STATEMENT OF PRIORITY

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 62/000,325, filed May 19, 2014, the entire contents of which are incorporated by reference herein.

STATEMENT OF FEDERAL SUPPORT

[0002] This invention was made with government support under Grant Nos.

AI095690, CA164029, and AI109965 awarded by the National Institutes of Health. The government has certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to replication competent clones of hepatitis C virus genotype la and the use thereof to produce virus, study the viral life cycle, and to facilitate drug development and the assessment of viral resistance to therapeutics.

BACKGROUND OF THE INVENTION

[0004] Chronic infection with hepatitis C virus (HCV) is a major public health concern, with an estimated 180 million individuals being infected worldwide (6). HCV- related liver diseases have a high mortality rate, which is typically associated with rapid progression of liver fibrosis, cirrhosis and hepatocellular carcinoma. Recent advances on antiviral therapeutics have enabled effective treatment of HCV infections by

administration of direct-acting antiviral agents (DAAs) (21), however, no effective vaccines are available to date and the mechanism of HCV persistence and carcinogenesis in vivo remain poorly understood.

[0005] HCV is classified within the family Flaviviridae in the genus Hepacivirus and has a positive-strand RNA genome, which encodes a large single polyprotein that is cleaved into 10 mature structural and nonstructural (NS) proteins (1). Replication of hepatitis C virus takes place in the double membrane structures (DMVs) derived from endoplasmic reticulum (ER) membrane (16), formation of which depends on phosphatidylinositol 4-phosphate (PI4P) catalyzed by phosphatidylinositol-4 kinase Ilia (2, 15, 22). HCV studies largely depend on HCV JFH1 or JFHl-based systems, which are chimeric viruses expressing JFH1 replicase composed of nonstructural proteins (NS3- 5B) (7, 13, 23, 28). However, the mechanistic aspects of the robust replication phenotype, in contrast to other typical HCV strains which do not replicate efficiently in cell culture, are not fully understood.

[0006] We have found that the R A replicase from JFH1 is unique among other HCV strains in that it is highly resistant to cellular lipid peroxidation, in contrast to those from other typical HCV strains that are extremely susceptible to it. HCV is unique among all other tested positive-strand RNA viruses in its susceptibility to lipid peroxidation. This sensitivity to lipid peroxidation therefore likely evolved as a viral strategy for down-regulating replication to low levels in order to facilitate long-term viral persistence. This suggests that JFH1 may be a loss-of- function mutant whose RNA replicase activity is no longer regulated by endogenous lipid peroxidation that results from host responses to infection (24), while that of other typical HCV is critically regulated by endogenous lipid peroxidation.

[0007] The present invention addresses previous shortcomings in the art by providing HCV genotype la clones that have a robust replication phenotype in cell culture.

SUMMARY OF THE INVENTION

[0008] The present invention is based on the development of HCV genotype la clones that are capable of robust replication in cultured cells and the use of such clones to produce virus, study the viral life cycle, and to facilitate drug development and the assessment of viral resistance to therapeutics. The invention is further based on HCV genotype la clones that are suitable for identification of additional clones having a robust replication phenotype.

[0009] Accordingly, in one aspect, the invention relates to an isolated polynucleotide encoding a replication competent HCV genotype la genome, said genome comprising: a 5' non-translated region (NTR), a 3' NTR, and a coding sequence present between the 5' NTR and 3' NTR and encoding a HCV polyprotein, wherein the polyprotein comprises an amino acid sequence having at least about 95% identity to amino acids 1-3011 of SEQ ID NO:2, and wherein the amino acid sequence of the polyprotein comprises the following mutations relative to the amino acid positions using SEQ ID NO: 2 as a reference sequence:

aspartic acid at about amino acid 476;

glycine at about amino acid 1226;

leucine at about amino acid 1464;

isoleucine at about amino acid 1655;

serine at about amino acid 1672;

arginine at about amino acid 1691;■

histidine at about amino acid 1773;

threonine at about amino acid 1927;

arginine at about amino acid 2040;

glycine at about amino acid 2979;

phenylalanine at about amino acid 2981; and

serine at about amino acid 2994;

and does not comprise an isoleucine at about amino acid 2204;

wherein the polyprotein further comprises one or more of the following mutations:

serine at about amino acid 1909

glycine at about amino acid 2416; and

aspartic acid at about amino acid 2963.

[0010] In another aspect, the invention relates to a replication competent HCV genotype la genome encoded by the isolated polynucleotide of the invention.

[0011] In a further aspect, the invention relates to a method for making an isolated polynucleotide encoding a replication competent HCV genotype la genome comprising: providing a polynucleotide encoding a replication competent HCV genotype la genome comprising a 5' NTR, 3' NTR, and a coding sequence present between the 5' NTR and 3' NTR and encoding a HCV polyprotein, wherein the polyprotein comprises an amino acid sequence having at least about 95% identity to amino acids 1-3011 of SEQ ID NO:2, and wherein the amino acid sequence of the polyprotein comprises:

aspartic acid at about amino acid 476;

glycine at about amino acid 1226; leucine at about amino acid 1464;

isoleucine at about amino acid 1655;

serine at about amino acid 1672;

arginine at about amino acid 1691;

histidine at about amino acid 1773;

threonine at about amino acid 1927;

arginine at about amino acid 2040;

glycine at about amino acid 2979;

phenylalanine at about amino acid 2981; and

serine at about amino acid 2994;

and does not comprise an isoleucine at about amino acid 2204;

wherein the polyprotein further comprises one or more of the following mutations:

serine at about amino acid 1909

glycine at about amino acid 2416; and

aspartic acid at about amino acid 2963.

[0012] In an additional aspect, the invention relates to a method of producing a replication competent HCV genotype la genome, the method comprising transcribing the isolated polynucleotide of the invention.

[0013] In another aspect, the invention relates to a method for replicating a replication competent HCV genotype la genome, comprising incubating a cell comprising the replication competent HCV genotype la genome of the invention under conditions suitable for the genome to replicate.

[0014] In a further aspect, the invention relates to a method for producing viral particles, comprising incubating a cell comprising the replication competent HCV genotype la genome of the invention under conditions suitable for the genome to replicate.

[0015] In an additional aspect, the invention relates to a method for identifying a compound that inhibits replication of a replication competent HCV genotype la genome, the method comprising contacting a cell comprising the replication competent HCV genotype la genome of the invention with a compound, incubating the cell under conditions wherein the replication competent HCV genotype la genome replicates in the absence of the compound; and detecting the replication level of the replication competent HCV genotype la genome, wherein a decrease in the replication level in the cell contacted with the compound compared to the replication level in a cell not contacted with the compound indicates the compound inhibits replication of the replication competent HCV genotype la genome.

[0016] In a further aspect, the invention relates to an isolated polynucleotide encoding a replication competent HCV genotype la genome, said genome comprising: a 5' NTR, a 3' NTR, and a coding sequence present between the 5' NTR and 3' NTR and encoding a HCV polyprotein, wherein the polyprotein comprises an amino acid sequence having at least about 95% identity to amino acids 1-3011 of SEQ ID NO:2, and wherein the amino acid sequence of the polyprotein comprises the following mutations relative to the amino acid positions using SEQ ID NO: 2 as a reference sequence:

aspartic acid at about amino acid 476;

glycine at about amino acid 1226;

leucine at about amino acid 1464;

isoleucine at about amino acid 1655;

serine at about amino acid 1672;

arginine at about amino acid 1691;

histidine at about amino acid 1773;

threonine at about amino acid 1927;

arginine at about amino acid 2040;

glycine at about amino acid 2979;

phenylalanine at about amino acid 2981; and

serine at about amino acid 2994;

and does not comprise an isoleucine at about amino acid 2204.

[0017] In another aspect, the invention relates to a replication competent HCV genotype la genome encoded by the isolated polynucleotide of the invention.

[0018] In a further aspect, the invention relates to a method for identifying a HCV genotype la genome with a robust replication phenotype, the method comprising incubating a cell comprising the replication competent HCV genotype la genome of the invention and detecting an increase in the replication level of the replication competent HCV genotype la genome.

[0019] In another aspect, the invention relates to use of the replication-competent clones of the invention to assess viral resistance to antiviral agents.

[0020] These and other aspects of the invention are set forth in more detail in the description of the invention below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 shows that genotype la TNcc has the lipid peroxidation-resistant phenotype. Huh-7.5 cells were transfected with H77S.3, TNcc or HJ3-5 RNAs and media containing DMSO, 1 μΜ sphingosine kinase inhibitor (SKI), 1 μΜ -tocopherol (VE), 50 μΜ linoleic acid (PUFA), PUFA plus 1 μΜ VE or 10 μΜ MK-0608 (DA A), an NS5B RNA polymerase inhibitor, added at 6 h. Culture media were replaced with fresh media containing compounds every 24 h and assayed for (left) HCV RNA abundance (GE = genome equivalents) or (right) infectious virus yields. Infectivity titers are expressed as focus forming units (FFU) per ml. Results shown are the mean ± s.e.m. values from triplicate cultures and are representative of 2 independent experiments. *P < 0.05, **P < 0.01 by multiple t-tests.

[0022] Figures 2A-2D show B WA4C is a potent inducer of lipid peroxidation. (A) Huh-7.5 cells were treated with various doses of BWA4C (0.01, 0.03, 0.3, 3 μΜ; orange line) or linoleic acid (3, 12.5, 50, 100 μΜ; black line) for 48 h, and analyzed for intracellular malondialdehyde abundance. (B) Huh-7.5 cells transfected with H77S.3 (left) or HJ3-5 (right) RNA encoding GLuc were treated with various concentrations of BWA4C. Data shown represent percent GLuc activity (red line) secreted between 48-72 h and cell viability (black line) relative to DMSO control. (C) Anti-H77S.3 effect is reversed by anti-lipid peroxidative agents, SKI and VE. Huh-7.5 cells transfected with H77S.3 encoding GLuc were treated with DMSO, 1 μΜ BWA4C or 1 μΜ BWA4C plus 1 μΜ SKI (BW+SKI) or VE (BW+VE). Data shown are mean ± s.e.m. of GLuc activity in supernatant fluids of two replicate cultures. (D) Effects of various lipophilic pro- oxidants, 50 μΜ linoleic acid (LA), 10 μΜ cumene hydroperoxide (CuOH) and 1 μΜ BWA4C, with and without 1 μΜ VE (+VE), or a DAA (MK-0608, 10 μΜ) on GLuc expression from Huh-7.5 cells transfected with either H77S.3/GLuc2A, TNcc/GLuc2A or HJ3-5/GLuc2A RNAs. GLuc secreted between 48-72 h after transfection is shown. Results represent the mean ± s.e.m. from 3 replicate experiments.

[0023] Figures 3A-3B show the NS3h-4B and NS5B segments of the TNcc genome are both required for replication-competent lipid peroxidation-resistant virus when swapped into the background of H77S.3/I2204S. All HCV mutants were constructed in the background of genomes that express Gaussia Luciferase fused to foot-and-mouth disease virus 2A autoprotease (GLuc/2A) as part of the HCV polyprotein. (A)

H77S.3/I2204S (H77S.3i _S ) mutants that express partial nonstructural (NS) protein(s) derived from TNcc and restriction enzyme sites used to construct the swap mutants are shown on the left. Media containing DMSO (vehicle control), 1 μΜ SKI, 1 μΜ VE, 1 μΜ BWA4C, 1 μΜ BWA4C plus VE (BW+VE), or 10 μΜ MK-0608 (DAA) were added to cells 6 h post RNA transfection and replaced at 24 and 48 h. GLuc activity secreted between 48-72 h after the addition of compounds is shown on the right. Black arrowheads indicate the I2204S mutation in NS5A, and those in yellow indicate where cell culture adaptive mutations of TNcc are located. A dotted line indicates the limit of detection (L.O.D.) of replication determined based on the GLuc values in DAA-treated samples. (B) H77S.3i _S mutants that contain various combinations of TNcc-derived adaptive mutations were assessed for sensitivity to lipid peroxidation as in (A). Results shown are the mean ± s.e.m. values from triplicate cultures and are representative of 2 or 3 independent experiments.

[0024] Figure 4 shows NS5A S2204 is a key residue for H77 compatibility with TNcc-derived adaptive mutations. H77S.3/GLuc2A mutants that express wild type NS5A (wt5A) or NS5A with either K2040R (KR) or S2204I (SI) or both mutations (KR/SI) were constructed in the background of H77S.3/GLuc2A or H77S.3/GLuc2A with the 8 mutations derived from TNcc (H77S _8mt ) and are shown on the left. Media containing DMSO (vehicle control), 1 μΜ SKI, 1 μΜ VE, 1 μΜ BWA4C, 1 μΜ BWA4C plus VE (BW+VE), or 10 μΜ MK-0608 (DAA) were added to cells 6 h post RNA transfection and replaced at 24 and 48 h. GLuc activity secreted between 48-72 h after the addition of compounds is shown on the right. L.O.D. = limit of detection. Results shown are the mean ± s.e.m. values from triplicate cultures and are representative of 3 independent experiments.

[0025] Figure 5 shows the spread of H77Sis/8mt virus over a month-long passage. Huh-7.5 cells were transfected with H77Sis/8mt RNA, and stained for core protein expression (green) at the indicated time points. Nuclei were counterstained with DAPI (blue). Images were taken at low magnification (100x).

[0026] Figures 6A-6C show robust replication of H77Sis/8mt with 3 compensatory mutations, G1909S, D2416G and G2963D, designated H77D. (A) The viral genomes with amino acid substitutions (red arrowheads) isolated from 3 independent culture plates are shown. Each HCV genome RNA was isolated from Huh-7.5 cells inoculated with supernatant fluids from day 32 (post-RNA transfection) sample for 4 days. Black and yellow arrowheads indicate I2204S (NS5A) and the mutations derived from TNcc, respectively. (B) H77S.3is mutants that contain each compensatory mutation or combinations of mutations constructed in the background of H77S.3is/8mt are shown on the left. Media containing DMSO (vehicle control), 1 μΜ SKI, 1 μΜ VE, 1 μΜ

BWA4C, 1 μΜ BWA4C and 1 μΜ VE (BW+VE), or 10 μΜ MK-0608 (DAA) were added to cells 6 h post RNA transfection and replaced at 24 and 48 h. GLuc activity secreted between 48-72 h after the addition of compounds is shown on the right. L.O.D. = limit of detection. (C) GLuc activities in supernatant fluids of cultures transfected with H77S.3 (black circle), H77D (red circle), TNcc (filled triangle) or HJ3-5 (gray square) RNAs in Huh-7.5 (left) or FT3-7 (right) cells. Supernatant fluids were collected to assay GLuc secreted into the medium between 0 and 6 h following HCV RNA transfection and over successive 24-h periods ending at 24, 48, 72 and 96 h posttransfection. Results shown are the mean ± s.e.m. values from triplicate cultures and are representative of 2 independent experiments.

[0027] Figures 7A-7C shows robust production of H77D virus does not require lipid peroxidation inhibitors. (A) Huh-7.5 cells were transfected with H77S.3, H77S.3is, H77S.3 _IS/8mt , H77S.3i _S/ 8 _m t/Gi 906S(GS), H77S.3 _{IS/l lmt} (H77D), TNcc, JFH1-QL (containing the cell culture-adaptive mutation Q221L in the NS3 helicase) or HJ3-5 RNAs and media containing DMSO (vehicle control) or 1 μΜ VE were added to cells 6 h post RNA transfection and replaced at 24 and 48 h. At 72 h, supernatant fluids were harvested for titration of HCV infectivity in a focus-forming assay using Huh-7.5 cells. Infectivity titers are expressed as focus forming units (FFU) per ml. (B) Infectivity of infectious virus particles of H77D vs. TNcc produced from viral RNA-transfected Huh-7.5 cells. Infectious virus particles produced between 48-72 h post transfection were inoculated on naive Huh-7.5 cells and stained for core protein expression (green) at 72 h post-infection. Nuclei were counterstained with DAPI (blue). Images were taken at low magnification (40x). (C) Focus-forming assay was carried out for H77S.3, H77D, TNcc and HJ3-5 viruses produced from FT3-7 cells as described in (A). Results shown are the mean ± s.e.m. values from triplicate cultures.

[0028] Figures 8A-8B show TNcc-derived mutations in NS3 helicase and NS4B are dispensable for the resistance to lipid peroxidation and that lipid peroxidation resistance is tightly linked to robust replication in cell culture. (A) TNcc mutations in NS3

(helicase) and NS4B are not required for lipid peroxidation resistance. Combinations of TNcc substitutions were introduced into H77S.3/GLUC!S/GS (NS proteins shown only) that contains the compensatory mutation G1909S (GS) in NS4B (red arrowhead, see Figure 10). Huh-7.5 cells were treated with DMSO, 1 μΜ SKI, 1 μΜ VE, 10 uM CuOH, 10 μΜ CuOH plus VE (CuOH+VE), or 30 μΜ sofosbuvir (DAA) beginning 6 h following RNA transfection. Data shown represent mean GLuc activity ± s.e.m. from at least two independent experiments. L.O.D. = limit of detection. (B) Structural models of (left) NS4A and (right) NS5B membrane interactions showing key residues that determine sensitivity to lipid peroxidation.

[0029] Figure 9 shows mapping of TNcc-derived mutations that confer resistance to lipid peroxidation. Cells transfected with H77Si _S mutants with individual TNcc-derived mutations or the different combinations of them were transfected with indicated RNAs encoding GLuc and treated with 1 μΜ VE, 10 μΜ CuOH, CuOH plus VE, or a DAA (sofosbuvir, 30 μΜ). Data shown represent GLuc activity secreted between 48-72 h. Results shown are the mean ± s.e.m. values from triplicate cultures and are representative of 2 or 3 independent experiments. L.O.D. = limit of detection.

[0030] Figure 10 shows mapping of TNcc-derived mutations that confer resistance to lipid peroxidation in the presence of a cell culture adaptive mutation, G1909S, in NS4B. Huh-7.5 cells were transfected with H77S.3 _IS RNAs into which compensatory mutations identified in panel A were introduced. Cells were treated with 1 μΜ SKI, 1 μΜ VE, 10 μΜ CuOH, 10 μΜ CuOH plus VE (CuOH+VE), 30 μΜ sofosbuvir (DAA), or DMSO (vehicle control) beginning 6 h post RNA transfection. Data shown represent mean ± s.e.m. GLuc secreted between 48-72 h from triplicate cultures and are representative of 2 independent experiments. L.O.D. = limit of detection.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0032] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0034] Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 C.F.R. §1.822 and established usage. [0035] Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual 2nd Ed. (Cold Spring Harbor, NY, 1989); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0036] All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented.

I. Definitions

[0037] As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0038] Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0039] The term "about," as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activity and the like, is meant to encompass variations of ± 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0040] The transitional phrase "consisting essentially of means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim, "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03.

[0041] The term "consists essentially of (and grammatical variants), as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide or polypeptide that consists of both the recited sequence {e.g., SEQ ID NO) and a total of ten or less {e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5' and/or 3' or N-terminal and/or C-terminal ends of the recited sequence such that the function of the polynucleotide or polypeptide is not materially altered. The total of ten or less additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids on both ends added together. The term "materially altered," as applied to polynucleotides of the invention, refers to an increase or decrease in ability to express the encoded polypeptide of at least about 50% or more as compared to the expression level of a polynucleotide consisting of the recited sequence. The term "materially altered," as applied to polypeptides of the invention, refers to an increase or decrease in epitope binding activity of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0042] As used herein, "nucleic acid," "nucleotide sequence," and "polynucleotide" are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mR A, synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA. The term polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain.

[0043] The term "isolated" can refer to a nucleic acid, nucleotide sequence or polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized). Moreover, an "isolated fragment" is a fragment of a nucleic acid, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found in the natural state. "Isolated" does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.

[0044] The term "fragment," as applied to a polynucleotide, will be understood to mean a nucleotide sequence of reduced length relative to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of, and/or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of oligonucleotides having a length of at least about 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive nucleotides of a nucleic acid or nucleotide sequence according to the invention.

[0045] The term "fragment," as applied to a polypeptide, will be understood to mean an amino acid sequence of reduced length relative to a reference polypeptide or amino acid sequence and comprising, consisting essentially of, and/or consisting of an amino acid sequence of contiguous amino acids identical or almost identical (e.g., 90%, 92%, 95%, 98%, 99% identical) to the reference polypeptide or amino acid sequence. Such a polypeptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and/or consist of peptides having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide or amino acid sequence according to the invention.

[0046] As used herein, the terms "protein" and "polypeptide" are used

interchangeably and encompass both peptides and proteins, unless indicated otherwise.

[0047] A "fusion protein" is a polypeptide produced when two heterologous nucleotide sequences or fragments thereof coding for two (or more) different

polypeptides not found fused together in nature are fused together in the correct translational reading frame. Illustrative fusion polypeptides include fusions of a polypeptide of the invention (or a fragment thereof) to all or a portion of glutathione-S- transferase, maltose-binding protein, or a reporter protein (e.g., Green Fluorescent Protein, β-glucuronidase, β-galactosidase, luciferase, etc.), hemagglutinin, c-myc, FLAG epitope, etc.

[0048] As used herein, a "functional" polypeptide or "functional fragment" is one that substantially retains at least one biological activity normally associated with that polypeptide (e.g. , target protein binding). In particular embodiments, the "functional" polypeptide or "functional fragment" substantially retains all of the activities possessed by the unmodified peptide. By "substantially retains" biological activity, it is meant that the polypeptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native polypeptide (and can even have a higher level of activity than the native polypeptide). A "non-functional" polypeptide is one that exhibits little or essentially no detectable biological activity normally associated with the polypeptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%). Biological activities such as protein binding can be measured using assays that are well known in the art and as described herein.

[0049] As used herein, the term "functional RNA" refers to RNA molecules that do not encode a protein and provide a functional activity as an RNA molecule. Examples include, without limitation, RNAi, microRNA, antisense RNA, and ribozymes.

[0050] As used herein, the term "replication competent" with respect to a HCV genome refers to a genome that replicates when present in a cell. In some aspects of the present invention, replication in a cell can include the production of infectious viral particles, i.e., viral particles that can infect a cell and result in the production of more infectious viral particles. The cells may specifically be cells that are maintained in culture ex vivo.

[0051] The terms "coding region" and "coding sequence" are used interchangeably and refer to a polynucleotide region that encodes a polypeptide or functional RNA and, when placed under the control of appropriate regulatory sequences, expresses the encoded polypeptide or functional RNA. The boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end. A coding region can encode one or more polypeptides or functional RNAs. For instance, a coding region can encode a polypeptide or functional RNA that is

subsequently processed into two or more polypeptides or functional RNAs. A regulatory sequence or regulatory region is a nucleotide sequence that regulates expression of a coding region to which it is operably linked. Nonlimiting examples of regulatory sequences include promoters, transcription initiation sites, translation start sites, internal ribosome entry sites, translation stop sites, and terminators. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A regulatory sequence is "operably linked" to a coding region when it is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence. [0052] The terms "5 ' non-translated RNA," "5 ' non-translated region," "5 ' untranslated region" and "5' noncoding region" are used interchangeably, and are terms of art (see Bukh et al, Proc. Nat. Acad. Sci. USA 89:4942-4946 (1992)). The term refers to the nucleotides that are at the 5' end of a replication competent polynucleotide and that do not encode a translated polypeptide but rather serve regulatory purposes in replication of the virus.

[0053] The terms "3 ' non-translated RNA," "3 ' non-translated region," and "3 ' untranslated region" are used interchangeably, and are terms of art. The term refers to the nucleotides that are at the 3' end of a replication competent polynucleotide and that do not encode a translated polypeptide but rather serve regulatory purposes in replication of the virus.

[0054] The term "robust replication phenotype" refers to a replication competent HCV genotype la genome that is capable of a replication level in cultured cells that is at least about 10-fold higher than the replication level of a wild-type HCV genotype la genome. In some embodiments, the term refers to a replication competent HCV genotype la genome that is capable of a replication level in cultured cells that is at least about 10-fold higher than the replication level of a previously disclosed HCV genotype la genome clone such as H77S.3 or TNcc.

[0055] As used herein "sequence identity" refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. "Identity" can be readily calculated by known methods including, but not limited to, those described in:

Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0056] As used herein, the term "substantially identical" or "corresponding to" means that two nucleic acid sequences have at least about 80% sequence identity. In some embodiments, the two nucleic acid sequences can have at least about 85%, 90%, 95%, 96%, 97%, 98%), 99% or 100% sequence identity.

[0057] An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) as compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions, or gaps totaling less than 20 percent of the reference sequence over the window of comparison). In some embodiments, "percent identity" can refer to the percentage of identical amino acids in an amino acid sequence.

[0058] Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., Burlington, Mass.). An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For purposes of this invention "percent identity" may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences. [0059] The percent of sequence identity can be determined using the "Best Fit" or "Gap" program of the Sequence Analysis Software Package™ (Version 10; Genetics Computer Group, Inc., Madison, Wis.). "Gap" utilizes the algorithm of Needleman and Wunsch (Needleman and Wunsch, J Mol. Biol. 48:443-453, 1970) to find the alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. "BestFit" performs an optimal alignment of the best segment of similarity between two sequences and inserts gaps to maximize the number of matches using the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math. 2:482 (1981); Smith et al, Nucleic Acids Res. 11:2205 (1983)).

[0060] Useful methods for determining sequence identity are also disclosed in Guide to Huge Computers (Martin J. Bishop, ed., Academic Press, San Diego (1994)), and Carillo, H., and Lipton, D., Applied Math 5:1073(1988)). More particularly, preferred computer programs for determining sequence identity include but are not limited to the Basic Local Alignment Search Tool (BLAST) programs which are publicly available from National Center Biotechnology Information (NCBI) at the National Library of Medicine, National Institute of Health, Bethesda, Md. 20894; see BLAST Manual, Altschul et al, NCBI, NLM, NIH; (Altschul et al, J. Mol. Biol. 275:403 (1990)); version 2.0 or higher of BLAST programs allows the introduction of gaps (deletions and insertions) into alignments; for peptide sequence BLASTX can be used to determine sequence identity; and, for polynucleotide sequence BLASTN can be used to determine sequence identity.

II. HCV genotype la clones and methods of use

[0061] One aspect of the invention relates to an isolated polynucleotide encoding a replication competent HCV genotype la genome, said genome comprising: a 5' NTR, a 3' NTR, and a coding sequence present between the 5' NTR and 3' NTR and encoding a HCV polyprotein, wherein the polyprotein comprises an amino acid sequence having at least about 95% identity to amino acids 1-3011 of SEQ ID NO:2, and wherein the amino acid sequence of the polyprotein comprises the following mutations relative to the amino acid positions using SEQ ID NO: 2 as a reference sequence:

aspartic acid at about amino acid 476; glycine at about amino acid 1226;

leucine at about amino acid 1464;

isoleucine at about amino acid 1655;

serine at about amino acid 1672;

arginine at about amino acid 1691;

histidine at about amino acid 1773;

threonine at about amino acid 1927;

arginine at about amino acid 2040;

glycine at about amino acid 2979;

phenylalanine at about amino acid 2981 ; and

serine at about amino acid 2994;

and does not comprise an isoleucine at about amino acid 2204;

wherein the polyprotein further comprises one or more of the following mutations:

serine at about amino acid 1909

glycine at about amino acid 2416; and

aspartic acid at about amino acid 2963.

[0062] The residue numbering of the mutations in the HCV polyprotein are based on the sequence of the HCV H77 genome found at GenBank Accession Mo. NC_004102. The nucleotide sequence of the H77 genome is disclosed herein as SEQ ID NO: l and the amino acid sequence of the H77 polyprotein is disclosed herein as SEQ ID NO:2.

[0063] In some embodiments, the polyprotein has at least about 95%, 96%, 97%, 98%, 99%, or 99.5% or more identity to amino acids 1-301 1 of SEQ ID NO:2. In some embodiments, the polyprotein has at least about 95%, 96%, 97%, 98%, 99%, or 99.5% or more identity to a portion of SEQ ID NO:2 of at least 2000 contiguous amino acids, e.g., at least about 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 contiguous amino acids. Percent identity, as used herein, refers to the percent of amino acid residues of the polyprotein of the invention that are identical to the amino acid residues of the corresponding portion of the amino acid sequence of SEQ ID NO:2. Thus, if the polyprotein of the invention is shorter than 3011 amino acids, the percent identity is calculated based on the corresponding portion of SEQ ID NO:2, not the full length of SEQ ID NO:2. [0064] The replication competent HCV genotype la genomes of the present invention each include a set of 12 adaptive mutations. As used herein, an adaptive mutation is a change in the amino acid sequence of the polyprotein that increases the ability of a replication competent HCV genotype la genome to replicate compared to a replication competent HCV genotype la genome that does not have the adaptive mutation. The mutation is relative to the amino acid present in most clinical HCV isolates and molecularly cloned laboratory HCV strains. The twelve mutations present in the genomes of the present invention are listed in Table 1.

Table 1 : HCV genotype la mutations

[0065] In addition to the 12 adaptive mutations listed in Table 1, the replication competent HCV genotype la genomes of the present invention include at least one of the additional adaptive mutations listed in Table 2. Table 2: Additional HCV genotype la mutations

[0066] In some embodiments, the polyprotein comprises one of the additional mutations (e.g., serine at about amino acid 1909 or glycine at about amino acid 2416 or aspartic acid at about amino acid 2963) or two of the additional mutations (e.g., serine at about amino acid 1909 and glycine at about amino acid 2416, or serine at about amino acid 1909 and aspartic acid at about amino acid 2963, or glycine at about amino acid 2416 and aspartic acid at about amino acid 2963) or all three of the additional mutations (serine at about amino acid 1909 and glycine at about amino acid 2416 and aspartic acid at about amino acid 2963).

[0067] In addition to the 12 adaptive mutations listed in Table 1 and the one or more adaptive mutations listed in Table 2, the replication competent HCV genotype la genome of the present invention does not comprise a mutation at about amino acid 2204. The amino acid residue at 2204 is most commonly serine. In some embodiments of the present invention, serine 2204 is not mutated. In certain embodiments, serine 2204 is not mutated to isoleucine, e.g., serine 2204 may be mutated to an amino acid residue other than isoleucine.

[0068] In some embodiments, the polyprotein comprises, consists essentially of, or consists of all of the cleavage products found in wild-type HCV polyprotein, e.g., core, El, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B. In other embodiments, the polyprotein is a subgenomic polyprotein comprising less than all of the cleavage products. For example, the polyprotein may comprise, consist essentially of, or consist of the nonstructural proteins (e.g., NS2, NS3, NS4A, NS4B, NS5A, and NS5B) or the minimal products necessary for replication (e.g., NS3, NS4A, NS4B, NS5A, and NS5B). In certain embodiments, the polyprotein may comprise, consist essentially of, or consist of El, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B; E2, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B; P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B; NS2, NS3, NS4A, NS4B, NS5A, and NS5B; or NS3, NS4A, NS4B, NS5A, and NS5B. In other aspects of the invention, the polyprotein does not include polypeptides present in an internal portion of a HCV polyprotein. Thus, a subgenomic polyprotein may comprise, consist essentially of, or consist of, for example, NS3, NS4A, NS4B, and NS5B.

[0069] In those aspects of the invention where the replication competent HCV genotype la genome includes a coding region that encodes less than a full length HCV polyprotein, the 5' end of the coding region encoding the HCV polyprotein may further include about 33 to about 51 nucleotides, or about 36 to about 48 nucleotides, that encode the first about 11 to about 17, or about 12 to about 16, amino acids of the core polypeptide. The result is a fusion polypeptide made up of amino terminal amino acids of the core polypeptide and the first polypeptide encoded by the first cleavage product of the polyprotein, e.g., El, or E2, or P7, or NS2, etc.

[0070] A polyprotein that can yield the core, El, E2, P7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B polypeptides (a full length polyprotein) is typically between about 3000 and 3033 amino acids in length, e.g., about 3011 amino acids in length. The relationship between such a polyprotein and the corresponding residues of the individual polypeptides resulting after post-translational processing is shown in Table 3. This numbering system is used herein when referring to a full length polyprotein, and when referring to a polyprotein that contains a portion of the full length polyprotein. A person of skill in the art recognizes that this numbering system can vary between members of different genotypes, and between members of the same genotype, thus the numbers shown in Table 4 are approximate, and can vary by 1, 2, 3, 4, or about 5.

Table 3 : Correspondence between amino acids of polyprotein and individual polypeptides after processing

810-1026 NS2

1027-1657 NS3

1658-171 1 NS4A

1712-1972 NA4B

1973-2420 NS5A

2421-301 1 NS5B

*Refers to the approximate amino acid number prior to cleavage of the polyprotein where the first amino acid is the first amino acid of the polyprot expressed by the HCV at GenBank Accession No. NC_004102.

[0071] In some embodiments, the genome further comprises a second coding sequence. The second coding sequence may encode a polypeptide or functional RNA of interest. In some embodiments, the second coding sequence may be located at either end of the polyprotein or within the polyprotein. The second coding sequence may be surrounded by cleavage sequences so that it is cleaved from the polyprotein after the polyprotein is expressed. In some embodiments, the cleavage sequences are those naturally found in the HCV genome or other cleavage sequences known to those of skill in the art.

[0072] In some embodiments, the second coding sequence may be present in the 3 ' NTR, for instance, in the variable region of the 3' NTR. In other embodiments, the second coding sequence is present downstream of the 5' NTR, and upstream of the first coding region, i.e., the coding region encoding a HCV polyprotein.

[0073] In embodiments where the second coding sequence is present downstream of the 5' NTR and upstream of the coding region encoding the HCV polyprotein, the replication competent HCV genotype la genome typically includes a regulatory region operably linked to the downstream coding region, e.g., the coding region encoding the HCV polyprotein. Preferably, the regulatory region provides for the translation of the downstream coding region. Typically, the regulatory region is an internal ribosome entry site (IRES). Examples of IRES elements are described herein.

[0074] In some embodiments, the second coding sequence encodes a marker (e.g., a detectable marker and/or a selectable marker) or a transactivator that can be used to detect the genome and/or the replication level of the genome. Detectable and selectable markers are well known in the art and any suitable marker can be used. Examples of detectable markers include molecules having a detectable enzymatic activity, for instance, secretory alkaline phosphatase, molecules having a detectable fluorescence, for instance, green or red or blue fluorescent protein, and molecules that can be detected by antibody. One example is Gaussia princeps luciferase, which may be secreted from the cell after replication of the genome and cleavage of the luciferase from the polyprotein. Luciferase activity may then be quantitated as is known in the art and described herein. Examples of selectable markers include molecules that confer resistance to antibiotics able to inhibit the replication of eukaryotic cells, including the antibiotics kanamycin, ampicillin, chloramphenicol, tetracycline, blasticidin, neomycin, and formulations of phleomycin Dl including, for example, the formulation available under the trade-name ZEOCIN (Invitrogen, Carlsbad, Calif.). Coding sequences encoding such markers are known to the art.

[0075] A transactivator is a polypeptide that affects in trans the expression of a coding region, e.g., a coding region integrated in the genomic DNA of a cell. Such coding regions are referred to herein as "transactivated coding regions." Transactivators useful in the present invention include those that can interact with a regulatory region, e.g., an operator sequence, that is operably linked to a transactivated coding region. As used herein, the term "transactivator" includes polypeptides that interact with an operator sequence and either prevent transcription from initiating at, activate transcription initiation from, or stabilize a transcript from, a transactivated coding region operably linked to the operator sequence. Examples of useful transactivators include the HIV tat polypeptide. The HIV tat polypeptide interacts with the HIV long terminal repeat (LTR). Other useful transactivators include human T cell leukemia virus tax polypeptide (which binds to the operator sequence tax response element, Fujisawa et al., J. Virol., 65, 4525- 4528 (1991)), and transactivating polypeptides encoded by spumaviruses in the region between env and the LTR, such as the bel-1 polypeptide in the case of human foamy virus (which binds to the U3 domain of these viruses, Rethwilm et al, Proc. Natl. Acad. Sci. USA, 88, 941-945 (1991)). Alternatively, a post-transcriptional transactivator, such as HIV rev, can be used. HIV rev binds to a 234 nucleotide RNA sequence in the env gene (the rev-response element, or RRE) of HIV (Hadzopolou-Cladaras et al, J. Virol., 63, 1265-1274 (1989)).

[0076] In some aspects of the invention, the second coding region may further include an operably linked regulatory region. Preferably, a regulatory region located 5' of the operably linked coding region provides for the translation of the coding region. An example of a regulatory region located 5' of an operably linked second coding region is an internal ribosome entry site (IRES). An IRES allows a ribosome access to mRNA without a requirement for cap recognition and subsequent scanning to the initiator AUG (Pelletier, et al, Nature, 334, 320-325 (1988)). An IRES is located upstream of the translation initiation codon, e.g., ATG or AUG, of the coding sequence to which the IRES is operably linked. The distance between the IRES and the initiation codon is dependent on the type of IRES used, and is known to the art. For instance, poliovirus IRES initiates a ribosome translocation/scanning process to a downstream AUG codon. For other IRES elements, the initiator codon is generally located at the 3' end of the IRES sequence. Examples of an IRES that can be used in the invention include a viral IRES, e.g., a picornaviral IRES or a flaviviral IRES. Examples of IRES elements include, for instance, poliovirus IRES, encephalomyocarditis virus IRES, or hepatitis A virus IRES. Examples of flaviviral IRES elements include HCV IRES, GB virus B IRES, or a pestivirus IRES, including but not limited to bovine viral diarrhea virus IRES or classical swine fever virus IRES. Other IRES elements with similar secondary and tertiary structure and translation initiation activity can either be generated by mutation of these viral sequences, by cloning of analogous sequences from other viruses (including picornaviruses), or prepared by enzymatic synthesis techniques.

[0077] In some embodiments, the replication competent HCV genotype la genome exhibits a robust replication phenotype in cultured cells. In certain embodiments, the replication competent HCV genotype la genome is capable of replication, e.g., robust replication, in a hepatic cell, e.g., a primary human hepatocyte, human hepatoma cell (such as Huh-7.5), or other hepatocyte-derived cell line. In some embodiments, the replication competent HCV genotype la genome is capable of a replication level that is about 2-fold higher than the replication level of a wild-type HCV genotype la genome, e.g., about 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more higher. In some embodiments, the replication competent HCV genotype la genome is capable of a replication level that is about 2-fold higher than the replication level of previously disclosed HCV genotype la genome clones such as H77S.3 or TNcc, e.g., about 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more higher.

[0078] In some embodiments of the invention, the polynucleotide is DNA. In other embodiments, the polynucleotide is RNA or a mixture of DNA and RNA.

[0079] In certain embodiments, the polynucleotide is in a vector, e.g., a plasmid vector or other type of vector. In some embodiments, the vector is suitable for in vitro replication for production of the replication competent HCV genotype la genome.

[0080] Methods for cloning and/or inserting HCV sequences into a vector are known to the art (see, e.g., Yanagi et al, Proc. Natl. Acad. Sci., USA, 94, 8738-8743 (1997); and Rice et al, (U.S. Pat. No. 6,127,116)). Such constructs are often referred to as molecularly cloned laboratory strains, and an HCV sequence that is inserted into a vector is often referred to as a cDNA clone of the HCV. If the RNA encoded by the HCV is able to replicate in vivo or after transfection into cultured cells, the HCV sequence present in the vector is referred to as an infectious cDNA clone. A vector is a replicating polynucleotide, such as a plasmid, phage, cosmid, or artificial chromosome to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. A vector can provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polypeptide or functional RNA encoded by the coding region, i.e., an expression vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is able to replicate in a prokaryotic host cell, for instance Escherichia coli. In certain embodiments, the vector can integrate in the genomic DNA of a eukaryotic cell.

[0081] An expression vector optionally includes regulatory sequences operably linked to the replication competent polynucleotide such that it is transcribed to produce RNA molecules. These RNA molecules can be used, for instance, for introducing a replication competent HCV genotype la genome into a cell that is in an animal or growing in culture. The terms "introduce" and "introducing" refer to providing a replication competent HCV genotype la genome to a cell under conditions that the replication competent HCV genotype la genome is taken up by the cell in such a way that it can then replicate. The replication competent HCV genotype la genome can be present in a virus particle, or can be a nucleic acid molecule, for instance, RNA. The invention is not limited by the use of any particular promoter, and a wide variety is known. Promoters act as regulatory signals that bind RNA polymerase in a cell to initiate transcription of a downstream (3' direction) HCV. The promoter used in the invention can be a constitutive or an inducible promoter. Examples of promoters for the production of replication competent HCV genotype la genome as an RNA molecule include the T7, T3, and SP6 promoters.

[0082] Another aspect of the invention is a replication competent HCV genotype la genome encoded by the isolated polynucleotide of the invention. The genome may be produced by in vitro transcription of the polynucleotide as is well known in the art and described herein. In other embodiments, the genome can be produced by introducing the polynucleotide of the invention into a cell under conditions where the polynucleotide is transcribed, e.g., in a plasmid or as part of a viral particle.

[0083] A further aspect of the invention relates to a method for making an isolated polynucleotide encoding a replication competent HCV genotype la genome comprising: providing a polynucleotide encoding a replication competent HCV genotype la genome comprising a 5' NTR, a 3' NTR, and a coding sequence present between the 5' NTR and 3' NTR and encoding a HCV polyprotein, wherein the polyprotein comprises an amino acid sequence having at least about 95% identity to amino acids 1-3011 of SEQ ID NO:2, and wherein the amino acid sequence of the polyprotein comprises:

aspartic acid at about amino acid 476;

glycine at about amino acid 1226;

leucine at about amino acid 1464;

isoleucine at about amino acid 1655;

serine at about amino acid 1672;

arginine at about amino acid 1691;

histidine at about amino acid 1773;

threonine at about amino acid 1927;

arginine at about amino acid 2040;

glycine at about amino acid 2979; phenylalanine at about amino acid 2981; and

serine at about amino acid 2994;

and does not comprise an isoleucine at about amino acid 2204;

wherein the polyprotein further comprises one or more of the following mutations:

serine at about amino acid 1909

glycine at about amino acid 2416; and

aspartic acid at about amino acid 2963.

[0084] Polynucleotides encoding an HCV polyprotein can be obtained from different sources, including molecularly cloned laboratory strains, for instance cDNA clones of HCV such as H77S.3 or TNcc, and clinical isolates. Examples of molecularly cloned laboratory strains include the HCV that is encoded by pCV-H77C (Yanagi et al, Proc. Natl. Acad. Sci. USA, 94, 8738-8743 (1997), Genbank accession number AF011751), and pHCV-H (Inchauspe et al, Proc. Natl. Acad. Sci. USA, 88, 10292-10296 (1991), Genbank accession number M67463). Clinical isolates can be from a source of infectious HCV, including tissue samples, for instance from blood, plasma, serum, liver biopsy, or leukocytes, from an infected animal, including a human or a primate. It is also expected that the polynucleotide encoding the HCV polyprotein present in a replication competent HCV genotype la genome can be prepared by recombinant, enzymatic, or chemical techniques. Such methods are routine and known to the art and include, for instance, PCR mutagenesis.

[0085] Further aspects of the invention relate to methods of using the replication competent HCV genotype la genomes of the invention to produce viral particles, to study aspects of the viral life cycle, for drug development, to study viral resistance to therapeutics, and for any other suitable use.

[0086] One aspect of the invention relates to a method for replicating a replication competent HCV genotype la genome, comprising incubating a cell comprising the replication competent HCV genotype la genome of the invention under conditions suitable for the genome to replicate. Any suitable method for delivering the replication competent HCV genotype la genome may be used as is well known in the art and described herein. Such methods include, for instance, liposome and non-liposome mediated transfection. Non-liposome mediated transfection methods include, for instance, electroporation. Conditions suitable for HCV replication are well known in the art and described herein.

[0087] An additional aspect of the invention relates to a method for producing viral particles, comprising incubating a cell comprising the replication competent HCV genotype la genome of the invention under conditions suitable for the genome to replicate. In some embodiments, the method further comprises isolating the viral particles from the cell. Conditions suitable for HCV replication and virus production are well known in the art and described herein. Any suitable cell can be used, e.g., a hepatic cell, e.g. , a primary human hepatocyte, human hepatoma cell (such as Huh-7.5, Huh-7, HepG2, IMY-N9, PH5CH8), or other hepatocyte-derived cell line.

[0088] The viral particles can be used as a source of virus particles for various assays, including evaluating methods for inactivating particles, excluding particles from serum, identifying a neutralizing compound, and as an antigen for use in detecting anti-HCV antibodies in an animal. An example of using a viral particle as an antigen includes use as a positive-control in assays that test for the presence of anti-HCV antibodies.

[0089] For example, the activity of compounds that neutralize or inactivate the particles can be evaluated by measuring the ability of the molecule to prevent the particles from infecting cells growing in culture or in cells in an animal. Inactivating compounds include detergents and solvents that solubilize the envelope of a viral particle. Inactivating compounds are often used in the production of blood products and cell-free blood products. Examples of compounds that can be neutralizing include a polyketide, a non-ribosomal peptide, a polypeptide (for instance, an antibody), a polynucleotide (for instance, an antisense oligonucleotide or ribozyme), or other organic molecules.

[0090] In some embodiments, the replication competent HCV genotype la genome of the invention may be used in screening assays to identify agents that modulate HCV replication and viral particle production.

[0091] In one embodiment, the invention relates to a method for identifying a compound that inhibits replication of a replication competent HCV genotype la genome, the method comprising contacting a cell comprising the replication competent HCV genotype la genome of the invention with a compound, incubating the cell under conditions wherein the replication competent HCV genotype la genome replicates in the absence of the compound; and detecting the replication level of the replication competent HCV genotype la genome, wherein a decrease in the replication level in the cell contacted with the compound compared to the replication level in a cell not contacted with the compound indicates the compound inhibits replication of the replication competent HCV genotype la genome.

[0092] A compound that inhibits replication of the genome includes compounds that completely prevent replication, as well as compounds that decrease replication. In certain embodiments, a compound inhibits replication of a replication competent HCV genotype la genome by at least about 50%, e.g., at least about 75%, e.g., at least about 95%.

[0093] The compounds added to a cell can be a wide range of molecules and is not a limiting aspect of the invention. Compounds include, for instance, a polyketide, a non- ribosomal peptide, a polypeptide, a polynucleotide (for instance an antisense

oligonucleotide or ribozyme), other organic molecules, or a combination thereof. The sources for compounds to be screened can include, for example, chemical compound libraries, fermentation media of Streptomycetes, other bacteria and fungi, and extracts of eukaryotic or prokaryotic cells. When the compound is added to the cell is also not a limiting aspect of the invention. For instance, the compound can be added to a cell that contains a replication competent HCV genotype la genome. Alternatively, the compound can be added to a cell before or at the same time that the replication competent HCV genotype la genome is introduced to the cell.

[0094] Detection of the replication level of the genome may be carried out by any method known in the art or described herein. In certain embodiments, detecting the replication level of the replication competent HCV genotype la genome comprises detecting the quantity of viral R A (e.g., using nucleic acid amplification assays such as PCR), detecting the quantity of viral protein (e.g., using immunoassays), detecting the quantity of a marker protein, or detecting the quantity of infectious virus particles (e.g., using an infectivity assay).

[0095] In one embodiment, the replication competent HCV genotype la genome further comprises a second coding sequence encoding a marker, and detecting the replication level of the replication competent HCV genotype la genome comprises detecting the marker. [0096] In another embodiment, the replication competent HCV genotype la genome further comprises a second coding sequence encoding a transactivator, wherein the cell comprises a polynucleotide comprising a transactivated coding sequence encoding a detectable marker and an operator sequence operably linked to the transactivated coding sequence, wherein the transactivator interacts with the operator sequence and alters expression of the transactivated coding sequence, and wherein detecting the replication level of the replication competent HCV genotype la genome in the cell comprises detecting the detectable marker encoded by the transactivated coding sequence.

[0097] In each of these methods, any suitable cell can be used, e.g., primate or human cells, e.g., a hepatic cell, e.g., a primary human hepatocyte, human hepatoma cell (such as Huh-7.5, Huh-7, HepG2, IMY-N9, PH5CH8), or other hepatocyte-derived cell line.

[0098] In some aspects of the invention, the cultured cell includes a polynucleotide that includes a coding region, the expression of which is controlled by a transactivator. Such a coding region is referred to herein as a transactivated coding region. A transactivated coding region encodes a marker, such as a detectable marker, for example, secretory alkaline phosphatase (SEAP). Typically, a cultured cell that includes a polynucleotide having a transactivated coding region is used in conjunction with a replication competent polynucleotide of the present invention that includes a coding region encoding a transactivator. The polynucleotide that includes the transactivated coding region can be present integrated into the genomic DNA of the cell, or present as part of a vector that is not integrated. Methods of modifying a cell to contain an integrated DNA are known to the art (see, for instance, Lemon et ah, U.S. Published Application US 2003 0125541, and Yi et ah, Virology 302, 197-210 (2002)).

[0099] A further aspect of the invention takes advantage of the clone of HCV genotype la genome comprising the combined mutations from H77S.3 and TNcc with the exception of S2204I. This clone is capable of replication in hepatic cells but at levels lower than either H77S.3 or TNcc. Importantly, this clone may be cultured to produce new mutants, including mutants exhibiting the robust replication phenotype. The culturing can take place in the absence of presence of selective pressure.

[0100] Thus, one aspect of the invention relates to an isolated polynucleotide encoding a replication competent HCV genotype la genome, said genome comprising: a 5' NTR, a 3' NTR, and a coding sequence present between the 5' NTR and 3' NTR and encoding a HCV polyprotein, wherein the polyprotein comprises an amino acid sequence having at least about 95% identity to amino acids 1-3011 of SEQ ID NO:2, and wherein the amino acid sequence of the polyprotein comprises the following mutations relative to the amino acid positions using SEQ ID NO: 2 as a reference sequence:

aspartic acid at about amino acid 476;

glycine at about amino acid 1226;

leucine at about amino acid 1464;

isoleucine at about amino acid 1655;

serine at about amino acid 1672;

arginine at about amino acid 1691;

histidine at about amino acid 1773;

threonine at about amino acid 1927;

arginine at about amino acid 2040;

glycine at about amino acid 2979;

phenylalanine at about amino acid 2981; and

serine at about amino acid 2994;

and does not comprise an isoleucine at about amino acid 2204.

[0101] The invention further relates to a replication competent HCV genotype la genome encoded by the isolated polynucleotide described above.

[0102] Another aspect of the invention takes advantage of the clone of HCV genotype la genome comprising the mutations NS4A (A1672S) and NS5B (D2979G), optionally without a S2204I mutation. The two mutations (A1672S and D2979G) are collectively responsible for resistance to lipid peroxidation. This clone is capable of replication in hepatic cells and importantly, may be cultured to produce new mutants, including mutants exhibiting the robust replication phenotype. The culturing can take place in the absence of presence of selective pressure.

[0103] Thus, one aspect of the invention relates to an isolated polynucleotide encoding a replication competent HCV genotype la genome, said genome comprising: a 5' NTR, a 3' NTR, and a coding sequence present between the 5' NTR and 3' NTR and encoding a HCV polyprotein, wherein the polyprotein comprises an amino acid sequence having at least about 95% identity to amino acids 1-301 1 of SEQ ID NO:2, and wherein the amino acid sequence of the polyprotein comprises the following mutations relative to the amino acid positions using SEQ ID NO: 2 as a reference sequence:

serine at about amino acid 1672; and

glycine at about amino acid 2979.

In some embodiments, the polyprotein does not comprise an isoleucine at about amino acid 2204.

[0104] The invention further relates to a replication competent HCV genotype la genome encoded by the isolated polynucleotide described above.

[0105] Another aspect of the invention relates to method of using the clones described above to identify new mutants having desired properties such as a robust replication phenotype. Thus, one embodiment relates to a method for identifying a HCV genotype la genome with a robust replication phenotype, the method comprising incubating a cell comprising the replication competent HCV genotype la genome of the invention and detecting an increase in the replication level of the replication competent HCV genotype la genome, thereby identifying a HCV genotype la genome with a robust replication phenotype.

[0106] Detection of the replication level of the genome may be carried out by any method known in the art or described herein. In certain embodiments, detecting the replication level of the replication competent HCV genotype la genome comprises detecting the quantity of viral RNA (e.g., using nucleic acid amplification assays such as PCR), detecting the quantity of viral protein (e.g., using immunoassays), detecting the quantity of a marker protein, or detecting the quantity of infectious virus particles (e.g., using an infectivity assay).

[0107] In one embodiment, the replication competent HCV genotype la genome further comprises a second coding sequence encoding a marker, and detecting the replication level of the replication competent HCV genotype la genome comprises detecting the marker.

[0108] In another embodiment, the replication competent HCV genotype la genome further comprises a second coding sequence encoding a transactivator, wherein the cell comprises a polynucleotide comprising a transactivated coding sequence encoding a detectable marker and an operator sequence operably linked to the transactivated coding sequence, wherein the transactivator interacts with the operator sequence and alters expression of the transactivated coding sequence, and wherein detecting the replication level of the replication competent HCV genotype la genome in the cell comprises detecting the detectable marker encoded by the transactivated coding sequence.

[0109] In each of these methods, any suitable cell can be used, e.g., a hepatic cell, e.g., a primary human hepatocyte, human hepatoma cell (such as Huh-7.5, Huh-7, HepG2, IMY-N9, PH5CH8), or other hepatocyte-derived cell line.

[0110] A cDNA molecule of a new mutant genome identified by the methods of the invention can be cloned using methods known to the art (see, for instance, Yanagi et ah, Proc. Natl. Acad. Sci., USA, 94, 8738-8743 (1997)). The nucleotide sequence of the cloned cDNA can be determined using methods known to the art, and compared with that of the input RNA. This allows identification of mutations that have occurred in association with passage of the replication competent polynucleotide in cell culture. For example, using methods known to the art, including long-range RT-PCR, extended portions of a variant replication competent HCV genome can be obtained. Multiple clones could be obtained from each segment of the genome, and the dominant sequence present in the culture determined. Mutations that are identified by this approach can then be reintroduced into the background of the cDNA encoding the parent or input polynucleotide.

[0111] One aspect of the invention relates to use of the replication-competent clones of the present invention to assess viral resistance to antiviral agents. HCV resistance to drugs that are used clinically is a growing issue. In one embodiment, the replication- competent clones of the invention may be exposed to an antiviral drug continuously in culture and the types and locations of mutations that are selected may be monitored and studied. In another embodiment, potential resistance-associated mutation(s) may be introduced into the replication-competent clones of the invention and the susceptibility of the virus to antiviral drugs may be assessed. In a further embodiment, one or more segments of the genome from a virus present in a patient may be cloned or amplified and used to replace the corresponding segment(s) in the replication-competent clones of the invention. The susceptibility of the resulting virus to antiviral drugs may then be assessed.

[0112] The present invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLE 1

Experimental Methods

[0113] Cells and reagents. Huh-7.5 cells were grown in Dulbecco's modified Eagle's medium (DMEM), High Glucose supplemented with 10% fetal bovine serum (FBS), 1 xPenicillin-Streptomycin, lxGlutaMAX and 1 *MEM Non-Essential Amino Acids Solution (Life Technologies). SKI [2-(p-Hydroxyanilino)-4-(p-chlorophenyl) thiazole] was obtained from Merck Millipore. BWA4C, a-tocopherol and linoleic acid were from Sigma- Aldrich, MK-886 was from Cayman Chemical, cumene hydroperoxide was from Santa Cruz Biotechnology, and BAY-X 1005 was from Tocris Bioscience. Cell viability assays were carried out using the WST-1 reagent (Millipore). Protein concentrations in samples were determined using the Protein Assay kit (Bio-Rad) with bovine serum albumin as standards.

[0114] Plasmids. pHJ3-5, pHJ3-5/GLuc2A, pHJ3-5/GND, pH77c, pH77S.3, pH77S.3/GLuc2A, and pH77S/GLuc2A-AAG have been described (19, 25, 27-29). pJFHl-QL has a cell culture adaptive mutation Q221L in the NS3 helicase (14). Point mutations were generated by standard primer-directed mutagenesis. The Gaussia princeps luciferase (GLuc)-coding sequence followed by the foot-and-mouth disease virus 2A protease-coding sequence was inserted between p7 and NS2 in the HCV genome constructs using a strategy applied previously to pH77S (19).

[0115] Gaussia luciferase assay. Cell culture supernatant fluids were collected at intervals following RNA transfection and cells refed with fresh media. Secreted GLuc activity was measured as described (20).

[0116] RNA transcription. RNA transcripts were synthesized in vitro using MEGAscript T7 Transcription Kit (Life Technologies) as described previously (19). [0117] HCV RNA transfection. RNA transfection was carried out by

electroporating 5 μg of HCV RNA into 2.5* 10 ⁶ Huh-7.5 cells using a Gene Pulser Xcell Total System (Bio-Rad) as described previously (20).

[0118] HCV infectivity assays. Huh-7.5 cells were seeded at 5x 10 ⁴ cells per well into 48-well plates 24 h before inoculation with 100 μΐ of culture medium. Cells were fed with media containing 1 μΜ VE 24 h later to facilitate visualization of core protein expression, fixed with methanol-acetone (1 :1) at -20°C for 10 min 72 h post-inoculation (48 h for JFH1-QL and HJ3-5), and stained for intracellular core antigen with mAb C7- 50 (Thermo Scientific; 1 :300 dilution). Clusters of infected cells identified by staining for core antigen were considered to constitute a single infectious focus, and the data expressed as focus-forming units (FFU)/ml.

[0119] Quantitative Real-time RT-PCR. One-step qRT-PCR analysis of HCV RNA in Huh-7.5 cells was carried out using iScript One-Step RT-PCR for Probes (Bio- Rad) as described (20).

[0120] RNA interference. ON-TARGETplus SMARTpool siRNAs targeting human ALOX5 and nontargeting control were purchased from Thermo Scientific. siRNA (20 nM) was transfected into cells using siLentfect Lipid Reagent (Bio-Rad) according to the manufacturer's protocol.

[0121] Immunoblots. Immunoblotting was carried out using standard methods with the following antibodies: mouse monoclonal antibodies (mAbs) to β-actin (AC-74; Sigma) and HCV NS3 (ab65407; abeam). Protein bands were visualized with an Odyssey Infrared Imaging System (Li-Cor Biosciences).

[0122] Lipid peroxidation assays. Malondialdehyde (MDA), a product of lipid peroxidation, was quantified by the thiobarbituric acid reactive substances (TBARS) Assay Kit (Cayman Chemical). Cells scraped into PBS containing complete protease inhibitor cocktail (Roche) were homogenized by sonication on ice using Sonic

Dismembrator (FB-120, Fisher Scientific). The amount of MDA in 100 μΐ of cell homogenates was analyzed by a fluorescent method as described by the manufacturer. Lipid peroxidation levels were expressed as the amount of MDA normalized to the amount of total protein. [0123] Statistical methods. Unless noted otherwise, all between-group comparisons were carried out by either multiple t-tests or 2-way ANOVA using Prism 6.0 software (GraphPad Software Inc.) and Prism 5.0c for Mac OS X software (GraphPad Software, Inc.). P < 0.05 was considered significant.

EXAMPLE 2

Resistance to endogenous lipid peroxidation is a common feature of HCV with robust replication phenotype

[0124] We have found that the robust replication phenotype of HCV expressing JFH1 replicase is associated with unique resistance to endogenous or chemically induced lipid peroxidation, while typical HCV replicase activity is downregulated by endogenous lipid peroxidation as reflected in the enhanced replication by lipophilic anti-peroxidative agents, such as sphingosine kinase inhibitor (SKI) or a-tocopherol (VE), and the attenuation by lipophilic pro-oxidants, such as polyunsaturated fatty acid (PUFA). Thus, we became interested in testing whether recently-developed genotype la TNcc strain that is capable of robust replication in Huh-7.5 cells (12), has the phenotype resistant to lipid peroxidation. We examined this by electroporating viral RNA in Huh-7.5 cells, and found that replication and production of TNcc was not enhanced by inhibition of lipid peroxidation by SKI or VE, and had relatively higher resistance to PUFA treatment like HJ3-5 (Fig. 1), indicating that TNcc replicase has the lipid peroxidation-resistant pheriotype. However, we noted that PUFA had a slight inhibitory effect on TNcc replication, and importantly, this inhibition was not reversed by VE supplementation unlike the case with H77S.3, indicating lipid peroxidation-independent suppression by PUFA. As PUFA regulates multiple cellular metabolic pathways via regulation of peroxisome proliferator-activated receptors (PPARs) and sterol regulatory element binding proteins (SREBPs) (5, 9, 11), we screened for more potent inducers of lipid peroxidation, and found that BWA4C, a 5-lipoygenase (5-LOX) inhibitor, induces the lipid peroxidation product malondialdehyde at approximately > 100-fold lower concentrations compared to PUFA (Fig. 2A). BWA4C has a potent antiviral activity against H77S.3, but not HJ3-5, with IC ₅₀ of 78 nM (Fig. 2B), while having no such activity in the presence of anti-lipid peroxidative compounds, SKI and VE, showing the lipid peroxidation-dependent inhibition of H77S.3 by BWA4C (Fig. 2C). Although BWA4C had more potent antiviral effect on H77S.3 than other lipophilic pro-oxidants, such as PUFAs and cumene hydroperoxide (CuOH), it had no detectable antiviral effect on HJ3-5 nor TNcc (Fig. 2D). Thus, we used BWA4C to assess the viral sensitivity to lipid peroxidation.

EXAMPLE 3

Mapping NS region(s) involved in the sensitivity to lipid peroxidation

[0125] We have found that multiple NS proteins, but not a single NS protein alone, determines the sensitivity/resistance to lipid peroxidation as swapping each individual protein between H77S.3 vs. JFHl did not confer resistance to lipid peroxidation, although many of the swap mutants were not able to replicate due to sequence incompatibilities between the two different viral genotypes. The fact that genotype la TNcc has a JFH1- like, lipid peroxidation resistant phenotype enabled us to study the viral determinants for the phenotype without concern for the intergenotypic incompatibility. To construct swap mutants containing partial NS region(s) from TNcc in H77S.3 background, we reverted one of the cell culture adaptive mutation 12204 (27) back to serine, designated H77S.3i _S , as this serine was required for compatibility of H77S-derived NS5A in JFHl background, and importantly, TNcc also has serine in the corresponding site. We attempted to swap the partial NS regions of TNcc to figure out which domain might be the determinant for the resistance to lipid peroxidation. Unexpectedly, H77S.3 _! s expressing partial TNcc NS3 helicase (NS3h) had slightly reduced replication fitness, and that carrying NS4AB, or partial NS5B from TNcc was not viable or barely replicated compared to the non- replicating control (DAA, 10 μΜ MK0608 targeting NS5B RNA polymerase). H77S.3i _S expressing TNcc NS4AB became viable only when TNcc NS3h and NS5B regions were simultaneously swapped (Fig. 3A). Although this H77S _1S/T N ₃ IV4/ _5B mutant had 10-fold less replication fitness compared to the parental H77S.3!s, it had a JFHl-like phenotype that is not enhanced by SKI or VE and resistant to BWA4C-induced lipid peroxidation (Fig. 3A).

[0126] These results indicated that multiple NS proteins are involved in the lipid peroxidation resistant phenotype. Importantly, we found that H77S.3is with all 8 TNcc mutations was completely resistant to lipid peroxidation. H77S.3i _S with mutations in each NS protein and NS3h+5B still responded to anti-lipid peroxidants, SKI and VE, and remained susceptible to chemically-induced lipid peroxidation and many of the combinations, including mutations in NS3h+NS4AB, NS3h+NS4A+NS5B, and

NS4AB+5B regions, were not compatible in the H77S.3 _1S background (Fig. 3B).

EXAMPLE 4

S2204I is required for compatibility with the cell culture adaptive mutations derived from TNcc

[0127] We wanted to confirm whether S2204 in NS5A is indeed required for the compatibility, and the other cell culture adaptive mutation in NS5A, K2040R, has any roles on the replication fitness of H77S carrying 8 mutations derived from TNcc

(H77S _8mu t). Compared to the wild type NS5A, K2040R mutation increased replication fitness by 2-3 fold in both H77S and H77S _8mut background, however, S2204I mutation had contrasting effects on H77S vs. H77S _8mut , promoting the replication of the former by 5-fold while being lethal to the latter (Fig. 4).

EXAMPLE 5

Robust replication of H77STs/s _m t with compensatory mutations

[0128] H77S.3is/8mt has acquired the lipid peroxidation-resistant phenotype, yet it has low replication fitness. We considered that some TNcc-derived mutations may not be compatible with other NS proteins for proper folding of the replicase complex. Thus, we attempted to select compensatory mutations that enable efficient replication by long term passage of Huh-7.5 cells transfected with H77S ₁ s _/8mt RNA in 3-independent culture plates. After a month-long incubation without positive selection, HCV core-positive cells increased to almost 100% in all 3 plates, coincidently with the appearance of severe cytopathic effects on day-32, with cells displaying condensed or fragmented nuclei (Fig. 5). We identified a G1909S (NS4B) mutation in viral RNA in all 3 plates, and 2 additional mutations, D2416G (NS5A) and G2963D (NS5B) in plate #3 (Fig. 6A). The mutation found in all 3 plates, G1909S, had the highest impact on replication fitness, resulting in approximately 70-fold increase compared to H77Sis _/8mt , and D2416G and G2963D increased 3- and 12-fold, respectively (Fig. 6B). The combination of D2416G and G2963D and that of all 3 mutations increased the fitness by 70- and 800-fold, respectively. Importantly, replication fitness of the mutants with these 3 individual mutations or the combinations of them was not enhanced by SKI or VE and remained resistant to lipid peroxidation (Fig. 6B). As our attempts to identify mutations that enable robust replication have been unsuccessful when H77S.3 was similarly transfected and passaged in Huh-7.5 cells over one and a half months, it is likely that the lipid peroxidation-resistant phenotype of H77Sis _/8mt was the key basis for successful selection of the robust replication phenotype. This newly developed clone, H77Sis with a total of 11 mutations, designated H77D (or H77S _1S/ i i _mt ), replicated as efficiently as HJ3-5 in Huh-7.5 cells and slightly lower than HJ3-5 in FT3-7 cells, a clonal derivative of Huh-7 cells (Fig. 6C). H77D replicated at 10-fold higher levels than TNcc or H77S.3 in both Huh-7.5 and FT3-7 cells.

EXAMPLE 6

Robust production of infectious H77D virus does not require anti-lipid peroxidative agents

[0129] We further investigated whether robust replication capacity of H77D leads to efficient production of infectious virus particles. Consistent with its replication level, H77D was capable of producing infectious virus particles to the similar level as HJ3-5 or JFH1-QL, a cell culture adapted JFH1 with Q221L mutation in NS3 helicase, and 100- and 10-fold higher than H77S.3 and TNcc, respectively, without VE supplementation in Huh-7.5 cells (Figs. 7A-7B). Efficient production of H77D virus was also confirmed with FT3-7 cells (Fig. 7C). Importantly, VE did not enhance H77D yield in both cell cultures, while it increased H77S.3 yield >5 fold in parallel experiments, demonstrating that H77D has the JFHl-like, lipid peroxidation-resistant phenotype for both replication and production of infectious virus particles. EXAMPLE 7

Mutations Essential For Lipid Peroxidation Resistance

[0130] To determine which TNcc-derived mutations are essential for resistance to lipid peroxidation, we inserted them individually or in combinations into H77S.3is containing the NS4B G1909S mutation (H77S.3 _1S / _GS )- The combination of mutations in NS4A+NS4B+NS5B conferred resistance, as did NS3+NS4A+NS5B, but not

NS3+NS4B+NS5B, pointing to a critical role for A1672S (NS4A) and the nonessential nature of mutations in NS3 and NS4B (Fig. 8A). Mutations in NS5B were essential for replication of these constructs (Fig. 8A), but neither these nor that in NS4A conferred resistance to lipid peroxidation on H77S.3is (Fig. 9). A combination of NS4A+NS5B mutations resulted in very low replication fitness but with continued minimal

enhancement by VE (Fig. 10). These results suggest that mutations in multiple nonstructural proteins are required for resistance to lipid peroxidation. The mutations in NS4A (A1672S) and near the C-terminus of NS5B are located within or in close proximity to the replicase membrane (Fig. 8B), consistent with direct involvement of these residues in resistance to lipid peroxidation. Additional studies carried out in a similar fashion confirmed that the NS4A (A1672S) and NS5B (D2979G) mutations were collectively responsible for resistance to lipid peroxidation.

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[0132] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.