WO1999051781A9 - Compositions contenant des sequences ns5b du virus de l'hepatite c et leurs procedes d'utilisation - Google Patents

Compositions contenant des sequences ns5b du virus de l'hepatite c et leurs procedes d'utilisation

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Publication number
WO1999051781A9
WO1999051781A9 PCT/US1999/007404 US9907404W WO9951781A9 WO 1999051781 A9 WO1999051781 A9 WO 1999051781A9 US 9907404 W US9907404 W US 9907404W WO 9951781 A9 WO9951781 A9 WO 9951781A9
Authority
WO
WIPO (PCT)
Prior art keywords
seq
protein
ns5b
hepatitis
sequence
Prior art date
Application number
PCT/US1999/007404
Other languages
English (en)
Other versions
WO1999051781A1 (fr
Inventor
Marc S Collett
Original Assignee
Viropharma Inc
Marc S Collett
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Viropharma Inc, Marc S Collett filed Critical Viropharma Inc
Priority to IL13880499A priority Critical patent/IL138804A0/xx
Priority to EP99921379A priority patent/EP1068362A4/fr
Priority to AU38610/99A priority patent/AU768177B2/en
Priority to CA002325016A priority patent/CA2325016A1/fr
Priority to JP2000542492A priority patent/JP2002510509A/ja
Publication of WO1999051781A1 publication Critical patent/WO1999051781A1/fr
Publication of WO1999051781A9 publication Critical patent/WO1999051781A9/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to hepatitis C virus (HCV) , and more specifically, to the HCV NS5B gene and the encoded protein.
  • HCV hepatitis C virus
  • the invention relates to novel compositions comprising HCV NS5B 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.
  • HCV Hepatitis C virus
  • interferon alpha For treatment of hepatitis due to HCV, interferon 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 (C, 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 daunting task.
  • HCV can not yet be efficiently propagated in vi tro .
  • 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 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.
  • 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.
  • NS3 protein possesses serine proteinase, nucleoside triphosphatase and helicase activities and the NS5B protein possesses RNA-dependent RNA polymerase (RdRp) activity.
  • RdRp RNA-dependent RNA polymerase
  • 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.
  • the present invention provides novel HCV NS5B nucleotide sequences from which recombinant HCV proteins 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 .
  • an isolated nucleic acid molecule 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.
  • 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 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 NS5B protein.
  • 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 NS5B.
  • 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.
  • 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.
  • the changes will not affect, or substantially affect, the structure or useful properties of the polypeptide.
  • HCV NS5B variants may suitably possess functional NS5B 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 to or otherwise affect the HCV NS5B activity.
  • HCV NS5B variants can be either naturally occurring (i.e., purified or isolated from a natural source) or synthetic (i.e., generated by biological expression of DNA 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.
  • 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 vi tro, 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.
  • 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 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.
  • 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 vi tro systems such as cell, tissue and organ cultures.
  • 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.
  • 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.
  • 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.
  • a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3 ' direction.
  • isolated nucleic acid 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.
  • an "isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.
  • isolated nucleic acid refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues) .
  • An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.
  • Natural allelic variants refer to nucleic acid sequences that are closely related to a particular sequence but which may possess, either naturally or by design, changes in sequence or structure.
  • closely related it is meant that at least about 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.
  • percent similarity when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program.
  • NS5B refers to a portion of the HCV genome located near the 3 ' end of the viral genome that specifies the region encoding a protein, termed the "NS5B 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) .
  • RdRp RNA-dependent RNA polymerase
  • the nucleic acid region encoding the NS5B protein may also be referred to as the "NS5B gene".
  • NS5B may refer to either a nucleic acid encoding the
  • NS5B polypeptide to an NS5B gene or to an NS5B polypeptide, or to any portions thereof, depending on the context in which the term is used.
  • NS5B may further refer to natural allelic variants, mutants and derivatives of either NS5B nucleic acid sequences or
  • NS5B polypeptides The NS5B nucleic acid, NS5B gene or NS5B protein referred to may be either functional or non-functional .
  • the present invention also includes active portions, fragments, derivatives and functional or non- functional mimetics of HCV NS5B polypeptides or proteins of the invention.
  • An "active portion" of HCV NS5B 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 NS5B 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 NS5B 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.
  • the HCV NS5B polypeptide or protein of the invention includes any analogue, fragment, derivative or mutant which is derived from a HCV NS5B polypeptide and which retains at least one property or other characteristic of the HCV NS5B polypeptide.
  • variants of the HCV NS5B polypeptide exist in nature. These variants may be alleles characterized by differences in the nucleotide sequences of the gene coding for the protein, or may involve different RNA processing or post-translational modifications. The skilled person can produce variants having single or multiple amino acid substitutions, deletions, additions or replacements.
  • variants may include inter alia: (a) variants in which one or more amino acids residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the HCV NS5B polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the HCV NS5B 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.
  • 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 .
  • nucleic or amino acid sequence is functional for the recited assay or purpose.
  • consisting essentially of when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID No:.
  • the phrase when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the basic and novel characteristics of the sequence.
  • a “replicon” is any genetic element, 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 (e.g., ATG or AUG codons) , polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
  • transcriptional and translational control sequences such as promoters, enhancers, translational start signals (e.g., ATG or AUG codons) , polyadenylation signals, terminators, and the like, and which facilitate the expression of a polypeptide coding sequence in a host cell or organism.
  • oligonucleotide, " 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.
  • oligonucleotide The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.
  • probe refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe.
  • a probe may be either single-stranded or double-stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method.
  • 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.
  • a non-complementary nucleotide fragment may be attached to the 5' or 3 ' end of the probe, with the remainder of the probe sequence being complementary to the target strand.
  • non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specfically.
  • the term “specifically hybridize” refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule of the invention, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
  • primer refers to an oligonucleotide, either RNA or DNA, either single-stranded or double-stranded, either derived from a biological system, generated by restriction enzyme digestion, or produced synthetically which, when placed in the proper environment, is able to functionally act as an initiator of tempiate-dependent nucleic acid synthesis.
  • suitable nucleoside triphosphate precursors of nucleic acids, a polymerase enzyme, suitable cofactors and conditions such as a suitable temperature and pH
  • the primer may be extended at its 3 ' terminus by the addition of nucleotides by the action of a polymerase or similar activity to yield an primer extension product.
  • the primer may vary in length depending on the particular conditions and requirement of the application.
  • the oligonucleotide primer is typically 15-25 or more nucleotides in length.
  • the primer must be of sufficient complementarity to the desired template to prime the synthesis of the desired extension product, that is, to be able anneal with the desired template strand in a manner sufficient to provide the 3 ' hydroxyl moiety of the primer in appropriate juxtaposition for use in the initiation of synthesis by a polymerase or similar enzyme. It is not required that the primer sequence represent an exact complement of the desired template.
  • a non-complementary nucleotide sequence may be attached to the 5 ' end of an otherwise complementary primer.
  • non-complementary bases may be interspersed within the oligonucleotide primer sequence, provided that the primer sequence has sufficient complementarity with the sequence of the desired template strand to functionally provide a template-primer complex for the synthesis of the extension product.
  • Amino acid residues described herein are preferred to be in the "L” isomeric form. However, residues in the "D" isomeric form may be substituted for any L-amino acid residue, provided the desired properties of the polypeptide are retained. All amino-acid residue sequences represented herein conform to the conventional left-to-right amino-terminus to carboxy-terminus orientation.
  • isolated protein or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. 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.
  • substantially pure refers to a preparation comprising at least 50-60% by weight of a given material (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75% by weight, and most preferably 90-95% by weight of the given compound. Purity is measured by methods appropriate for the given compound
  • “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 protoelytic processing from a polyprotein precursor.
  • the first amino of the mature protein sequence is designated as amino acid residue 1.
  • the first amino acid residue of the mature NS5B protein represents a serine residue for all known HCV sequences.
  • 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 -1, -2, -3 and so on.
  • a methionine initiator codon is often utilized for purposes of efficient translation.
  • 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.
  • tag refers to a chemical moiety, either a nucleotide, oligonucleotide, polynucleotide or an amino acid, peptide or protein or other chemical, that when added to another sequence, provides additional utility or confers useful properties, particularly in the detection or isolation, to that sequence.
  • a homopolymer nucleic acid sequence or a nucleic acid sequence complementary to a capture oligonucleotide may be added to a primer or probe sequence to facilitate the subsequent isolation of an extension product or hybridized product.
  • histidine residues may be added to either the amino- or carboxy-terminus of a protein to facilitate protein isolation by chelating metal chromatography.
  • amino acid sequences, peptides, proteins or fusion partners representing epitopes or binding determinants reactive with specific antibody molecules or other molecules (e.g., flag epitope, c-myc epitope, transmembrane epitope of the influenza A virus hemaglutinin protein, protein A, cellulose binding domain, calmodulin binding protein, maltose binding protein, chitin binding domain, glutathione S-transferase, and the like) may be added to proteins to facilitate protein isolation by procedures such as affinity or immunoaffinity chromatography.
  • Chemical tag moieties include such molecules as biotin, which may be added to either nucleic acids or proteins and facilitates isolation or detection by interaction with avidin reagents, and the like. Numerous other tag moieties are known to, and can be envisioned by, the trained artisan, and are contemplated to be within the scope of this definition.
  • reporter 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, e.g., by biological assay, immunoassay, radioimmunoassay, or by colorimetric, fluorogenic, chemiluminescent or other methods.
  • the nucleic acid may be either RNA or DNA, linear or circular, single or double stranded, antisense or sense polarity, and is operatively linked to the necessary control elements for the expression of the reporter gene product .
  • the required control elements will vary according to the nature of the reporter system and whether the reporter gene is in the form of DNA or RNA, but may include, but not be limited to, such elements as promoters, enhancers, translational control sequences, poly A addition signals, transcriptional termination signals and the like.
  • transfection 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, PEG- fusion and the like.
  • the introduced nucleic acid may or may not be integrated (covalently linked) into nucleic acid of the recipient cell or organism.
  • the introduced nucleic acid may be maintained as an episomal element or independent replicon such as a plasmid.
  • the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism and further passed on or inherited to progeny cells or organisms of the recipient cell or organism.
  • the introduced nucleic acid may exist in the recipient cell or host organism only transiently.
  • a "clone” or "clonal cell population” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell or cell population that is capable of stable growth in vi tro for many generations.
  • Immune response signifies any reaction produced by an antigen, such as a viral antigen, in a host having a functioning immune system.
  • Immune responses may be either humoral in nature, that is, involve production of 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 vi tro and in various cellular or animal systems . Such immune responses may be important in protecting the host from disease and may be used prophylactically and therapeutically.
  • viral antigen shall be any peptide, polypeptide or protein sequence, segment or epitope that is derived from a virus that has the potential to cause a functioning immune system of a host to react to said viral antigen.
  • 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.
  • antibody or antibody molecule contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunloglobulin molecule such as those portions known in the art as Fab, Fab', F(ab')2 and F(v) .
  • the term "living host” shall mean any non-human autonomous being.
  • Figure 1 shows an alignment of the amino acid sequences of several NS5B proteins of the invention.
  • FIG. 2 shows the purification of the HCV NS5B 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 NS5B sequence of the invention with the genotype la consensus NS5B sequence derived from an infectious clone (GenBank Accession Number AF009606) and a genotype la NS5B 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 NS5B sequence of the invention with a genotype lb "consensus" amino acid sequence derived from sequences of HCV NS5B 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 : 5.
  • 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.
  • HCV is the major causative agent of transfusion- associated 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.
  • the present invention provides HCV genetic material derived from an HCV- infected patient.
  • the invention further provides HCV recombinant NS5B proteins expressed from these sequences and sequences that have RdRp activity.
  • 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.
  • 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 NS5B 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 NS5B protein.
  • 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.
  • HCV NS5B 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 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.
  • NS5B 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.
  • Nucleic acid molecules encoding the HCV NS5B proteins of the invention may be prepared by two general methods: (1) They may be synthesized from appropriate chemical starting materials, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art.
  • nucleotide sequence information such as that provided herein for HCV NS5B 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 Radios
  • Biosystems 38A DNA Synthesizer or similar devices may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC) .
  • HPLC high performance liquid chromatography
  • 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 with the protein coding region of the DNA molecules of the present invention may be identified by using hybridization and washing conditions of appropriate stringency.
  • hybridizations may be performed, using a hybridization solution comprising, for example, 5X SSC, 5X Denhardt ' s reagent, 1.0% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide. Hybridization is carried out at 37-42°C for at least six hours.
  • filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65°C in IX SSC and 1% SDS, changing the solution every 30 minutes.
  • T m 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex
  • clones are maintained in plasmid cloning/expression vectors, such as pBluescript plasmids (Stratagene, La Jolla, CA) or recombinant baculovirus transfer vectors such as pFastBac vectors (Gibco-BRL, Gaithersburg, MD) that are propagated in suitable E. coli host cells.
  • plasmid cloning/expression vectors such as pBluescript plasmids (Stratagene, La Jolla, CA) or recombinant baculovirus transfer vectors such as pFastBac vectors (Gibco-BRL, Gaithersburg, MD) that are propagated in suitable E. coli host cells.
  • nucleic acids of the invention may also be used as starting materials for the generation of sequence variants of the nucleic acids of the invention using any number of synthetic and molecular biologic procedures well known in the art including but not limited to site-directed mutagenesis techniques. Particular mutations may give rise to 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.
  • 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 NS5B encoding nucleic acids.
  • 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, e.g., 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.
  • a cDNA or gene may be cloned into an appropriate in vi tro transcription vector, such as pSP64 or pSP65 for in vi tro 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.
  • vi tro transcription and translation systems are commercially available (e.g., Promega Biotech, Madison, WI ; Gibco-BRL, Gaithersburg, MD) .
  • 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.
  • 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 elements necessary for expression of the DNA in the host cell (e.g., E. coli or insect cell), positioned in such a manner as to permit expression of the DNA in the host cell.
  • regulatory elements required for expression may include promoter sequences, transcriptional initiation and termination sequences, enhancer sequences, translational control sequences and the like.
  • 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 .
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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) .
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • Appropriate primers pairs may be further used in nested PCR applications.
  • Such primers, primer pairs and probes may represent any portion of the NS5B sequences of the invention. The actual sequence of the NS5B gene used will vary according to the specific application.
  • 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.
  • oligonucleotides of from 10 to 80 nucleotides in length that are either the same as or complementary to the sequences of the invention are useful as hybridization probes or as primers in RT-PCR applications.
  • the entire NS5B sequence may be employed as a capture hybridization probe, for example.
  • oligonucleotide primers and probes of the invention comprise sequences set forth in Table 1, sequences complementary to these and potions of these sequences.
  • nucleic acid sequences of the invention may be used as primers for the generation of variants or mutants of the sequences of the invention using a variety of methodologies known in the art, including site-directed mutagenesis procedures.
  • nucleic acid sequences of the invention may be used in the construction or generation of, or incorporated into, infectious viruses, vectors or replicons. Provision or substitution of the functionally superior NS5B sequences of the invention for poorly functional or non-functional counterparts will serve to improve the infectious and replicative characteristics of the resulting virus or replicating unit.
  • Rice et al . (PCT WO 98/39031) provide an infectious nucleic acid of HCV.
  • the NS5B 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 .
  • 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 vi tro systems. Use in other systems in which the function of the HCV
  • NS5B 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.
  • the nucleic acid sequences of the invention may be used in methods to elicit immune responses to the HCV NS5B protein.
  • the NS5B-encoding nucleic acid sequences of the invention operationally linked to an expression operon may introduced directly into cells, particularly into antigen presenting cells such as dendritic cells, of a living host or human possessing a functioning immune system.
  • Introduction of the sequences may utilize transfection, transformation, or transduction methods, or involve the physical uptake of particles coated with the NS5B nucleic acid sequences, such as plasmid-coated gold particles.
  • the NS5B sequences 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.
  • 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.
  • RNA synthesis that is directly or indirectly dependent on the HCV NS5B polymerase activity may be quantified.
  • the amount of a radiolabeled precursor of RNA e.g., 3 H- uridine
  • trichloroacetic acid- precipitable RNA that is dependent on NS5B activity
  • such cell systems may utilize a reporter system in which the production of the reporter signal is dependent on RNA synthesis by the HCV NS5B polymerase.
  • 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) .
  • 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, ⁇ - galactosidase, alkaline phosphatase, fluorescent green protein and the like.
  • the cell systems that may be used in this method of the invention may be of bacterial, fungal, insect, avian, mammalian or plant origin.
  • nucleic acid sequences of the invention may be used in assays to identify agents or materials capable of interacting with or affecting the HCV NS5B nucleic acid sequences.
  • assays may be established in which nucleic acid sequences of the invention are provided and then contacted with agents or materials suspected of interacting with such sequences.
  • Agents identified in such interaction assays would then have potential diagnostic utility and uses in the detection of HCV in, for example, biological samples.
  • 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 vi tro 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.
  • the NS5B proteins, or peptides thereof may be used in assays for the detection of immune responses to the same.
  • protein sequences or peptides of the invention may be used in assays in which said sequences are immobilized on a matrix and used to capture antibodies directed to said sequences.
  • protein sequences or peptides of the invention may be used to detect or measure cell -mediated immune responses to the protein, such as in immune cell proliferation assays.
  • the HCV NS5B protein compositions of the invention also have potential utility in the elicitation of immune responses, such as in vaccines.
  • 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 NS5B.
  • NS5B protein may be immobilized and used to bind antibodies specific to the NS5B protein, and thus enrich for such antibodies.
  • the NS5B 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 NS5B protein, whether polyclonal or monoclonal, may be further evaluated for their ability to affect the enzymatic activity of the NS5B RdRp activity.
  • NS5B protein sequences useful in the above applications comprise sequences or portions thereof, provided in Table 2.
  • 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.
  • agents or materials suspected of interacting with such sequences For example, upon provision of the HCV NS5B protein of the invention, or fragment or portion thereof, contacted agents may be assessed for their ability to bind specifically to the protein.
  • binding agents would then have potential diagnostic utility and uses in the detection of HCV in, for example, biological samples.
  • 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 vi tro systems such as cell, tissue and organ cultures or in biological materials.
  • Agents that enhance the function of the NS5B 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 vi tro systems such as cell, tissue and organ cultures.
  • 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 vitro cell-free expression system or in an enriched or purified form.
  • NS5B- 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.
  • the incorporation of a precursor of RNA into a polymer of RNA is measured, such as the incorporation of a radiolabeled NTP into trichloroacetic acid-precipitable RNA, which may then be quantified by scintillation spectrometry or phosphorimaging technologies .
  • Such precursors may alternatively be tagged with other moieties to allow their ready detection such as with biotin for detection with avidin reagents including various avidin conjugates such alkaline phosphatase and the like or with fluorescently-labeled NTPs for detection using fluorescent technologies such as fluorescence polarization.
  • NS5B-dependent RNA synthesis may also be assessed by measuring the extension of a pre-labeled or tagged primer of RNA synthesis such as a radiolabeled or biotin-tagged oligonucleotide that is used by the polymerase to initiate RNA synthesis on a template RNA molecule.
  • Extension of the primer may be assessed by quantifying the addition of nucleoside triphosphates to the primer, by determining the length of the primer product , or by other methods known in the art .
  • the product of NS5B RdRp activity may be detected and quantified by capture of the product RNA using hybridization techniques.
  • an oligonucleotide complementary to the product of the NS5B 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 .
  • 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.
  • 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, peptide- nucleic acid conjugates, ribozymes, polynucleotides and the like.
  • agents or materials identified using the compositions and assay methods of the invention will be broad and will include uses for the detection and isolation of 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 vi tro systems such as cell, tissue and organ cultures, as well as for other uses the may be envisioned once the nature of the agent is clear.
  • kits to facilitate the use of the compositions and methods disclosed herein include kits to facilitate the use of the compositions and methods disclosed herein.
  • Exemplary kits would include HCV NS5B nucleic acids and polypeptides of the invention, and/or variants thereof, alone or in suitable vectors.
  • Such reagents may include, but not be limited to, buffers, solvents, media and solutions, substrates and cofactors, vectors and host cells, and detection or reporter reagents.
  • Accessory items may include vials, vessels, reaction chambers and instruction sheets.
  • RNA isolated from the serum of an HCV-infected patient was used to amplify the HCV NS5B gene using an RT-nested PCR protocol.
  • 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°C for 50 minutes followed by a 1 minute 94°C denaturation, followed by 40 cycles of first round PCR, 15 cycles of 94 °C for 30 seconds, 50°C for 30 seconds 68°C for 2 minutes and 25 cycles of 94°C for 30 seconds, 50°C for 30 seconds, 68°C for 2 minutes.
  • 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 Notl restriction sites, respectively (underlined) for subsequent cloning purposes.
  • the second round PCR involved an initial denaturation at 94°C for 1 minute, followed by 10 cycles of 94°C for 45 seconds, 50°C for 30 seconds, 68°C for 2 minutes and 20 cycles of 94°C for 30 seconds, 50°C for 30 seconds, 68°C for 2 minutes.
  • the resultant PCR product was purified, digested with EcoRI and Notl and ligated to pFastBac plasmid (Gibco-BRL) previously digested with EcoRI and Notl.
  • 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 DHlOBac cells several colonies containing bacmid DNA were transfected into Sf9 insect cells according to the protocol supplied by the manufacturer.
  • NS5B protein expression in recombinant baculovirus-infected cells was verified by Western immunoblot analysis with antiserum specific to the HCV NS5B sequences .
  • 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 NS5B 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.
  • 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.
  • 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 NaCl, 1 mM EDTA, 2% Triton X-100) supplemented with a Complete Protease Inhibitor tablet (Boehringer Mannheim) . MgCl 2 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 was clarified by centrifugation at 35,000 rpm for 35 minutes at 4°C.
  • lysis buffer 50% glycerol, 20 mM Tris-HCl, pH 7.5 , 10 mM dithiothreitol (DTT) , 0.5 M NaCl, 1 mM EDTA, 2% Triton X-100
  • the clarified lysate was diluted to a final NaCl concentration of 0.3 M with elution buffer (20% glycerol, 20 mM Tris-HCl, pH 7.5 , 10 mM DTT, 1 mM EDTA, 0.5% 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 NaCl 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.
  • elution buffer 20% glycerol, 20 mM Tris-
  • NS5B-containing fractions were pooled and loaded onto a Cibacron Blue column equilibrated in elution buffer containing 0.4 M NaCl. Bound proteins were eluted with a linear gradient of NaCl (400 mM to 4M NaCl in elution buffer) while fractions were collected.
  • NS5B-containing 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°C. Typical purification results are shown in Figure 2.
  • HCV NS5B RdRp activity uses a purified recombinant NS5B protein in an in vi tro RdRp assay.
  • 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.
  • PCT WO 97/12033 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.
  • 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.
  • 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 ⁇ L 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 ⁇ g pOF1213 RNA at 30°C for 60 minutes. The reaction is terminated by the addition of cold trichloroacetic acid (TCA) and sodium pyrophosphate . The TCA-precipitable radioactivity was then quantified to determine the extent of RdRp activity.
  • TCA cold trichloroacetic acid
  • 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 NS5B proteins of the invention and the consensus sequence protein of Kolykhalov.
  • Protein activity* relative to clone 4 cclloonnee 44 (SSEEQQ I IDD N NOO : 2 2 )) 9 9..33 cclloonnee 1144 ( (SSEEQQ I IDD N NOO: 8 8)) 1 1..77 2 clone 21 (SEQ ID NO 9) 1.9 5 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 A AFF000099660066 0 0..99 71
  • This example demonstrates a range of functional activities among cloned and expressed NS5B sequences, from very active to inactive.
  • 6 NS5B 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.
  • the data presented in this example demonstrate that the NS5B RdRp activity derived from clone 4 has dramatically superior activity over that of the NS5B derived from the infectious clone identified by GenBank Accession Number AF009606. This result indicates that the mere generation of a consensus sequence of an NS5B protein does not necessarily provide an NS5B protein with optimal functionality.
  • 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 5.
  • the sequence disclosed by Kolykhalov et al . 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 4) .
  • This demonstrates that the clone 4 NS5B 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 nature of the sequences of the invention.
  • 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 -1) , the clone 4 sequence differs from this genotype lb consensus sequence by 13 amino acids. Alignment of this genotype lb consensus sequence with all other sequences of the invention similarly demonstrates the distinct nature of the sequences of the invention.
  • Genome 4 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.
  • 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); GenBank Accession Number AF054247] .
  • the clone 4 sequence (SEQ ID NO: 2) differs by 25 amino acid residues, excluding the methionine residue at position Dl in clone 4.
  • the NS5B sequences of the invention do not represent a consensus sequence.
  • clone 4 is a functional RdRp or a functionally superior RdRp.
  • 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.
  • NS5B sequences of the invention 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 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 (V) residue at position -1 of the clone 20 NS5B protein instead of an initiator methionine (M) 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) .
  • 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 -1) 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.
  • Protein# specific activit clone 20 (SEQ ID NO 12) ⁇ 0 1 clone 20 (V-1 ) (SEQ ID NO 13) 23 5 clone 4 (SEQ ID NO 2) 9 3 clone 4 (A75V) (SEQ ID NO 54) 3 5 clone 4 (N177D) (SEQ ID NO 55) 7 2 clone 4 (S543P) (SEQ ID NO 56) 3 5
  • 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 .
  • amino acid substitutions may be either conservative or non- conservative in nature .
  • 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) ] .
  • M2A alanine
  • M2L leucine
  • M2T threonine
  • 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 (e.g., 8, 9, 10, 11 and 13, those of Tables 4 and 5) , 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.
  • nucleic acid sequences encoding variant NS5B protein sequences useful in the practice of the invention comprise sequences provided in Table 6.
  • 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, i.e., SEQ ID NOS: 3, 4, 5, 6 and 7. Such altered nucleic acid sequences are also contemplated for use in the present invention.
  • Table 6 demonstrate various beneficial changes in the nucleic acid sequence of SEQ ID NO: 1.
  • the changes exemplified in SEQ ID NO:l will also have similar utility, either singly or in various combinations, in other NS5B sequences, including sequences of the invention (e.g., 3, 4, 5, 6 and 7,), 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.
  • an assay for polymerase activity that is capable of high volume screening, in other words, a high throughput assay, is desirable.
  • assay methodologies well known to the trained artisan that allow the efficient screening of large numbers of samples [see, for example, Cole, JL, in Meth. Enzymology 275:310-328 (1996)] , 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.
  • 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 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.
  • TCA trichloroacetic acid
  • 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 (1) and last (12) columns of the plate contain activity and background controls and a titration of a reference inhibitor compound.
  • wells Al , Bl, A12 and B12 show the activity of the enzyme in the absence of any test compound (100% of the expected activity) .
  • 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.
  • 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.
  • HCV NS5B proteins may be used to advantage to identify and assess agents or materials that may affect the HCV RdRp and HCV replication.
  • the disclosure of the present invention demonstrates that simple knowledge of an HCV NS5B sequence is insufficient to allow one to conclude, or even reliably predict that such sequence is a functional sequence or has functional utility.
  • an HCV NS5B consensus sequence is not the only, and not necessarily the optimal, functional NS5B protein encoding sequence.
  • the present invention provides novel NS5B sequences with unanticipated functionality.
  • the present invention demonstrates that particular sequence changes in HCV NS5B can lead to unanticipated and significantly improved functionality.
  • the NS5B sequences of the invention represent novel sequences with demonstrated functionality and utility and unique improvements over prior art.
  • the HCV NS5B sequences of the invention have broad utility in research, diagnostic, therapeutic and pharmaceutical applications.

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Abstract

L'invention concerne des acides nucléiques et des protéines de NS5B de HCV. Elle concerne également des procédés et des trousses mettant en application ces compositions.
PCT/US1999/007404 1998-04-02 1999-04-02 Compositions contenant des sequences ns5b du virus de l'hepatite c et leurs procedes d'utilisation WO1999051781A1 (fr)

Priority Applications (5)

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IL13880499A IL138804A0 (en) 1998-04-02 1999-04-02 Hepatitis c virus ns5b nucleic acids and proteins
EP99921379A EP1068362A4 (fr) 1998-04-02 1999-04-02 Compositions contenant des sequences ns5b du virus de l'hepatite c et leurs procedes d'utilisation
AU38610/99A AU768177B2 (en) 1998-04-02 1999-04-02 Hepatitis C virus NS5B compositions and methods of use thereof
CA002325016A CA2325016A1 (fr) 1998-04-02 1999-04-02 Compositions contenant des sequences ns5b du virus de l'hepatite c et leurs procedes d'utilisation
JP2000542492A JP2002510509A (ja) 1998-04-02 1999-04-02 Ｃ型肝炎ウイルスｎｓ５ｂ組成物およびその使用方法

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US6461845B1 (en) * 1995-09-27 2002-10-08 Emory University Recombinant hepatitis C virus RNA replicase
JP2003514562A (ja) * 1999-11-23 2003-04-22 バイロファーマ・インコーポレイテッド ポリメラーゼ組成物およびその使用方法
CA2389691A1 (fr) * 2000-04-07 2001-10-18 Shionogi & Co., Ltd. Methode de pre-incubation pour le dosage d'un inhibiteur d'une arn polymerase arn-dependante liee au vhc
AU2001256745A1 (en) * 2000-05-15 2001-11-26 Chugai Seiyaku Kabushiki Kaisha Vector for analysing replication mechanism of rna virus and use thereof
WO2002070739A2 (fr) * 2001-03-08 2002-09-12 Boehringer Ingelheim (Canada) Ltd Dosage d'inhibiteurs de la polymerase vhc
EP1256628A3 (fr) * 2001-05-10 2003-03-19 Agouron Pharmaceuticals, Inc. ARN polymerase NS5B du virus de la hepatite C et mutants derivés de la polymerase
EP1711516A4 (fr) * 2004-01-09 2009-12-30 Merck & Co Inc Arn polymerase dependant de l'arn du vhc
WO2009022939A1 (fr) * 2007-08-09 2009-02-19 Uchrezhdenie Rossiiskoi Akademii Nauk Institut Molekulyarnoi Biologii Im. V.A. Engelgardta Ran (Imb Ran) Procédé d'identification du génotype et du sous-type du virus de l'hépatite c sur une biopuce

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US5372928A (en) * 1989-09-15 1994-12-13 Chiron Corporation Hepatitis C virus isolates
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JP2002510509A (ja) 2002-04-09
AU768177B2 (en) 2003-12-04
EP1068362A4 (fr) 2005-07-20
AU3861099A (en) 1999-10-25

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