NZ619294B2 - Hcv genotype 4 replicons - Google Patents

Abstract

Disclosed is a genotype 4a hepatitis C viral (HCV) RNA construct comprising a S'NTR, an internal ribosome entry site (IRES), a sequence encoding NS4A, and a 3 'NTR, wherein the RNA construct further comprises a mutation, as compared to a wild-type NS4A HCV 4a sequence, wherein the mutation is selected from Q34K, Q34R, or ES2V in NS4A, or combinations thereof. Also disclosed is an NS4A protein of HCV genotype 4a that comprises a mutation, as compared to the wild-type HCV 4a NS4A protein, selected from Q34K, Q34R, or E52V or combinations thereof. ed from Q34K, Q34R, or ES2V in NS4A, or combinations thereof. Also disclosed is an NS4A protein of HCV genotype 4a that comprises a mutation, as compared to the wild-type HCV 4a NS4A protein, selected from Q34K, Q34R, or E52V or combinations thereof.

Description

HCV GENOTYPE 4 REPLICONS CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of United States Provisional Applications Serial Number 61/504,853 filed July 6, 2011, Serial Number 61/509,984 filed July 20, 2011, and Serial Number 61/589,789 filed January 23, 2012, the content of each of which is incorporated by reference in its entirety into the present disclosure.

FIELD OF THE DISCLOSURE The disclosure is directed to hepatitis C ons of pe 4 and s of preparing and using the replicons.

STATE OF THE ART Chronic hepatitis C virus (HCV) infection remains a significant global heath burden with an estimated 160 n people infected world wide. The current standard of care is 24 to 48 week courses of pegylated interferon plus ribavirin. Due to the partial y and poor tolerability of this regimen, the discovery and development ofnew antiviral agents has been intensely d. Recently, these efforts have culminated in the FDA approval of two NS3 protease inhibitors (boceprevir and telaprevir) for use in combination with pegylated interferon and ribavirin for the treatment of chronic genotype 1 HCV infection. Many other inhibitors are in advanced clinical development, however, the majority are being developed to treat genotype 1 infections.

HCV is a positive-strand RNA virus that exhibits extraordinary genetic diversity.

Six major genotypes (i. e. genotype l-6) along with le subtypes (e.g. genotype la, lb, lc etc.) have been reported. pes l, 2 and 3 have worldwide distributions.

Genotypes la or lb are generally predominant in North a, South a, Europe and Asia. However, genotypes 2 and 3 are common and can constitute 20 to 50% of ions in many of these areas. Genotype 4a is the predominant in the Middle East and many African countries; up to 15% of the population of Egypt is ed with HCV and 93% of infections are genotype 4. Genotype 5 is prevalent in South Africa, while Genotype 6 is most common in Asia. Although most continents and countries have a “dominant” genotype, infected populations are almost universally made up of a mixture of multiple genotypes. Furthermore, the geographical distribution and diversity miology) ofHCV infection is continuously evolving, due to large-scale ation and widespread enous drug use. For instance, genotype 4a has noticeably spread into central and northern . This presents a clinical challenge, since it is well documented that individual genotypes respond differently to both direct antivirals and immunomodulatory therapies, including the t standard of care.

HCV replicons are self-replicating RNA ces derived from the HCV genome and have served as workhorses both for molecular virology studies and drug discovery. To date, replicons have been established from two genotypes and three subtypes (genotypes la, lb and 2a). These replicons have been crucial in multiple s of drug discovery and development including the identification of novel inhibitor classes, the zation of clinical candidates and the characterization of al resistance.

Recently, there has been increasing interest in developing next-generation drugs that are active against all major HCV genotypes. Ideally, the approval of “pan-genotypic” drugs and regimens will greatly simplify the treatment of HCV.

[0006] A key step in the pursuit of pan-genotypic treatment regimens will be the development of in vitro tools that allow the study of all major genotypes and subtypes.

Replicons derived from sequences of additional major genotypes (z'. e. those other than genotype la, lb or 2a), however, have not been generated. In particular, e the worldwide prevalence of genotype 4 HCV in the Middle East, North Africa and Europe, no genotype 4 replicons have been described.

SUMMARY It has been discovered, ctedly, that clonal cell lines stably replicating Genotype 4 replicons were ed by transcribing and electroporating subgenomic genotype 4 cDNAs into HCV permissive cell lines. Adaptive mutations have been identified from these clones, as compared to the wildtype virus. When these mutations were engineered by site-directed mutagenesis and introduced into the cell lines, HCV genotype 4 replications ensued.

These adaptive mutations for genotype 4 were located in N83 (T343K/R, A200E, or T51 lK), NS4A (Q34K/R, or E52V) or NSSA (Ll79P). The establishment of robust genotype 4 replicon systems provides powerful tools to facilitate drug ery and development efforts.

Accordingly, one embodiment of the t disclosure provides a genotype 4 tis C viral (HCV) RNA construct that is capable of replication in a eukaryotic cell, wherein the RNA sequence comprises a S’NTR, an internal ribosome entry site (IRES), sequences encoding one or more ofNS3, NS4A, NS4B, NSSA or NSSB, and a 3’NTR.

In one aspect, the construct ses one or more adaptive mutations in NS3, NS4A, NS4B, NSSA or NSSB. Non-limiting examples include (1) an isoleucine at location 2204, (2) a glutamic acid at residue 200, a lysine or an arginine at residue 343, an arginine at residue 51 l, or combinations thereof in NS3, (3) a lysine or an arginine at residue 34, a valine at residue 52, or combinations thereof in NS4A, or (4) a e at residue 179 in NSSA. It is also contemplated that the construct includes at least two, or alternatively three or four ve mutations. In one aspect, the adaptive ons come from different genes. In some aspects, the construct is a subgenomic or full-length HCV replicon.

Moreover, DNA that transcribes to the RNA construct, viral particles that include the RNA construct, and cells containing such DNA or RNA are also provided.

Also provided, in one embodiment, are individual NS3, NS4A or NSSA ns that include one or more of the corresponding adaptive mutations. Polynucleotides encoding these proteins and antibodies that specifically recognize the proteins are also provided.

[0013] In another embodiment, the present disclosure provides an isolated cell comprising a genotype 4 hepatitis C viral (HCV) RNA that ates in the cell. In one aspect, there is an absence, in the cell, of a DNA construct encoding the RNA. In another aspect, the cell ses at least 10 copies, or alternatively at least about 100, 500, 1000, 2000, 5000, , 1 x 105, 1 x 106, 1 x 107, 1 x 108 or 1 x 109 copies ofthe RNA. In any of such aspects, the RNA can be a subgenomic HCV sequence or a full-length HCV sequence and can include one or more of the adaptive mutations described above.

In one aspect, the cell is a mammalian cell which can be, for instance, a ma cell, in particular a Huh7 lC cell.

Methods of improving the capability of a genotype 4 HCV viral RNA to replicate in a otic cell are also provided, comprising one or more of (a) substituting 2012/045592 residue 200 ofNS3 with a glutamic acid, (b) substituting residue 343 ofNS3 with a lysine or an arginine, (c) substituting residue 5 ll ofNS3, with an arginine, (d) substituting residue 34 ofNS4A with a lysine or an ne, (e) substituting residue 52 of NS4A with a valine, or (f) tuting residue 179 ofNSSA with a proline.

Still provided, in one embodiment, is a method of identifying an agent that inhibits the ation or ty of a genotype 4 HCV, comprising contacting a cell of any of the above embodiments with a candidate agent, wherein a decrease of replication or a decrease of the actiVity of a protein encoded by the RNA indicates that the agent inhibits the replication or activity of the HCV. Alternatively, the method comprises ting the lysate of a cell of any of the above embodiments with a candidate agent, wherein a decrease of the ty of a protein encoded by the RNA indicates that the agent ts the activity of the HCV.

BRIEF DESCRIPTION OF THE DRAWINGS The disclosure is best understood from the ing detailed description when read in conjunction with the accompanying drawings. Included in the drawings are the following figures: is a schematic diagram of genotype 4a replicon constructs. HCV replicons used to generate novel genotype 4a stable replicon cell lines. ED43 4a strain replicons encode either a nenomycin otransferase II (A) or a Renilla luciferase (Rluc)-neomycin phosphotransferase II fusion reporter (B). The sized replicons incorporated the following elements from 5’ to 3’: the ED43 S’UTR; the neomycin phosphotransferase II gene (neo) or Rluc-Neo gene; the encephalomyocarditis virus (EMCV) IRES; the NS3 - NSSB polyprotein region of ED43 including an NSSA adaptive mutation (822041) and the 3’UTR of ED43. Solid black boxes indicate HCV core sequence. Dot shaded boxes indicate HCV polyprotein sequence. “+” indicates the S2204I adaptive mutation. The 5’ and 3 ’ non-translated regions (NTR), and EMCV IRES are indicated. is a schematic m of genotype 4a replicon establishment strategy. shows the numbers of surviving colonies in three different cell lines.

Huh-7 Lunet, 51C and 1C cells were transfected with the GT4a replicon RNA WO 06721 respectively as described in the Materials and Methods. The numbers of surviving es were counted for each selection. The data represent an average of at least 6 independent transfections. Huh7-lunet was obtained from ReBLikon GmbH (Mainz, Germany). The derivation of 5 1C cells was previously described (Robinson et al., Antimicrob Agents Chemother 54:3099-106 (2010)). 1C cells were derived by curing a GSresistant pe la replicon clone derived from 5 1C cells. GS-5885 is an NSSA inhibitor, available from Gilead Sciences, Inc. Foster City, CA. The figure shows that Huh7 lC cells were more permissive than Huh7-Lunet or 5 1C cells to GT4a replicon replication.

[0021] shows that selected GT4a replicon clones acquired adaptive genetic changes. Total cellular RNA was extracted from a primary genotype 4a replicon cell clone then oporated into Huh-7 Lunet cells at the indicated amounts. Transfected cells were resuspended in complete DMEM medium and plated at multiple densities g from 2 x 105 to 2 x 106 cells in a 100 mm-diameter dish. Forty-eight hours after plating, medium was replaced with complete DMEM supplemented with 0.5 mg/ml G418 which was refreshed twice per week. Three weeks later, colony plates were fixed with 4% formaldehyde and stained with 0.05% crystal violet in H20. In vitro transcribed GT4a replicon RNA was transfected in parallel as a control. The greatly enhanced colony ion efficiency of the RNA extracted from the primary pe 4 replicon indicates that the replicons in that clone had acquired adaptive changes that d robust replication in vitro. shows robust NS5A and NS3 expression in GT4a replicon cell lines (A).

A GT4a replicon cell pool was d with anti-NS5A antibody (Apath, Brooklyn, New York; upper panel, light gray) and Hoechst 33342 (Invitrogen; l ug/ml) (lower panel, dark gray indicates nuclei). lC cells were stained as a negative control (lower panel).

GT4a replicon cells were y positive for NS5A indicating active replication. (B) Selected GT4a replicon cell lines were measured for their intracellular NS3 protease activity as bed in Materials and Methods. GTla and Gle stable replicon cells were included for comparison of the NS3 protease activity. lC cells were included as a ve control. Robust NS3 ty, indicating robust replicon activity, was observed in the GT4a on cell lines with some GT4a replicon cell lines exceeding the NS3 signal produced by standard GTla and lb replicon cells. confirms robust NSSA expression in GT4a replicon cell lines. Stable GT4a and Gle replicon cells, 0.5 x 106 each, were pelleted and completely lysed in 100 ul SDS g buffer. 12 ul lysates were subjected to SDS-PAGE and Western blot is. The blot was d with primary anti-NSSA antibody (Apath; 1:10000 dilution) and ary anti-mouse antibody (IRDye 800CW Goat anti-Mouse IgG (H + L) from LI-COR, l:l0,000 dilution). The staining was then analyzed by Odyssey Imaging (LI-COR. Lincoln, Nebraska). The blot was also co-stained with anti-BiP antibody (Abcam; 1:1000 dilution) and secondary anti-rabbit antibody (IRDye 800CW Goat anti-Rabbit IgG (H + L) from LI-COR, l:l0,000 dilution) as a loading control.

Strong expression ofNSSA was detected in the GT4a on cell clones, confirming that these cells stably and robustly replicate this replicon, either exceeding or being comparable to the NSSA expression level by standard Gle replicon cells. shows that NS4A Q34R is a cell culture adaptive mutation for GT4a replication. The Neo gene of the GT4a ED43-neo construct was replaced with a Rluc-neo filSlOI‘l reporter to facilitate the measurement of replicon ation in the cell culture (by luciferase). The Q34R mutation in the NS4A gene was then introduced into the GT4a ED43-RlucNeo uct by site-directed mutagenesis. All three replicon RNAs were transfected into Huh7-Lunet (left panel) and 1C (right panel) cells respectively. The number of surviving colonies was counted for each selection. The data represent an e of at least two independent transfections. The Q34R on enabled the GT4a ED43-RlucNeo to establish colonies whereas the same replicon t this mutation does not establish colonies. A clone of GT4a RlucNeoQ34R was selected due to its higher Rluc signal and amplified for antiviral assays. presents data to show that the NS3 A200E, T343R and T343K and NS4A Q34R, Q34K and E52V mutations are cell e adaptive mutations for GT4a replication. The Neo gene of the GT4a ED43-neo construct was replaced with a Rluc-neo filSlOI‘l reporter to facilitate the measurement of on replication in the cell culture (by luciferase). Mutations AZOOE, T343R and T343K in the NS3 gene and Q34K, Q34R and E52V in the NS4A gene were then introduced into the GT4a ED43-RlucNeo uct by site-directed mutagenesis respectively. All replicon RNAs were ected into lC cells individually and l x 104 transfected cells were plated into a well in a 96-well plate. At 4h and day l to day 8 daily post transfection, cells were analyzed for renilla luciferase 2012/045592 ty. Cells were passaged and replated at day 4. At each time point, quadruple wells were assayed for each transfection and the data represents an average of two independent experiments with error bars. All tested mutations, A200E, T343R and T343K in the NS3 gene and Q34K, Q34R and E52V in the NS4A gene, significantly enhanced GT4a ED43- RlucNeu replication as evidenced by the increase of Rlu signal from day 2 after initial decrease of the signal derived from the direct translation of input RNA that was independent of RNA replication. In st, the same replicon without a mutation did not show any meaningful replication.

DETAILED DESCRIPTION

[0026] Prior to describing this disclosure in greater detail, the following terms will first be .

It is to be understood that this sure is not limited to particular ments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be ng, since the scope of the present disclosure will be d only by the appended claims.

It must be noted that as used herein and in the appended claims, the singular forms “ 3, (C , an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, nce to “a thread” includes a plurality of threads. 1. Definitions Unless defined otherwise, 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 disclosure belongs. As used herein the following terms have the following meanings.

As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others.

“Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a ition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel teristic(s) of the claimed disclosure. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these tion terms are within the scope of this disclosure.

The term ” when used before a numerical designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may varyby(+)or(-)lO%,5%orl%.

The term “protein” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the t may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein’s or peptide’s ce. As used herein the term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical s, amino acid analogs and peptidomimetics. Single letter and three letter abbreViations of the naturally occurring amino acids are listed below. A e of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein. l-Letter 3-Letter Amino Acid Y Tyr L-tyrosine G Gly L-glycine F Phe L-phenylalanine M L-methionine A Ala L-alanine S L-serine I L-isoleucine L L-leucine T L-threonine V L-Valine P L-proline K Lys L-lysine H L-histidine Q Gln amine E L-glutamic acid W L-tryptohan R L-arginine D L-aspartic acid N L-asparagine C Cys L-cysteine The terms “polynucleotide” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any fianction, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, s, messenger RNA (mRNA), er RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched cleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can se modif1ed nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the tide structure can be imparted before or after ly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be fithher modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded les. Unless otherwise specified or required, any ment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0034] A polynucleotide is composed of a specific sequence of four tide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the cleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical entation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as fianctional genomics and gy searching.

“Homology” or “identity” or “similarity” refers to ce similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be d for purposes of comparison.

When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a fianction of the number of matching or homologous positions shared by the sequences. An ated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present invention.

In one embodiment, the homologous peptide is one that shares the same onal characteristics as those described, including one or more of the adaptive mutations.

A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when d, that percentage of bases (or amino acids) are the same in comparing the two sequences.

This alignment and the percent homology or sequence ty can be determined using software ms known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non- redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following et address: http://www.ncbi.nlm.nih.gov/blast/Blast.cgi, last accessed on July 15, 2011. Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological ty.

The term “a homolog of a nucleic acid” refers to a nucleic acid having a nucleotide ce having a certain degree of homology with the nucleotide sequence of the c acid or complement thereof. A homolog of a double stranded nucleic acid is intended to include c acids having a nucleotide sequence which has a certain degree of homology with or with the complement thereof. In one aspect, homologs of nucleic acids are capable of hybridizing to the nucleic acid or complement thereof

[0038] A “gene” refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular ptide or protein after being transcribed and translated. Any of the cleotide or polypeptide sequences described herein may be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. s of isolating larger fragment sequences are known to those of skill in the art.

The term “express” refers to the production of a gene product.

As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and/or the s by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or ns. If the cleotide is derived from genomic DNA, expression may include splicing of the mRNA in an eukaryotic cell.

The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a ptide if, in its native state or when lated by methods well known to those skilled in the art, it can be transcribed and/or translated to e the mRNA for the ptide and/or a fragment thereof The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

“Eukaryotic cells” comprise all of the life kingdoms except monera. They can be easily distinguished h a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. A eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells, or alternatively from a prokaryotic cells as described above.

Non-limiting examples include simian, bovine, porcine, , rats, avian, reptilian and human.

[0043] As used herein, an “antibody” includes whole antibodies and any antigen binding fragment or a single chain thereof. Thus the term “antibody” includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin le. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding n thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any n thereof, or at least one portion of a binding protein. The antibodies can be polyclonal or monoclonal and can be isolated from any suitable biological source, e.g., murine, rat, sheep and canine.

The terms lonal antibody” or “polyclonal antibody composition” as used herein refer to a preparation of antibodies that are derived from different B-cell lines.

They are a mixture of immunoglobulin molecules secreted against a specific antigen, each izing a different epitope.

The terms “monoclonal antibody” or “monoclonal antibody ition” as used herein refer to a preparation of antibody molecules of single molecular composition.

A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

The term “isolated” as used herein refers to molecules or biological or cellular materials being substantially free from other materials or when referring to proteins or polynucleotides, infers the breaking of covalent bonds to remove the protein or cleotide from its native environment. In one , the term “isolated” refers to nucleic acid, such as DNA or RNA, or protein or polypeptide, or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source. The term ted” also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when ally synthesized. Moreover, an ted nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides which are isolated from other ar proteins and is meant to encompass both purified and recombinant polypeptides. In other ments, the term “isolated or recombinant” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, e, polypeptide, n, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype. An isolated polynucleotide is separated from the 3 ’ and 5 ’ contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome. As is nt to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or nt(s) thereof, does not require “isolation” to distinguish it from its lly occurring counterpart. The term “isolated” is also used herein to refer to cells or s that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues.

Hepatitis C virus or “HCV” is a small (55-65 nm in size), enveloped, positive- sense single-stranded RNA virus of the family Flavivz'rz'dae. Hepatitis C virus is the cause of hepatitis C in humans. The hepatitis C virus particle consists of a core of genetic material (RNA), surrounded by an edral protective shell of n, and further encased in a lipid (fatty) envelope of cellular origin. Two viral envelope glycoproteins, El and E2, are embedded in the lipid envelope.

Hepatitis C virus has a positive sense single-stranded RNA . The genome consists of a single open reading frame that is 9600 nucleotide bases long. This single open reading frame is translated to produce a single protein product, which is then r processed to produce smaller active proteins.

At the 5 ’ and 3 ’ ends of the RNA are the UTR, that are not translated into proteins but are important to translation and replication of the viral RNA. The 5 ’ UTR has a me binding site (IRES - Internal ribosome entry site) that starts the translation of a very long protein containing about 3,000 amino acids. This large pre-protein is later cut by cellular and viral proteases into the 10 smaller proteins that allow viral replication within the host cell, or assemble into the mature viral particles.

Structural proteins made by the hepatitis C virus include Core protein, El and E2; nonstructural proteins include NS2, NS3, NS4, NS4A, NS4B, NS5, NSSA, and NSSB.

[0051] Based on genetic differences between HCV isolates, the hepatitis C virus species is classified into six genotypes (1-6) with several subtypes within each genotype sented by letters). Subtypes are fithher broken down into pecies based on their genetic diversity. The preponderance and distribution ofHCV genotypes varies globally. For example, in North America, genotype la predominates ed by lb, 2a, 2b, and 3a. In Europe, genotype lb is inant followed by 2a, 2b, 2c, and 3a.

Genotypes 4 and 5 are found almost exclusively in Africa. Genotype is clinically ant in determining potential response to interferon-based therapy and the required duration of such therapy. Genotypes l and 4 are less responsive to eron-based treatment than are the other genotypes (2, 3, 5 and 6). on of standard interferon- based therapy for pes l and 4 is 48 weeks, whereas treatment for genotypes 2 and 3 is completed in 24 weeks.

Sequences from different HCV genotypes can vary as much as 33% over the whole viral genome and the sequence variability is buted equally throughout the viral genome, apart fiom the highly conserved 5’ UTR and core regions and the hypervariable envelope (E) region.

HCV genotypes can be identified with various methods known in the art. PCR- based genotyping with pe-specific s was first introduced in 1992, in particular with primers targeting the core region. Commercial kits (6.g.

, InnoLipa® by Innogenetics ndre, Belgium)) are also available. Direct sequencing, in the vein, can be used for more reliable and sensitive genotyping.

[0054] Serologic genotyping uses genotype-specific antibodies and identifies genotypes indirectly. Two commercially available serologic genotyping assays have been introduced, including a RIBA SIA assay from Chiron Corp. and the Murex HCV serotyping enzyme immune assay from Nurex Diagnostics Ltd.

Sequences of genotype 4 HCV have been identified. For ce, k accession # GU814266 represents a subgenomic pe 4a replicon based on the ED43 infectious clone. Further discussion of the genotype 4 and their sequences are clinical impacts can be found at Zein Clin. Microbiol. Rev. l3(2):223-35 (2000).

The term “replicon” refers to a DNA molecule or RNA molecule, or a region of DNA or RNA, that replicates from a single origin of replication. For most prokaryotic chromosomes, the replicon is the entire some. In some aspects, a replicon refers to a DNA or RNA construct that replicates in a cell in vitro. In one aspect, a replicon can replicate to produce at least about 10, or alternatively, at least about 100, 500, 1000, 2000, 5000, 10,000, 1 x 105, 1 x 106, 1 x 107, 1 x 108 or 1 x 109 copies ofthe replicon in a cell in vitro. Alternatively, a replicon’s replication efficiency can be ed by producing certain amount of viral RNA in total RNA that includes cellular RNA. In one aspect, a replicon can produce at least about 1000, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, l x 1010, l x 1011, or I x 1012 copies ofthe on per microgram oftotal RNA or cellular RNA.

A “subgenomic” HCV sequence refers to a HCV sequence that does not include all ces of a wild-type HCV. In one aspect, a subgenomic HCV or a subgenomic HCV replicon does not include the El, E2 or C regions. In another aspect, a subgenomic HCV or a subgenomic HCV replicon es all or part of the 5’ UTR, NS3, NS4A, NS4B, NSSA, NSSB and 3’ UTR sequences. In contrast, a “full-length” or “full genome” HCV or HCV replicon includes El, E2 and C regions. In some aspects, both a subgenomic and a full-length HCV replicon can e one or more of a reporter gene (e.g, luciferase), a marker gene (e.g., Neo), and an IRES (e.g., EMCV IRES) sequence.

A virus particle (or virion) ts of the c material made from either DNA or RNA of a virus and a protein coat that protects the genetic material. In one aspect, an envelope of lipids surrounds the protein coat when they are outside a cell.

The term “adaptive mutation” of a HCV replicon of a certain genotype refers to a mutation, as compared to a wild-type HCV ce of the genotype, that enables the wild-type replicon to ate in a cell, in particular in a eukaryotic cell such as a mammalian cell and in vitro, or es a HCV replicon’s ability to replicate. It is contemplated that an adaptive on can bly influence assembly of the replicase complex with host cell-specific protein, or alternatively promote interactions of the protein that includes the adaptive mutation (e.g., NS3, NS4A, NS4B, NSSA etc) with cellular proteins involved in host cell antiviral defenses.

A “reporter gene” refers to a gene that can be attached to a regulatory sequence of another gene of interest in cell culture, animals or plants, to facilitate fication of this other gene. Reporter genes are often used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population. Non-limiting examples of reporter gene include the rase gene and the green fluorescent protein gene.

A “marker gene” or “selectable marker” refers to a gene that protects the organism from a selective agent that would ly kill it or prevent its growth. One non-limiting example is the neomycin phosphotransferase gene (Neo), which upon expression confers resistance to G418, an aminoglycoside antibiotic similar in structure to gentamicin Bl.

HCVgenotype 4 replicon constructs The present disclosure s, in general, to the unexpected discovery that clonal cell lines stably replicating genotype 4 replicons can be obtained by transcribing and oporating subgenomic genotype 4 cDNAs into HCV permissive cell lines. From the clonal cells, adaptive mutations are then identified.

These adaptive mutations were located in NS3 (T343K/R, A200E, or T51 lK), NS4A (Q34K/R, or E52V) or NS5A (Ll79P). The S2204I mutation is also applicable in either genotypes. fication of these mutations suggests that these ons contribute to the HCV’s capability to replicate in cells in vitro, a phenomenon not observed with Wild-type HCV genotype 4 RNA. Such contribution has then been confirmed by ering the ons, by site-directed mutagenesis, into genotype 4 RNA and introducing them into the cell lines. Genotype 4 HCV RNA, With such mutations, successfully replicated in the cell lines. Therefore, the Applicant has demonstrated that the Applicant has prepared HCV genotype 4 replicons capable of replication in vitro and has identified adaptive mutations leading to such capabilities.

Accordingly, in one embodiment, the present disclosure provides a genotype 4 hepatitis C viral (HCV) RNA is capable of ation in a host cell. In one aspect, the replication is in vitro. In another aspect, the replication is productive. In another aspect, the cell is a eukaryotic cell such as a mammalian cell or a human cell. In yet r aspect, the cell is a hepatoma cell. In some aspects, the RNA can replicate to produce at least 10 copies of the RNA in a cell. In another aspect, the number of copies is at least about 100, 500, 1000, 2000, 5000, 10,000, 1 x 105, 1 x 106, 1 x 107, 1 x 108 or 1 x 109.

[0065] The HCV RNA can be a subgenomic HCV sequence. It is specifically contemplated that a filll-length HCV replicon containing any or more of such adaptive mutations is also capable to replicate. Still further, an entire HCV virus of the corresponding genotype containing the ve mutation(s) would be infectious and capable to replicate. In any such case, RNA can include one or more of 5’NTR, an internal ribosome entry site (IRES), sequences encoding NS3, NS4A, NS4B, NS5A and NSSB, and a 3 ’NTR. In one aspect, the RNA includes, from 5’ to 3’ on the positive-sense nucleic acid, a filnctional HCV 5’ non-translated region (5 ’NTR) comprising an extreme inal conserved sequence; an HCV polyprotein coding ; and a fianctional HCV 3’ non-translated region (3’NTR) comprising an extreme 3’-terminal conserved SGQUGIICG.

In any of the above embodiments, the HCV RNA can include an adaptive mutation that enables the RNA to replicate in the cell. Such adaptive mutations can include an isoleucine at location 2204 at NS5A.

Non-limiting examples of adaptive mutation for genotype 4 also include a glutamic acid at residue 200, a lysine or an arginine at residue 343, an arginine at residue l l, or combinations thereof for NS3, or a lysine or an arginine at e 34, a valine at residue 52, or combinations thereof for NS4A, or yet a proline at e 179 for NS5A.

Non-limiting examples of adaptive mutation for genotype 4 also include a serine at residue 607 for NS3.

[0069] In one embodiment, provided are replicons listed in Table 1. It is specifically contemplated that the HCV RNA can include one or more of the described mutations. In one aspect, the HCV RNA includes at least an adaptive mutation in NS3 and at least an adaptive on in NS4A. In another aspect, the HCV RNA includes at least an adaptive on in NS3 and at least an adaptive mutation in NS5A. In yet another aspect, the HCV RNA includes at least an adaptive mutation in NS4A and at least an adaptive mutation in NS5A.

Also plated are that the HCV RNA can be a RNA ce that has at least about 75%, or about 80%, 85%, 90%, 95%, 98%, 99%, or about 99.5% sequence identity to any of the disclosed sequences, so long as it retains the corresponding adaptive on(s) and/or ties.

Thus, in one aspect, a pe 4 HCV RNA construct is provided, comprising a ’NTR, an internal ribosome entry site (IRES), sequences encoding NS3, NS4A, NS4B, NS5A and NS5B, and a 3’NTR, wherein the construct is capable to replicate in a eukaryotic cell. In one aspect, the construct comprises an adaptive mutation in NS3, NS4A, NS4B, NS5A or NS5B.

In one aspect, the mutation comprises an isoleucine at location 2204 in NS5A.

In another aspect, the mutation comprises, in NS3, a glutamic acid at residue 200, a lysine or an ne at residue 343, an arginine at residue 5 l l, or ations thereof. Yet in another aspect, the mutation comprises, in NS4A, a lysine or an arginine at residue 34, a valine at residue 52, or ations thereof. Further in an aspect, the mutation comprises, in NSSA, a proline at residue 179. In some aspect, the genotype 4 is genotype In any of the above embodiments, the HCV RNA can further comprise a marker gene for selection. A non-limiting example of such marker gene is a neomycin phosphotransferase gene. Other examples are well known in the art.

In any of the above embodiments, the HCV RNA can further comprise a reporter gene. A miting example of such marker gene is a luciferase gene. Other examples are well known in the art.

The RNA construct of any of the above embodiment can further comprise sequences encoding one or more of C, E1 or E2. In one aspect, the RNA construct is a full-length HCV replicon.

The disclosure also provides a single or double-stranded DNA that can be transcribed to a RNA construct of any of the above embodiment, a viral particle comprising a RNA construct of any of the above embodiment, or an isolated cell comprising a RNA construct of any of the above embodiment.

In one embodiment, the present disclosure provides an NS3 protein ofHCV genotype 4 that comprises a glutamic acid at residue 200, a lysine or an arginine at e 343, an arginine at residue 51 l, or combinations thereof.

In one ment, the present disclosure provides an NS4A n ofHCV genotype 4 that comprises a lysine or an arginine at residue 34, a valine at residue 52, or combinations thereof.

In one ment, the present sure provides an NSSA protein ofHCV pe 4 that comprises a e at residue 179.

In one aspect of any such embodiments, the genotype 4 is genotype 4a. In yet another aspect, provided is a polynucleotide encoding the protein of any of such embodiments. The polynucleotide can be RNA or DNA. In r aspect, provided is an RNA or DNA construct comprising the polynucleotide. In yet another aspect, provided is a cell sing the polynucleotide. Still in one aspect, provided is an antibody that specifically izes a protein of any of the above embodiments.

HCVGenotype 4 Replicons and Cells Containing the ons Another embodiment of the present disclosure es an isolated cell comprising a pe 4 hepatitis C Viral (HCV) RNA that replicates in the cell. In one aspect, there is an absence, in the cell, of a DNA construct ng the RNA and thus copies of the HCV RNA are not transcribed from a DNA, such as cDNA, construct.

In one , the cell comprises at least 10 copies of the RNA. In another aspect, the cell comprises at least 100, 500, 1000, 2000, 5000, 10,000, 1 X 105, l X 106, l x 107, 1 x 108 or 1 x 109 copies ofthe RNA.

The HCV RNA can be subgenomic HCV sequence or a ength HCV sequence. In either case, RNA can include one or more of 5 ’NTR, an internal ribosome entry site , sequences encoding NS3, NS4A, NS4B, NS5A and NS5B, and a 3’NTR.

In any of the above embodiments, the HCV RNA can include an adaptive mutation that enables the RNA to replicate in the cell. Such adaptive mutations can include an isoleucine at location 2204 at NS5A. miting examples of adaptive mutation for genotype 4 also include a glutamic acid at residue 200, a lysine or an arginine at e 343, an arginine at residue l l, or combinations thereof for NS3, or a lysine or an arginine at residue 34, a valine at residue 52, or combinations thereof for NS4A, or yet a proline at residue 179 for NS5A.

[0086] In one embodiment, provided are replicons listed in Table 1. It is specifically contemplated that the HCV RNA can include one or more of the bed mutations. In one aspect, the HCV RNA includes at least an adaptive mutation in NS3 and at least an adaptive mutation in NS4A. In another aspect, the HCV RNA includes at least an adaptive mutation in NS3 and at least an adaptive mutation in NS5A. In yet another aspect, the HCV RNA includes at least an adaptive mutation in NS4A and at least an adaptive mutation in NS5A.

Also contemplated are that the HCV RNA can be a RNA sequence that has at least about 75%, or about 80%, 85%, 90%, 95%, 98%, 99%, or about 99.5% sequence identity to any of the disclosed sequences, so long as it retains the corresponding adaptive mutation(s). 2012/045592 In one aspect, the cell is a otic cell such as a mammalian cell and in particular a human cell. In r aspect, the cell is hepatoma cell, such as but not limited to a Huh7 cell (e.g., Huh7-Lunet, 51C and 1C). It is herein discovered surprisingly that Huh7 lC cell is particularly permissive to the genotype 4 replicons and thus in one aspect, the cell is a Huh7 lC cell. In some aspects, the cell is placed at an in vitro or ex vivo condition.

Methods ofPreparing Genotype 4 Replicons After HCV genotype 4 ons are fied, as shown in Example 1, introduction of the relevant adaptive mutation into a corresponding genotype HCV RNA can result in the RNA’s capability to replicate, in particular in a mammalian cell in vitro.

Accordingly, the present disclosure provides a method of improving the capability of a genotype 4 HCV viral RNA to replicate in a eukaryotic cell, comprising one or more of: (a) substituting residue 200 ofNS3 with a glutamic acid, (b) substituting residue 343 ofNS3 with a lysine or an arginine, (c) substituting residue 511 ofNS3, with an arginine, (d) substituting residue 34 ofNS4A with a lysine or an arginine, (e) substituting residue 52 ofNS4A with a , or (f) substituting residue 179 ofNS5A with a proline. In one aspect, the method comprises at least two substitutions of (a) — (f).

[0090] In any of the above methods, an S2204I on can filrther be introduced into the RNA.

Methods ofScreening HCVInhibitors Targeting Genotype 4 Numerous known and unknown HCV inhibitors have been tested for their efficiency in inhibiting the genotype 4 HCV, in comparison with genotype lb (Example 1). Some showed higher efficacy for genotype 4, and some were not as efficacious. The ness of the new identified genotype 4 replicons, therefore, is adequately demonstrated.

Thus, the present disclosure also provides, in one embodiment, a method of identifying an agent that ts the ation or activity of a genotype 4 HCV, comprising contacting a cell of any embodiment of the present disclosure with a candidate agent, wherein a decrease of replication or a decrease of activity of a n encoded by the RNA indicates that the agent inhibits the replication or activity of the HCV. In some aspects, the protein is a protease, such as any or more ofN83, NS4A, NS4B, NSSA or NSSB. Replication of the RNA, in one aspect, can be measured by a reporter gene on the RNA, such as the luciferase gene. ed in another embodiment is a method of identifying an agent that the activity of a genotype 4 HCV, sing contacting the lysate of a cell of any embodiment of the present disclosure with a candidate agent, wherein a decrease of the activity of a protein encoded by the RNA tes that the agent inhibits the activity of the HCV. In one aspect, the n is a protease, such as any or more ofNS3, NS4A, NS4B, NSSA or NSSB. In another aspect, the method fiarther comprises measuring the replication of the RNA or the activity of the protein encoded by the RNA.

A HCV inhibitor (or “candidate agent”) can be a small molecule drug that is an organic compound, a peptide or a protein such as antibodies, or nucleic acid-based such as siRNA. In May 2011, the Food and Drug Administration approved 2 drugs for Hepatitis C, evir and evir. Both drugs block an enzyme that helps the virus uce.

Boceprevir is a protease inhibitor that binds to the HCV NS3 active site on hepatitis C genotype 1. Telaprevir inhibits the hepatitis C virus NS3.4A serine se.

More conventional HCV treatment includes a combination of pegylated interferon-alpha-2a or ted interferon-alpha-2b (brand names s or PEG- Intron) and the antiviral drug ribavirin. Pegylated interferon-alpha-2a plus ribavirin may increase sustained virological response among patients with chronic hepatitis C as compared to pegylated interferon-alpha-2b plus ribavirin according to a systematic review of ized controlled trials.

All of these HCV inhibitors, as well as any other candidate agents, can be tested with the disclosed methods for their y in inhibiting HCV genotype 4. The cells are then incubated at a suitable temperature for a period time to allow the replicons to ate in the cells. The replicons can include a reporter gene such as luciferase and in such a case, at the end of the incubation period, the cells are assayed for luciferase activity as markers for replicon levels. Luciferase expression can be quantified using a commercial luciferase assay.

Altemately, y of the HCV inhibitor can be measured by the sion or activity of the proteins encoded by the replicons. One example of such proteins is the NS3 protease, and detection of the protein expression or activity can be carried out with methods known in the art, e.g., Cheng et al., Antimicrob Agents Chemother 7-205 (201 l).

Luciferase or NS3 protease ty level is then converted into percentages relative to the levels in the controls which can be untreated or treated with an agent having known activity in inhibiting the HCV. A decrease in HCV replication or decrease in NS3 ty, as ed to an untreated control, indicates that the candidate agent is capable of inhibiting the corresponding genotype of the HCV. Likewise, a larger se in HCV replication or larger decrease in NS3 activity, as compared to a control agent, indicates that the candidate is more efficacious than the control agent.

EXAMPLES The present disclosure is further defined by reference to the following examples.

It will be apparent to those skilled in the art that many ations, both to threads and methods, may be practiced without departing from the scope of the current disclosure.

Abbreviations Unless otherwise stated all temperatures are in degrees Celsius (0C). Also, in these examples and elsewhere, iations have the following gs: uF = MicroFaraday ug = Microgram uL = Microliter uM = Micromolar g = Gram hr = Hour mg = Milligram mL = Milliliter mM = Millimolar mmol = Millimole nM = Nanomolar nm = Nanometer pg = pictograms DMEM = Dulbecco’s modified Eagle’s medium EMCV = encephalomyocarditis virus FBS = fetal bovine serum IRES _ —=revolutions per minute RT-PRC H reverse transcription-polymerase chain reaction Example 1: Generation of Robust Genotype 4 Hepatitis C Virus Subgenomic Replicons This example shows that adaptive mutations were identified from pe 4 HCV viral replicons capable of replication in Huh7 cells and that HCV replicons with these adaptive mutations are useful tools for antiViral drug screening.

Materials and Methods Cell Culture Three HCV permissive cell lines were used during these studies: unet, 51C, and 1C. Huh7-lunet was obtained from ReBLikon GmbH (Mainz, Germany) (Friebe et al., J Virol 79:380-92 ). The tion of 51C cells, and stable pe 1a H77 and genotype 1b Con-1 Rluc-Neo replicon cells were previously described (see Robinson et al., crob Agents Chemother 54:3099-106 (2010)). 1C cells were derived by curing a 5-resistant genotype 1a replicon clone d from 51C cells (id.). This clonal line showed the highest sivity to GT1a and lb replicons out of screened 50 clones and was 5-10 folds more permissive than Huh7-Lunet and 51C cells overall. All cell lines were propagated in Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX-I (Invitrogen, ad, CA) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT), 1 unit/ml penicillin (Invitrogen), 1 ug/ml streptomycin (Invitrogen), and 0.1 mM sential amino acids (Invitrogen); this media formulation is referred to as complete DMEM. Replicon cell lines were selected and maintained in complete DMEM containing 0.5 mg/ml G418 (also known as Geneticin®, an aminoglycoside antibiotic, Invitrogen).

Construction ofPlasmids Encoding Genotype 4a HCV Subgenomic Replicons

[0103] A plasmid (pGT4aED43SG) encoding a subgenomic genotype 4a replicon based on the ED43 infectious clone (GenBank accession # GU814266) (Chamberlain et al., J Gen Virol 78 (Pt 6):1341-7 (1997); Gottwein et al., J Virol 84:5277-93 (2010)) was prepared by DNA synthesis and cloning (Genescript, Piscataway, NJ). The synthesized replicon incorporated following elements from 5’ to 3’ (: (1) the ED43 5’UTR, extending to the first 48 tides of core, (2) a linker with the nucleotide sequence, 5’- GGCGCGCCA-3’ (SEQ ID NO: 1) which introduces the AscI restriction site (underlined), (3) the neo gene, (4) a linker with nucleotide sequence, 5’- CCGCGGCCGCAA-3’ (SEQ ID NO: 2) which introduces FseI and Not I ction sites (underlined), (5) the encephalomyocarditis virus (EMCV) IRES, (6) a linker with nucleotide sequence 5’-ACGCGTATG-3’ (SEQ ID NO: 3) which introduces the MluI ction site (underlined) and an ATG start codon for HCV polyprotein sion, (7) the NS3 — NSSB otein region of ED43 including an NSSA adaptive mutation (S2204I) and (8) the 3’UTR of ED43. The synthetic DNA fragment encoding the ED43 replicon was ed into PUCl9 between EcoRI and XbaI restriction sites.

[0104] Another plasmid (pGT4aED43RlucSG) encoding a subgenomic replicon that incorporated the humanized Renilla luciferase er gene was generated as s: The pGT4aED43SG plasmid (described above) was cut using AscI and MluI restriction enzymes (to remove the neo gene) and gel purified using a commercial kit (Qiagen). A gene fragment encoding the humanized Renilla luciferase gene fused with the neo gene along with the EMCV region, were PCR amplified by using Accuprime super mix I (Invitrogen) with the following primers from the thlucNeoSG2a plasmid described below: 2aRlucNeoAscIFor: 5 ’- AACACCAACGGCGCGCCAATGGCTTCCAAGGTGTAC-3’ (SEQ ID NO: 4, AscI site is introduced by the primer and is underlined), 2aEMCVIRESMluIRev: 5 ’- TGGGCATAAGCAGTGATGGGAGCCATACGCGTATCG -3’ (SEQ ID NO: 5, MluI site underlined).

Plasmid eoSG2a was derived from the plasmid pLucNeo2a (Cheng et al., Antimicrob Agents Chemother 55:2197-205 (201 l)). The hRenilla Luciferase- Neomycin filSlOIl gene (thuc-Neo) was PCR amplified fiom pF9 CMV thuc-neo Flexi(R) (Promega, Madison, WI) by PCR using Accuprime Super Mix I (Invitrogen) and a primer set of AfeI hRLuc Fwd and NotI Neo Rev. These two primers had the following sequence and introduced ction sites for uent cloning: AfeI hRLuc: 5’ ATAGCGCTATGGCTTCCAAGGTGTACGA 3’ (SEQ ID NO: 6, AfeI site underlined), NotI Neo Rev: 5’ AATGCGGCCGCTCAGAAGAACTCGTCA 3’ (SEQ ID NO: 7, NotI site underlined). The thuc-Neo amplification product was subcloned into pCR2.l-TOPO (Invitrogen). The resulting plasmid was digested with AfeI and NotI, and the excised nt (thuc-Neo) was ligated with T4 DNA ligase (Promega) into pLucNeo2a digested with the same enzymes. The resulting vector, thlucNeoSG2a, was sequenced to ensure correct orientation and sequence of the thuc-Neo fusion gene.

The subsequent PCR fragment was cut with AscI and MluI and gel d using a commercial kit (Qiagen). The vector and insert pieces were ligated using LigaFast Rapid DNA Ligation System per manufacturer’s protocol (Promega). The resulting , pGT4aED43RlucSG was sequenced to confirm the correct orientation and sequence of the thuc-Neo.

Construction ofMutant Replicons Adaptive mutations were uced into the pGT4aED43RlucNeoSG replicon by site directed mutagenesis using a QuikChange Lightening kit agene, La Jolla, CA). All mutations were confirmed by DNA sequencing by TACGen (Hayward, CA).

RNA Transcription Plasmids encoding genotype 4a subgenomic HCV replicons were linearized with XbaI and purified using a PCR purification kit (Qiagen). RNA was synthesized and purified with T7 MEGAScript (Ambion, Austin, TX) and RNeasy kits, respectively, according to the manufacturer’s instructions. RNA concentrations were measured using optical density at 260 nm and confirmed by 0.8% agarose gel electrophoresis (Invitrogen).

RNA Transfection and ion of Stable Replicon Cell Lines Ten micrograms of in vitro-transcribed RNA were transfected into Huh7-Lunet, 51C, or lC cells by electroporation as previously bed (Robinson et al., Antimicrob Agents Chemother 54:3099-106 (2010)). Briefly, cells were collected by trypsinization and centrifugation, then washed twice with ice-cold phosphate buffered saline (PBS) and resuspended in Opti-MEM medium (Invitrogen) at a concentration of 107 cells/ml.

Replicon RNA was added to 400 ul of cell suspension in a Gene Pulser (BioRad, es, CA) cuvette (0.4-cm gap). Cells were electroporated at 270 V and 960 uF, incubated at room temperature for 10 minutes, ended in 30 ml complete DMEM and then plated into 100-mm-diameter dishes. Forty-eight hours after plating, medium was ed with complete DMEM supplemented with 0.5 mg/ml G418 which was refreshed twice per week. Cell clones were isolated after approximately three weeks of G418 ion, expanded, and eserved at early passages.

Replicon Colony ion Assays To determine the ency of G418-resistant colony formation, cells were electroporated with ted amounts of replicon RNA or cellular RNA extract, and plated at multiple densities ranging from 2 x 105 to 2 x 106 cells/100mm dish. Forty-eight hours after plating, medium was replaced with complete DMEM supplemented with 0.5 mg/ml G418 which was refreshed twice per week. Three weeks later, colony plates were used for cell expansion or G418-resistant foci were fixed with 4% formaldehyde and stained with 0.05% crystal violet in H20.

Extraction, amplification, and genotypic analysis ofHCV RNA

[0111] HCV RNA isolation, , and sequencing were performed by TACGen (Hayward, CA). HCV replicon cellular RNA was ted and ed using an RNeasy kit (Qiagen) according to the manufacturer’s protocol. RT-PCR was performed using the SuperScript III first-strand sis system (Invitrogen). PCR products were sequenced by TACGen (Hayward, CA).

Detection ofNS5A protein by indirect immunofluorescence Replicon cells were plated in 96-well plates at a density of 1 x 104 cells per well.

After cultured for 24 hours, cells were then stained for NS5A protein as described previously (Cheng et al., Antimicrob Agents Chemother 55 :2197-205 ). Briefly, cells were fixed in 4% paraformaldehyde for 20 minutes. Cells were then washed three times with PBS, blocked with 3% bovine serum albumin, 0.5% Triton X-100, and 10% FBS and then stained with anti-NS5A dy. Staining was performed using a 1:10,000 on of mouse monoclonal antibody 9E10 (Apath, yn, NY). After washing in PBS three times, a secondary anti-mouse antibody conjugated to Alexa Fluor 555 was used to detect anti-NS5A antibody labeled cells (Invitrogen). Nuclei were stained with 1 [Lle Hoechst 33342 (Invitrogen). Cells were washed with PBS and imaged with a Zeiss fluorescence microscope (Zeiss, Thomwood, NY).

Replicon cell NS3 protease assayfor replicon RNA ation Genotype 4a clonal replicons cells were seeded in 96-well plates at a concentration of 1 x 104 cells per well. The cells were incubated for 24 hours, after which culture media were removed. The replicon cells were then lysed with 90 ul of 1x Promega luciferase lysis buffer supplemented with 150 mM NaCl at room temperature for 20 min on a plate shaker. 10 ul of 1 uM europium-labeled NS3 substrate in the above lysis buffer was added to each well. Protease activity data were collected and analyzed as previously described (Cheng et al., Antimicrob Agents Chemother 55:2197-205 (2011)).

Replicon Antiviral Assays 2,000 cells/well were seeded in 384-well plates in 90 ul ofDMEM culture medium, ing G418. HCV inhibitors (Compounds A-E, available from Gilead Sciences, Inc, Foster City, CA) were added to cells at a 1:225 dilution, achieving a final concentration of 0.44% in a total volume of 90.4 ul. Three-fold serial drug ons with concentrations were used, and ng concentrations were 4.4 uM or 0.44 uM for all the tested compounds, except Compound A whose starting concentrations was 44.4 nM.

Cell plates were incubated at 37°C for 3 days, after which culture medium was removed and cells were assayed for luciferase ty as markers for replicon levels. Luciferase expression was fied using a commercial luciferase assay (Promega). Luciferase or NS3 protease activity levels were converted into percentages relative to the levels in the untreated controls (defined as 100%), and data were fitted to the logistic dose response equation y _ a/[1_(x/b)c] using XLFit4 software (IDBS, Emeryville, CA) ()2 is the amount of normalized rase signal, x is the drug concentration, a represents the curve’s amplitude, 19 is the x value at its tion center [EC 50], and c is a parameter which defines its transition width).

Adaptive Mutations Using the strategy as illustrated in a number of GT4a colonies were obtained. RNA was then extracted from these colonies. As shown in Huh7 1C cells were more permissive than Huh7-Lunet or 51C cells to GT4a replicon replication.

Using Huh7-Lunet cells, the colony formation capabilities of the GT4a replicons were tested and compared to the original GT4a RNA. As shown in y enhanced colony formation efficiency of the RNA extracted from the GT4a es indicates that the replicons acquired adaptive changes that allowed robust replication in vitro.

The expression ofNSSA and NS3 proteins were then examined to confirm the replication of the GT4a replicons. Stained with anti-NSSA dies, GT4a on cells were clearly positive for NSSA which indicated active replication (). In the same vein, robust NS3 activity, indicating robust replicon activity, was observed in the GT4a replicon cell lines with some GT4a replicon cell lines ing the NS3 signal produced by standard GTla and lb replicon cells, which were used as positive controls (). Apparently, the GT4b replicons were actively replicating in the cells. er, when the GT4a colonies were lysed, strong expression ofNSSA was detected in the cell lysates (, confirming that these cells stably and robustly replicated GT4a on, either exceeding or being comparable to the NSSA expression level of rd Gle replicon cells.

Selected GT4a replicon cell lines or pooled cell lines were expanded and subjected to genotypic analysis. Total RNA was extracted and purified using an RNeasy kit (Qiagen) according to the cturer’s protocol. RT-PCR was performed using the SuperScript III first-strand synthesis system (Invitrogen). PCR products were sequenced by TACGen. Novel mutations that emerged during adaptation of the GT4 replicon are presented in Table 1.

Table 1. Mutations identified in GT4a on cells These mutations were then tested by introducing them, by site-directed mutagenesis, into the original GT4a RNA. shows that, in both Huh7-Lunet (left panel) and 1C (right panel) cells, the GT4a RNA with the Q34R mutation enabled the GT4a ED43-RlucNeo to establish colonies whereas the same replicon without this mutation does not establish colonies.

Likewise, the ability ofNS3 A200E, T343R and T343K and NS4A Q34R, Q34K and E52V mutations to enable GT4a to ish colonies were also confirmed in Huh7 lC cells (. shows that all tested mutations, A200E, T343R and T343K in the NS3 gene and Q34K, Q34R and E52V in the NS4A gene, significantly enhanced GT4a ED43-RlucNeo replication as evidenced by the se of Rluc signal from day 2 after l decrease of the signal derived from the direct translation of input RNA that was independent ofRNA replication. In contrast, the same replicon without a mutation did not show any meaningful replication. ing the identification of the genotype 4 replicons containing ve mutations, the usefillness of these replicons in screening antiviral agents were evaluated with a variety of anti-HCV agents. Different classes of HCV inhibitors that target NSSA, NSSB active site, NS3 se, NSSB non-active sites, NS4A and host factors, were ted for their ral activities against stable genotype lb and genotype 4a Rluc- Neo replicon cells carrying NS4A Q34R mutation.

Like in stable genotype lb replicon cells, EC50 values against the genotype 4a replicon were generated successfully for all the inhibitors in a high throughput 384-well format by measuring renilla luciferase activity. The inhibition data are listed in Table 2 and indicate that nd B was potent against both genotype lb and 4a replicons with comparable EC50 values. Further, Compound A remained potent though it lost d potency against GT4a.

However, Compound D and Compound E lost their activities approximately lOOO-and lO-folds respectively. Compound C remained potent against genotype 4a replicon, with a minor loss (1.5-3 fold) of their potency compared to their activities against genotype 1b replicon.

These results demonstrate this novel genotype 4a Rluc-Neo replicon could serve as a valuable tool for drug discovery and lead compound optimization against HCV genotype 4a.

Table 2. Comparison of antiviral activities or HCV inhibition t genotype lb and 4a replicons Compounds Gle RLucNeo EC50 (nM) GT4a RlucNeo EC50 (nM) Compound A 0.002 0.105 Compound B 117.3 0.61 nd C 7.0 10.1 Compound D 0.47 469.4 Compound E 0.55 6.4

[0125] Here the Applicant reports the isolation of the first genotype 4 replicons that efficiently replicate in vitro. It is demonstrated that robust replication requires adaptive mutations in NS3 or NS4A in conjunction with NSSA. By incorporating adaptive mutations into luciferase encoding constructs, Applicant was able to generate genotype 4 replicon cell clones that will enable one to profile antiviral compounds. These replicon cells should also serve as valuable tools for molecular Virology s and the characterization of resistance mutations emerging in HCV genotype 4 ts.

In summary, subgenomic replicon cDNAs based on the genotype 4a strain ED43 were synthesized, , transcribed and electroporated into HCV permissive cell lines.

Clonal cell lines stably replicating genotype 4a replicons were selected with G418.

Adaptive mutations were fied by RT-PCR amplification and DNA cing and engineered into the parental replicons by site-directed mutagenesis.

Numerous electroporations into multiple different permissive cell lines allowed the fication of a few colonies that replicated genotype 4 replicons. Expansion and sequencing of these replicons clones revealed adaptive mutations in Viral proteins. These adaptive mutations were located in NS3 (T343K/R, A200E, or T51 1K), NS4A (Q34K/R, or E52V) or NSSA ). These adaptive mutations were engineered back into the parental ED43 strain and were able to greatly enhance replication and colony formation efficiency.

The establishment of robust genotype 4 replicon systems provides powerful tools to tate drug discovery and development efforts. Use of these novel replicons in conjunction with those derived from other genotypes will aid in the development of pan- pic HCV regimens.

Example 2. Screening of New HCV Inhibitors for Genotype 4 Example 1 shows that agents known to be HCV inhibitors for other genotypes, such as genotype 1, can be tested with the genotype 4 replicons for their efficacy in inhibiting genotype 4 HCV. It is also plated that agents not yet known to be inhibitory of HCV can be screened with these genotype 4 ons as well.

The ate HCV inhibitor can be a small molecule drug, a peptide or a protein such as antibodies, or nucleic acid-based such as siRNA. The candidate HCV inhibitor is incubated with cells that contain a genotype 4 replicon, at a suitable ature for a period time to allow the replicons to replicate in the cells. The ons can include a reporter gene such as luciferase and in such a case, at the end of the incubation period, the cells are assayed for luciferase activity as markers for replicon levels. Luciferase expression can be quantified using a commercial luciferase assay.

Alternately, efficacy of the HCV inhibitor can be measured by the expression or activity of the proteins encoded by the ons. One example of such proteins is the NS3 protease, and detection of the protein sion or activity can be carried out with methods known in the art, e.g., Cheng et al., Antimicrob Agents Chemother 55:2197-205 (201 1).

Luciferase or NS3 protease activity level is then converted into percentages relative to the levels in the controls which can be untreated or treated with an agent having known activity in inhibiting the HCV. A decrease in HCV replication or decrease in NS3 ty, as compared to an untreated control, indicates that the candidate agent is capable of ting the corresponding genotype of the HCV. Likewise, a larger decrease in HCV ation or larger decrease in NS3 ty, as compared to a control agent, indicates that the candidate is more efficacious than the l agent.

[0132] It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown , embody the principles of the disclosure and are included within its spirit and scope. Furthermore, all conditional language recited herein is principally intended to aid the reader in understanding the principles of the disclosure and the concepts contributed by the inventors to filrthering the art, and are to be construed as being without limitation to such specifically recited ions. Moreover, all statements herein reciting principles, aspects, and embodiments of the disclosure are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known lents and equivalents developed in the fiJture, z'.e., any elements developed that perform the same function, regardless of structure. The scope of the present disclosure, therefore, is not intended to be limited to the exemplary ments shown and described herein. Rather, the scope and spirit of present disclosure is ed by the ed claims.

Claims (35)

1. A genotype 4a tis C Viral (HCV) RNA construct comprising a S’NTR, an internal ribosome entry site (IRES), a sequence encoding NS4A, and a 3’NTR, wherein the RNA construct further comprises a mutation, as compared to a Wild—type NS4A HCV 4a ce, wherein the mutation is selected from Q34K, Q34R, or E52V in NS4A, or combinations thereof.

2. The pe 4a HCV RNA construct of claim 1, wherein the construct r comprises a ce encoding one or more of N83, NS4B, NSSA or NSSB.

3. The RNA construct of claim 2, wherein the construct further comprises a S2321 mutation in NSSA as compared to a wild—type NSSA HCV 4a sequence. 10

4. The RNA construct ol’claim 2 or 3, wherein the construct comprises a further mutation selected from T343K, T343R, A200E, or T51 1R in N83 or combinations thereof.

5. The RNA construct of any one of claims 2 to 4, wherein the construct comprises a further mutation ofL179P in NSSA.

6. The RNA construct of claim 2, wherein the uct comprises at least a 15 mutation in N83 and at least a mutation in NS4A.

7. The RNA construct of claim 2, wherein the construct ses at least a mutation in NS4A and at least a mutation in NSSA.

8. The RNA construct of claim 2, wherein the construct comprises at least a mutation in NS3, at least a mutation in NS4A, and at least a mutation in NSSA. 20

9. The RNA construct of any one of the preceding claims, further comprising a marker gene for selection.

10. The RNA construct of claim 9, wherein the marker gene is a neomycin phosphotransferase gene.

11. The RNA construct of any one of the preceding , further sing a reporter gene.

12. The RNA construct of claim 11, wherein the reporter gene is luciferase.

13. The RNA construct of any one of claims 2 to 12, wherein the construct comprises, from 5’ to 3’, the S’NTR, the IRES, sequences encoding NS3, NS4A, NS4B, NSSA and NSSB, and the 3’NTR.

14. The RNA construct of any one of the preceding claims, further comprising a sequence encoding one or more of C, E1 or E2.

15. A single or double—stranded DNA that can be transcribed to a RNA construct of any one of the preceding claims.

16. A Viral particle comprising a RNA construct of any one of claims 1 to l4.

17. An ed cell comprising a RNA construct 01’ any one of claims 1 to 14 or DNA ofclaim 15.

18. An NS4A protein of HCV genotype 4a that comprises a mutation, as compared to 15 the wild—type HCV 4a NS4A protein, selected from Q34K, Q34R, or E52V or combinations thcreof.

19. A polynuclcotide ng the protein m 18.

20. The cleotide of claim 19, wherein the polynucleotide is RNA or DNA.

21. A RNA or DNA construct comprising the polynuclcotide of claim 19 or 20. 20

22. An isolated cell comprising a polynuclcotide of claim 19 or 20, or an RNA or DNA construct of claim 21.

23. An antibody that specifically recognizes the protein of claim 18.

24. An isolated cell comprising a genotype 4a tis C viral (HCV) RNA construct of any one of claims 1 to 14, n the construct replicates in the cell.

25. The cell of claim 24, wherein there is an absence, in the cell, of a DNA construct encoding the RNA. 5

26. The cell of claim 24 or 25, wherein the cell comprises at least 10 copies of the RNA construct.

27. The cell of any one of claims 24 to 26, n the cell is a mammalian cell.

28. The cell of claim 27, wherein the cell is a hepatoma cell.

29. The cell of claim 28, wherein the cell is a Huh7 1C cell. 10

30. A method of identifying an agent that inhibits the replication or activity ofa genotype 4a HCV, comprising contacting a cell of any one of claims 24 to 29 with a candidate agent, wherein a se of replication or a decrease of the activity ofa protein encoded by the RNA indicates that the agent inhibits the replication or ty of the HCV.

31. A method of identifying an agent that inhibits the activity of a genotype 4a HCV, 15 comprising contacting the lysate ofa cell of any one of claims 24 to 29 with a candidate agent, wherein a decrease of the activity of a protein encoded by the RNA indicates that the agent inhibits the activity of the HCV.

32. The method of claim 30 or 31, wherein the protein is a protease.

33. The method of claim 32, further comprising measuring the replication of the RNA 20 or the activity of the protein d by the RNA.

34. The genotype 4a HCV RNA construct of claim 1, ntially as hereinbefore described.

35. The NS4A protein ofHCV genotype 4a of claim 18, substantially as hereinbefore described.