HCV N55B protein comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61,.62, 64, and ii) contacting said protein with a test compound suspected of antagonizing HCV NS5B activity; and iii) measuring antagonism of said HCV NS5B activity by said test compound.

21.. The method of claim 20, wherein said HCV NS5B protein 15 further comprises a tag sequence. oo :22. A method for assaying a test compound, for agonistic activity against hepatitis C virus comprising: i) providing an enzymatically active HCV NSSB protein comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and ii) contacting said protein with a test compound suspected of agonizing hepatitis C virus 25 activity; and iii) measuring agonism of said ICV NS5B activity by said •test compound.

23. The method of claim 22, wherein said HCV NS5B protein further comprises a tag sequence. 69 COMS ID No: SMBI-00448164 Received by IP Australia: Time 15:26 Date 2003-10-10 O lO-10-03;03 T17PMPIZZEYS ;61 7 32213077 12/ 59

24. A method for assaying a test compound for interaction with an HCV NS5B protein sequence comprising: i) providing an enzymatically active HCV protein comprising a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and ii) contacting said protein with a test compound suspected of interacting with the HCV NS5B protein sequence; and iii) measuring the interaction of said test S .compound with said enzymatically active HCV NS5B protein. 15 25. The method of claim 24, wherein said enzymatically active HCV NS5B protein further comprises a tag sequence.

26. A method for assaying a test compound, for interaction with a HCV NS5B nucleic acid sequence comprising: i) providing a HCV NSSB nucleic acid, said nucleic acid encodes an amino acid sequence e lselected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 63, and 64; ii) contacting said nucleic acid with a 25 test compound suspected of interacting with the HCV NS5B nucleic acid sequence; and iii) measuring the interaction of said test compound with said HCVNS5B nucleic acid. COMS ID No: SMBI-00448164 Received by IP Australia: Time 15:26 Date 2003-10-10 17-10-03;II:21AM;PIZZEYS 6 1 7 32218077 5/ 16

27. A method for detecting the presence of HCV in a biological sample, comprising nucleic acid amplification that effectively amplifies a selected nucleotide sequence from said sample and detection of said selected nucleotide sequence; said selected nucleotidesequence encodes an HCV NS5B protein having a sequence selected from the group consisting of SEQ ID NOs; 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and

28. The method of claim 27, wherein the nucleic acid amplification method is performed employing a series of nested primers.

29. The method of claim 27, wherein said nucleotide sequence is detected using an oligonucleotide comprising a sequence set forth in Table 1.

30. A method for detecting immunological 20 interactions between viral polypeptides and antibodies *."*directed toward hepatitis C virus in a biological sample, said method comprising isolating said antibodies using an NS5B amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and

31. An antibody immunologically specific for a HCV .polypeptide, said polypeptide having a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and 0 9 71 COMS ID No: SMBI-00457254 Received by IP Australia: Time 11:20 Date 2003-10-17 17-10-03'11'21AM:PIZZEYS 61 7 3 2 218 0 77 6 16

32. An antibody immunologically specific for a polypeptide comprising a sequence set forth in Table 2.

33. A method for introducing the nucleic acid of claim into a host, wherein said nucleic acid is delivered to said host via a process selected from the group consisting of transformation, transfection, transduction, transgenetics, surgically and by physical bombardment with nucleic acid- coated particles. I0

34. An isolated viral antigen comprising an amino acid sequence selected from the group consisting of SEQ ID NOs; 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and

35. The viral antigen of claim 34, further comprises a protein tag sequence.

36. A method of raising an immune response in a mammalian subject comprising administering to said subject the viral antigen encoded by the nucleic acid of claim

37. A method of raising an immune response in a mammalian subject comprising administering to said subject a viral antigen of claim 34.

38. A method of amplifying an HCV nucleic acid that encodes an HCV NS5S protein having a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and 65, said method employs primers comprising sequences selected from the oligonucleotide sequences set forth in Table 1. 72 COMS ID No: SMBI-00457254 Received by IP Australia: Time 11:20 Date 2003-10-17 0 10-10-03:03:17 PM: PI ZZEYS3 6 1 .7 3 22 1 S0"/77 15/ 59

39. An isolated host cell comprising a HCV nucleic acid that encodes a HCV NS5B protein, said NS5B protein having a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and The host cell of claim 39, wherein said host cell is selected from the group of cells consisting of bacterial cells, fungal cells, insect cells, mammalian cells, and plant cells.

41. The host cell of claim 40, wherein said host cell is a hepatocyte.

42. A cultured cell line comprising a nucleic acid that encodes a HCV NS5B protein, said NS5B protein having a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and

43. The cell line of claim 42, which is selected from the group consisting of a hepatocyte cell line, Chang liver cells, Hela cells, U937 cells, HepG2 cells, MT-4 cells and clonal. cells generated from said cell lines. 0 S. S SO' SO

44. The cell line of claim 43, functional NS5B protein. The cell line of claim 44, reporter system wherein a reporting reporter system is dependent on the functional HCV NS5B protein in said which expresses a further comprising a signal which from said polymerase activity of the cell line. COMS ID No: SMBI-00448164 Received by IP Australia: Time 15:26 Date 2003-10-10 1 0-10-03:03:ISPM P I ZZEYS 61 7 322 18077 16/ 59

46. A cell line of claim 45, wherein said reporter system includes a nucleic acid comprising the antisense strand of an RNA molecule encoding a functional reporter molecule, said NS5B polymerase acting on said antisense molecule to generate a translatable mRNA sequence in said host cell, said translated reporter molecule sequence being capable of producing an increase in signalwhich is indicative of the presence of a functional NS5B polymerase.

47. The cell line of claim 46 wherein said functional mRNA molecule encodes a reporter molecule selected from the group consisting of luciferase, beta-galactosidase, alkaline phosphatase or fluorescent green protein host cell is a hepatocyte. .9

48. A method for assessing the functionality of a hepatitis C NS5B protein, comprising: i) providing a cell line as claimed in claim 42; ii) transforming said cell line with a S• nucleic acid encoding an antisense molecule, the complement of which encodes a functional reporter molecule, said NS5B polymerase acting on said antisense 25 molecule to generate a translatable mRNA sequence in said host cell; and iii) assessing said cells for an increase in signal produced by said translated reporter molecule sequence, said increase indicating the presence of a functional NS5B protein. 74 COMS ID No: SMBI-00448164 Received by IP Australia: Time 15:26 Date 2003-10-10 17-10-03 1 1:21AM;PI ZZEYS ;61 7 32218077 7/ 16

49. The method of claim 48, wherein said functional reporter molecule is selected from a group consisting of luciferase, 13-galactosidase, alkaline phosphatase and fluorescent green protein. A method for assaying a test compound for antagonist activity against hepatitis C virus comprising: i) providing a cell line according to claim 42; ii) contacting said cell line with a test compound suspected of having antagonistic hepatitis C virus NS5B activity; and iii) measuring the antagonistic effect, if any, of said test compound on hepatitis C virus NS5B activity.

51. A method for assaying a test compound for agonist activity against hepatitis C virus comprising: 20 i) providing a cell line according to claim ii) contacting said cell line with a test compound suspected of having agonistic hepatitis C virus activity; and iii) measuring the agonistic effect, if any, of said test compound on hepatitis C virus activity. COMS ID No: SMBI-00457254 Received by IP Australia: Time 11:20 Date 2003-10-17 l0-03;03:ISPM:;PIlZZEYS 6 1 7 32218077

52. A method of preparing a hepatitis C virus protein comprising a sequence selected from a group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, and 64, comprising: i) expressing said protein in a biological system; ii) solubilizing said protein; iii) extracting said protein and iv) enriching for said NS5B protein.

53. The method of claim 52, wherein said HCV *see osee protein further comprising a protein tag sequence. koo0o0 0 Oe 15 54. The method of claim 52, wherein said NS5B protein is enriched by a process selected from the group consisting of cell disruption, cell fractionation, partitioning, reverse micelle partitioning, aqueous two-phase extraction, precipitation, chromatography, ion exchange chromatography, chelation chromatography, affinity chromatography, immunoaffinity chromatography, high pressure liquid 00 00 Ichromatography, hydrophobic interaction chromatography, Goe- centrifugation, membrane filtration, gel filtration, 0000 'immunoprecipitation, electrophoresis, isoelectric focusing, O eo 25 and isotachophoresis. 0000 0 0

55. The method of claim 52, wherein said biological system is selected from the group consisting of bacterial cells, fungal cells, insect cells, mammalian cells, and plant cells. 76 18/ 59 COMS ID No: SMBI-00448164 Received by IP Australia: Time 15:26 Date 2003-10-10 17-10-03;II:21AM;PIZZEYS 6 I 7 32218077 8/ 16

56. The method of claim 52, wherein said biological system is cell free.

57. A method for isolating an HCV NS5B protein having a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, and 65, said method comprises: i) contacting an extract of a biological system containing the NS5B protein with a solid matrix possessing a diethylaminoethyl (DEAE) moiety as a functional group in a salt-containing buffer solution; ii)removing said NS5B protein from said solid matrix and further contacting said protein with a solid matrix comprising heparin as a functional group; iii) exposing said heparin matrix containing bound NS5B protein to a buffer solution of continuously increasing levels of salt; iv) collecting NS5B protein-containing fractions and contacting said collected fractions with a 20 solid matrix containing Cibacron Blue as a functional group; exposing said Cibacron Blue matrix containing bound NS5B protein to a buffered solution containing increasing levels of salt; and vi) collecting said enriched NS5B containing fractions. *9 A method of generating an infectious viral S. ~vector, comprising incorporating a HCV NS58 nucleic acid that 30 encodes a HCV NS5B protein having a sequence selected from the S* group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and 65 into said viral vector. 77 COMS ID No: SMBI-00457254 Received by IP Australia: Time 11:20 Date 2003-10-17 1 0-10-03;03:IPM'PIZZEYS ;61 7 32218077 20/ 59

59. A method of generating an infectious viral vector comprising substituting an HCV NS5B homologous gene sequence derived from any HCV sequence with a hepatitis C virus NS5B nucleic acid that encodes a HCV protein having a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and

60. A host animal comprising a nucleic acid sequence that encodes an HCV N5B protein having a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and o 15 61. A method of isolating antibodies to HCV protein comprising screening a human or murine antibody library for reactivity to NS5S proteins having a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and and selecting a clone from said library which expresses the :reactive antibody and isolating said clone. .62. A method for propagating HCV in an in vitro system comprising culturing a cell line in which 25 functional HCV NSSB sequences are provided, said sequences encode amino acid sequences selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 64, and 65, under conditions that facilitate the replication of HCV. 78 COMS ID No: SMBI-00448164 Received by IP Australia: Time 15:26 Date 2003-10-10 I0-1O-03;03'IgPM:PIZZEYS 61 7 32218077 21/ 59

63. A method for infecting a cell culture with hepatitis C virus comprising administering a viral vector which is generated as claimed in claim 58.

64. A method for infecting an animal with hepatitis C virus comprising administering a viral vector which is generated as claimed in claim 58.

65. A method of propagating hepatitis C virus in vitro comprising: i) contacting a cell line with a functional HCV NS5B protein consisting essentially of a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 15 54, 55, 56, 67, 68, 69, 6, 62, 63 and 64; and 'ii) said cell line being further contacted with an HCV nucleic acid.

66. The method of claim 65, wherein said NS5B nucleic acid is provided within a hepatitis C virus replicon.

67. A method of propagating hepatitis C virus in a living host, comprising: i) delivering a nucleic acid encoding a 25 hepatitis C virus NS5B protein having a sequence selected from the group consisting SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 63, and 64, to a living host, said nucleic acid encoding a functional NSSB protein and being operably linked to an expression operon; and ii) contacting said living host with an HCV molecule selected from the group consisting of HCV particles, HCV nucleic acids, and HCV replicons. 79 COMS ID No: SMBI-00448164 Received by IP Australia: Time 15:26 Date 2003-10-10 17-10-03;II:22AM;PIZZEYS 6 1 7 32218077 9/ 16

68. A method of claim 67, wherein said nucleic acid is delivered to said. host via a process selected from the group consisting of transformation, transfection, transduction, transgenetics, surgically or by physical bombardment with nucleic acid-coated particles.

69. A method according to claim 67 wherein said NS58 encoding nucleic acid of step i) is provided within a replicon. A kit when used for detecting the presence of an HCV nucleic acid in a biological sample, said HCV nucleic acid encodes an HCV NS5B protein having a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, and 65, comprising: i) oligonucleotides comprising a sequence set forth in Table I, said oligonucleotides being 20 hybridizable to said HCV encoding nucleic acid; ii) reaction buffer; and iii) an instruction sheet.

71. The kit of claim 70 when used for detecting the presence of an HCV nucleic acid in a biological sample, said nucleic acid encoding an NS5B protein having a sequence selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, and COMS ID No: SMBI-00457254 Received by IP Australia: Time 11:20 Date 2003-10-17 17-10-03;II:22AM;PI ZZEYS 61 7 3 2 218 0 ?7 iiI

72. A kit for detecting the presence of HCV in a biological sample, comprising: i) antibodies immunologically specific for HCV proteins having sequences selected from the group consisting of SEQ ID NOs: 2, 8, 9, 13, 54, 55, 56, 57, 58, 59, 61, 62, 63, 64, and ii) a solid support with immobilized HCV antigens as a positive control; and iii) an instruction sheet.

73. A kit of claim 72, wherein said antibody contains a tag. 20 9 o 09* 9 DATED THIS SEVENTEENTH DAY OF OCTOBER 2003 VIROPHARMA INCORPORATED BY PIZZEYS PATENT AND TRADEMARK ATTORNEYS 9 9 COMS ID No: SMBI-00457254 Received by IP Australia: Time 11:20 Date 2003-10-17 WO 99/54 781 PCTIUS99/07404 1/12 MSMSYTWTGAL ITPCAAEESK LPINALSNSL LRRHNMVYAT TSRSASQRQK 50 Clone 4; SEQ ID NO: 2 Clone 14; SEQ ID NO: 8 Clone 21; SEQ ID NO: 9 Clone 11; SEQ ID NO: Clone 16; SEQ ID NO: 11 Clone 20; SEQ ID NO: 12 KVTFDRLQVL DDHYRDVLKE MKAKASTVKA SVWKDIJLEDTI FGYGAKDVRN LSSKAVNHIH KLLSVEEACK LTPPHSARSK 100 II TPQATM YGEFYSPQ 250 1I KGGRKPARLI VYPDLGVRVC r.EKM.uxaYNWSb D-I- 0D--- 0D--- IV/A VEFLVNAWKS KKCPMGFAYD TRCFDST'E p QAIRSLTERL I AACRAAKLQD YIGGPLTNSK NDIRVEESIY QCCDLAPF-A-R 250 V/B GQSCGYRRCR ASGVLTTSCG NTLTCYLICAS 300 VI/ C C 'MLVCGDDL VVICESAGTQ EDAASLRVFT EAMTRYSAPP 350 VII/D VIII I I GDPPQPEYDL ELITSCSSNV SVAHDASGKR VYYLTRDPTT PLARAAWETA 400 Fig. lA WO 99/51781 PCT/US99/07404 2/12 RHTPVNSWLG NI IHYAPTLW ARMILMTHFF SILLAQEQLE KALDCEIYGA 450 HYSIEPLDLP RHRARSVRAK FVAGYSGGDI OX IQRLHGLS LLSQGGRAAT YHSLSRARPR AFSLHSYSPG CGKYLFNWAV WFMWCLLLLS EINRVASCLR RTKLKLTPIP VGVGIYLLPN KLGVPPLRVW 500 AASQLDSGW 591 Clone 4 SEQ ID NO: 2 Clone 14; SEQ ID NO: 8 Clone 21; SEQ ID NO: 9 Clone 11; SEQ ID NO: Clone 16; SEQ ID NO: 11 Clone 20; SEQ ID NO: 12 Fig. lB