Novel HCV Reporter Replicon Cell Lines Enable Efficient. Antiviral Screening against Genotype 1a

Transcription

1 AAC Accepts, published online ahead of print on 1 June 0 Antimicrob. Agents Chemother. doi:./aac.00- Copyright 0, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Novel HCV Reporter Replicon Cell Lines Enable Efficient Antiviral Screening against Genotype 1a Margaret Robinson, Huiling Yang, Siu-Chi Sun, Betty Peng, Yang Tian, Nikos Pagratis, Andrew E. Greenstein, and William E. Delaney IV*. Gilead Sciences, Lakeside Dr. Foster City, CA 0, USA. * Corresponding Author: Lakeside Drive Foster City, CA 0 Tel: 0 Fax: 0 0 William.Delaney@gilead.com Running title: Replication of HCV Genotype 1a Replicons in Novel Cell Lines Downloaded from on January, 01 by guest 1

2 ABSTRACT The hepatitis C Virus (HCV) subgenomic replicon is the primary tool for evaluating the activity of anti-hcv compounds in drug discovery research. Despite the prevalence of HCV genotype 1a (~0% of U.S. HCV patients), few genotype 1a reporter replicon cell lines have been described; this is presumably due to the low replication capacity of such constructs in available Huh- cells. In this report, we describe the selection of highly permissive Huh- cell lines that support robust replication of genotype 1a subgenomic replicons harboring luciferase reporter genes. These novel cell lines support replication of multiple genotype 1a replicons (including H and SF strains), are significantly more permissive to genotype 1a HCV replication than parental Huh- Lunet cells, and maintain stable genotype 1a replication levels suitable for antiviral screening. We found that the sensitivity of genotype 1a luciferase replicons to known antivirals was highly consistent between individual genotype 1a clonal cell lines but could vary significantly between genotypes 1a and 1b. Sequencing the nonstructural region of twelve stable replicon cell clones suggested that the enhanced permissivity is likely due to cellular component(s) in these new cell lines rather than the evolution of novel adaptive mutations in the replicons. These new reagents will enhance drug discovery efforts targeting genotype 1a and facilitate the profiling of compound activity among different HCV genotypes and subtypes. Downloaded from on January, 01 by guest

3 Introduction With million people infected, Hepatitis C virus (HCV) represents a significant and immediate global health burden (). No vaccine is available, and the current standard of care of pegylated-interferon-α/ribavirin combination therapy is poorly tolerated, only partially effective and contraindicated in many patient populations () (). If untreated, HCV infection leads to an increased risk of liver cancer and/or cirrhosis (). HCV exhibits considerable genetic diversity and can be divided into seven major genotypes (i.e. genotype 1-) and several subtypes (e.g genotype 1a, 1b etc) (0) () (). Genotype 1a is the predominant genotype in North America (representing up to 0% of infections) while genotype 1b is predominant in Europe and Japan () (). The HCV subgenomic replicon, the primary tool for discovering and characterizing inhibitors of HCV replication, is a self-replicating, bicistronic viral RNA (1). The first cistron typically encodes a reporter gene and/or a selectable marker and expression is driven by the HCV internal ribosome entry site (IRES). The second cistron encodes the HCV nonstructural proteins and expression is generally driven by the Encephalomyocarditis virus (EMCV) IRES. The nonstructural proteins are translated as a polyprotein, proteolytically processed by the viral NS/A protease and form a replication complex in which the NSB RNA dependent RNA polymerase replicates the entire replicon RNA () (1). By transfecting replicon RNA into a permissive cell line (such as Huh-), clonal cell lines that stably replicate HCV replicons can be selected. Downloaded from on January, 01 by guest

4 With respect to drug discovery, replicon cell lines enable the identification of smallmolecule inhibitors, the selection of resistance mutations, and characterization of inhibitor mechanism of action. Historically, replication levels were monitored by measuring HCV RNA (i.e. Northern blotting or RT-PCR), but this approach is labor intensive and, consequently, not ideal for high-throughput screening (HTS). Incorporation of reporter genes, such as secreted alkaline phosphatase (SEAP), beta-lactamase, or firefly luciferase, facilitated antiviral screening efforts by increasing assay throughput and improving assay statistics (e.g. signal-to-noise and Z- factor) (1) () (). The humanized Renilla reniformis luciferase gene (hrluc) is being increasingly utilized in HCV the infectious virus system due to its enhanced signal-to-noise and decreased gene length (1, 1, ). More recently, a humanized Gaussia princeps luciferase gene (hgluc) has been described with even higher substrate turnover rates and smaller gene size than hrluc (). Despite these advances in reporter gene technology, few stable 1a luciferase replicon cell lines have been described, presumably due to the low replication capacity of such constructs in available Huh- cells. Drug discovery efforts involving genotype 1a, including direct potency comparisons of antiviral agents between genotypes 1a and 1b and phenotypic characterization of resistant mutant 1a replicons, remain low-throughput in the absence of genotype 1a reporter replicon cell lines and cell lines highly permissive for genotype 1a replication. Two distinct strategies have been used to facilitate robust HCV replication in Huh- cells. In one approach, combinations of cell culture adaptive mutations are engineered into the replicon genome prior to transfection () (1) (0). The second approach (1) () () selects an Downloaded from on January, 01 by guest

5 enriched population of Huh- cells that are highly permissive for HCV replication through curing cell lines that stably replicate HCV replicons. For example, Huh-Lunet and Huh. cells are both derivatives of Huh- cells that exhibit markedly enhanced permissivity to replication of genotype 1b replicons () (1). Selection of cells based on enhanced permissivity to genotype 1a replication, however, has not been previously described. Here we report the successful isolation of novel stable cell lines robustly replicating luciferase-encoding genotype 1a HCV replicons. To achieve this, it was necessary to first generate novel cell lines with enhanced permissivity to genotype 1a replication. Downloaded from on January, 01 by guest

6 Materials and Methods Cell Culture. Huh-luc and Huh-Lunet cells were obtained from ReBLikon GmbH (Mainz, Germany) () All Huh-Lunet derived replicon cell lines (including those described below) were propagated in Dulbecco's Modified Eagle Medium (D-MEM) with GlutaMAX -I (Invitrogen, Carlsbad, CA) supplemented with % FBS (HyClone, Logan, UT), 1 unit/ml Penicillin (Invitrogen), 1 µg/ml Streptomycin (Invitrogen), and 0.1 mm non-essential amino acids (Invitrogen). Replicon cell lines were selected and maintained in 0. mg/ml G-1 (Invitrogen). Generation of stable replicon cell lines. µg of in vitro-transcribed RNA were transfected into Huh-Lunet, 1C, or C cells (described below) by electroporation. Briefly, subconfluent cells were detached by trypsin treatment, collected by centrifugation, washed twice with ice-cold phosphate-buffered saline (PBS), and resupsended at cells/ml in Opti-MEM (Invitrogen). Replicon RNA was added to 00 µl of cell suspension in a Gene Pulser cuvette (0. cm gap). Cells were electroporated at 0 V and 0 µf (Bio-Rad Gene Pulser system, Bio-Rad, Hercules, CA). Pulsed cells were incubated at room temperature for minutes after electroporation and then were resuspended in 0 mls DMEM. Cells were plated into 0 mmdiameter dishes for G1 selection. Cell clones were isolated, expanded, and cryopreserved at early passage levels. To determine the efficiency of G1-resistant colony formation, transfected cells were plated at multiple densities. Twenty-four hours after plating, the media was replaced with DMEM-% FBS supplemented with 1.0 mg/ml G1 and refreshed twice per wek. Three weeks later, colony plates were either used for cell expansion or G1-resistant Downloaded from on January, 01 by guest

7 foci were fixed with % formaldehyde and stained with 1% crystal violet and 0% (v/v) methanol Generation of Cured Cells. The cell lines 1a H-, 1a H-1, 1a H-, 1a H-, and 1a H- are Huh-Lunet clones stably transfected with the genotype 1a ph/sg-neo(l+i) replicon (Apath, Brooklyn, NY). To cure the cells of HCV RNA, they were cultured in the presence of IFN-α (0 IU/ml), BILN-01 (0 nm) and GS-10 (0 nm) for one month. Cells were passaged in media containing the three drugs once they reached 0% confluence twice a week for a total of passages. Cured cell lines were expanded and cryopreserved at early passage levels, and no more than additional passages occurred before subsequent transfection. Cured cells were designated C, 1C, C, C and C. Two methods were used to confirm that the cured cells lacked detectable HCV replicon. First, cells were subjected to G-1 selection (00 µg/ml) for three weeks and determined to be fully sensitive to this cytotoxic agent. Second, the cells were found to lack any NS protease activity as determined by a sensitive fluorescent NS/A protease assay (1). Plasmids Encoding Luciferase Reporter Replicons. Plasmids ph/sg-glucneo(l+i) and ph/sg- RlucNeo (L+I) were generated from plasmid ph/sg-neo(l+i), which encodes a genotype 1a (H strain) subgenomic replicon () and was obtained from Apath. A novel hgluc- Neomycin fusion gene was designed without the wild type secretion signal and with a amino acid (GlyAlaGlyAla) linker, between the hgluc and the Neomycin genes. The hgluc-neo fusion gene was synthesized by Integrated DNA Technologies (Coralville, IA) and provided in the plasmid pidt-gluc-neo DNA. This plasmid was digested with AscI and AflII and the excised Downloaded from on January, 01 by guest

8 fragment was ligated with T DNA ligase (Promega, Madison, WI) into ph/sg-neo(l+i) digested with the same enzymes. The resulting plasmid, ph/sg-glucneo(l+i), was sequenced to confirm the correct orientation and sequence of the hgluc-neo gene. The hrluc-neomycin fusion gene (hrluc-neo) was amplified from pf CMV hrluc-neo Flexi(R) (Promega) by PCR using Accuprime Super Mix I (Invitrogen) and a primer set of AflII hrluc Fwd and AscI Neo Rev. These two primers have the following sequence and introduce restriction sites for subsequent cloning: AflII hrluc: GTC TTA AGT ACA ACC ATG GCT TCC AAG GTG (AflII site underlined), AscI Neo Rev: GGC GCG CCT CAG AAG AAC TCG TCA AGA AG (AscI site underlined). The hrluc-neo amplification product was subcloned into pcr.1- TOPO (Invitrogen). The resulting plasmid was digested with AscI and AflII, and the excised fragment (hrluc-neo) was ligated with T DNA ligase (Promega) into ph/sg-neo (L+I) digested with the same enzymes. The resulting vector ph/sg-hrluc-neo (L+I) was sequenced to confirm the correct orientation and sequence of the hrluc-neo fusion gene. Plasmid phcv-sf-rlucneo (K+Y+I) was generated from phcv-sf (K+Y+I), which encodes a genotype 1a (SF strain) subgenomic replicon and was obtained from Dr. Robert Lanford (Southwest Foundation for Biomedical Research, San Antonio, TX). The hrluc-neomycin fusion gene (hrluc-neo) was amplified from pf CMV hrluc-neo Flexi(R) (Promega) by PCR using Accuprime Super Mix I (Invitrogen) and a primer set of BstBI hrluc Fwd and EcoRI Neo Rev. These primers have the following sequence and introduce restriction sites for subsequent cloning: BstBI hrluc Fwd has the sequence: GAC TTC GAA CAT GGC TTC CAA GGT GTA CGA C (BstBI site underlined). EcoRI Neo Rev: CTG AAT TCC GGA CGC GTT CAG AAG AAC TCG TC (EcoRI site underlined). The hrluc-neo amplification product was Downloaded from on January, 01 by guest

9 subcloned into pcr.1-topo (Invitrogen). The resulting plasmid was digested with BstBI and EcoRI, and the excised fragment (hrluc-neo) was ligated with T DNA ligase (Promega) into phcv-sf (K+Y+I) cut with the same enzymes. The resulting vector phcv-sf-rlucneo (K+Y+I) was sequenced to ensure correct orientation and sequence of the hrluc-neo fusion gene. Plasmid pfk-rep PI-luc/.1, which encodes a genotype 1b (Con1 strain) replicon and firefly luciferase reporter driven by the polio IRES, was obtained from ReBLikon GMBH (). The plasmid pcon1/sg-hrlucneo was generated from the plasmid Iluc-ubi-neo/NS- /ET, which encodes a genotype 1b (Con1 strain) sub-genomic replicon and was obtained from ReBLikon GmbH. The hrluc-neo gene was PCR amplified from pf CMV hrluc-neo Flexi(R) by PCR using Accuprime Super Mix I and the primers AscI hrluc Fwd and NotI hrluc Rev. These two primers have the following sequence and carry restriction sites for subsequent cloning: AscI hrluc Fwd: ACT GAC GGC GCG CCA TGG CTT CCA AGG TGT ACG (AscI site underlined), NotI hrluc Rev: GTC AGT GCG GCC GCT CAG AAG AAC TCG TCA AGA (NotI site underlined). The hrluc-neo amplification product was subcloned into pcr.1-topo. The resulting plasmid was digested with AscI and NotI, and the excised fragment (hrluc-neo) was ligated using T DNA ligase into Iluc-ubi-neo/NS- /ET cut with the same enzymes. The resulting vector, pcon1/sg-hrlucneo, was sequenced to confirm the correct orientation and sequence of the hrluc-neo fusion gene. Downloaded from on January, 01 by guest 1 Plasmid plucneoa was derived from pjfh1, a plasmid containing the full-length genotype a (JFH-1 strain) genome (Toray Inc., Japan) as follows: the HCV non-structural genes along with

10 the plasmid backbone in pjfh1 were amplified and self-ligated to generate pasg by PCR using a primer set of HCVaCoreAfeIrev and HCVMluaNSfw. HCVaCoreAfeIrev has the sequence: TCTAGA AGCGCT tgggcg acggtt ggtgtt tctttt gg (HCV sequence in lower case) and encodes an AfeI site (underlined). HCVMluaNSfw has the sequence: GAGCTT ACGCGT atggct cccatc actgct tatg (HCV sequence in lower case) and a MluI site (underlined). The AfeI and MluI sites were introduced at the twentieth residue of core protein and upstream of NS, respectively, to allow the insertion of the luciferase reporter and the neomycin phosphotransferase (neo) gene. The fragment encoding the reporter, the neo gene, and EMCV IRES were amplified by PCR from the pfkilucubineons- _ET replicon using primers with AfeI and MluI sites at the ends and subsequently cloned into a pta TOPO vector. An XbaI site was knocked out of the luciferase/neo/ires fragment by site directed mutagenesis so that the final replicon construct would have only one XbaI site. The plasmid plucneoa was then generated by ligation of the cloned AfeI-MluI fragment containing luciferase/neo/ires into the plasmid pasg after digestion with AfeI and MluI. The sequence was confirmed by DNA sequencing. RNA transcription. Plasmid DNAs containing genotype 1a subgenomic HCV replicon sequences were linearized with either HpaI (H replicons), XbaI (SF replicons), or AseI and ScaI (genotype 1b replicons) and purified using a PCR Purification Kit (Qiagen, Valencia, CA). RNA was synthesized with T MEGAScript reagents (Ambion, Austin, TX) following the manufacturer's suggested protocol, and reactions were stopped by digestion with RNase-free DNase. RNA was purified using RNA Easy Kit (Qiagen) in accordance with the manufacturer's protocol. RNA concentrations were determined by measurement of the optical density at 0 nm, Downloaded from on January, 01 by guest

11 and RNA integrity was verified by a 0.% agarose gel electrophoresis and ethidium bromide staining Extraction, amplification, and genotypic analysis of HCV RNA. HCV RNA isolation, RT- PCR, and sequencing were performed by Tacgen (Hayward, CA). RNA was purified using RNA Easy Kit (Qiagen) in accordance with the manufacturer's protocol. RT-PCR was performed using SuperScript III First-Strand Synthesis System (Invitrogen). DNA samples were sequenced using ABI BigDye V.1 chemistry on an ABI PRISM 00 DNA Analyzer. Antiviral Compounds VX-0, BILN-01, a Bristol-Myers Squibb monomeric NSA inhibitor (BMS-Am), and - C-methyl adenosine ( CMA) were purchased from Acme Bioscience (Belmont, CA). Interferon-α was purchased from Sigma-Aldrich (St. Louis, MO). The Wyeth HCV NSB Site IV inhibitor HCV- was synthesized by Curragh Chemistries (Cleveland, OH). An Abbott benzothiadiazine NSB Polymerase inhibitor (A-) was synthesized by ChemALong Laboratories (Lemont, IL). Antiviral assays. Replicon cells were seeded in -well plates at a density of 000 cells per well in 0 µl of DMEM culture medium, excluding G-1. Compounds were serially diluted in 0% DMSO and added to cells at a 1:00 dilution, achieving a final concentration of 0.% DMSO in a total volume of 00 µl. In well assays, -fold serial drug dilutions with concentrations were used and starting concentrations were as follows: 0 µm (VX-0, CMA, A-), µm (BILN-01, HV-), or 0 units (IFN-α). Alternately, 000 cells/well were Downloaded from on January, 01 by guest

12 seeded in well plates in 0 ul of DMEM culture medium, excluding G-1. Compounds were added to cells at a 1: dilution, achieving a final concentration of 0.% in a total volume of 0. ul. -fold serial drug dilutions with concentrations were used and starting concentrations were as follows:. µm (A- and BMS-Am),. µm ( CMA),. µm (VX-0),. µm (BILN-01), or 0. µm (HCV-). Cell plates were incubated at C for days, after which culture media were removed and cells were assayed for luciferase activity as markers for replicon levels. Luciferase expression was quantified using a commercial luciferase assay (Promega). Luciferase levels were converted into percentages relative to the untreated controls (defined as 0%) and data were fit to the logistic dose response equation y = a/(1+(x/b) c ) using XLFit software (IDBS, Emeryville, CA). Z Determination To determine the Z -factor, eight wells of a -well plate were treated for hours with 0. µm of the protease inhibitor BILN-01 (positive control) and eight wells were treated with 0.% DMSO (negative control) in triplicate assays. The Z -factor was calculated for the antiviral luciferase endpoint using the equation (): ( σ p+ σn) Z' = 1 µ µ Where σ is the standard deviation of the positive ( p ) or negative ( n ) control and µ is the mean of the positive control ( p ) or negative control ( n ). p n Downloaded from on January, 01 by guest 1

13 Results Design and construction of HCV 1a replicon constructs While a variety of reporter replicon constructs encoding genotype 1b and genotype a nonstructural proteins have been described () (1) (1), there are few examples of reporter replicons encoding genotype 1a nonstructural genes. We designed and constructed subgenomic replicons with either the hrluc or hgluc reporter gene fused to neomycin in the first cistron, and genotype 1a nonstructural genes from the H or 1a SF strains (,, 1) in the second cistron to enable the selection of stable genotype 1a luciferase reporter replicon cell lines (Fig. 1). Adaptive mutations, shown to enhance replication of subgenomic H or SF replicons in tissue culture (EK, DY, P1L, S0I), were included in the nonstructural genes (). Selection of cell lines highly permissive to genotype 1a HCV replication. We initially attempted to establish a stable genotype 1a luciferase replicon cell lines in Huh- Lunet cells. However, transfection of these cells with the luciferase replicons described above failed to generate Geneticin (G-1)-resistant colonies in multiple independent experiments. To overcome this, we adopted a strategy used by others to generate cell lines highly permissive to genotype 1b replication. Specifically, we sought to select new cell lines highly permissive to genotype 1a replication by curing cell lines harboring 1a replicons without reporter genes. We established stable genotype 1a replicons without reporter genes by transfecting Huh- Lunet cells with 1a H-neo RNA which encodes an H 1a replicon carrying both P1L and Downloaded from on January, 01 by guest 1

14 S0I adaptive mutations (). Individual G1-resistant colonies were isolated, expanded, and replication levels were quantified by measuring NS protease activity (Figure.) (). Five clones exhibiting high levels of NS activity as well as morphologic and growth characteristics similar to the original parent cell line (Huh-Lunet) were further expanded (1a H-, 1a H- 1, 1a H-, 1a H-, and 1a H-),. These five 1a H replicon cell lines were then cured of the replicon by prolonged treatment with a combination of IFN-α, the NS protease inhibitor BILN-01, and the novel NSB polymerase inhibitor GS-10. The resulting cured cell lines, designated C, 1C, C, C, and C, were shown to lack detectable HCV replication by two independent methods: first, NS protease activity (data not shown) was at or below the level of naïve Huh-Lunet cells and, second, each clone was fully sensitive to the cytotoxic effects of G-1. To assess the ability of the cured cell lines to support genotype 1a replication, we transfected 1a H-neo RNA into the five cured cell lines and the parental Huh-Lunet cells. After three weeks of G1 selection, the number of G1-resistant colonies observed in the cured cells was significantly higher than that with Huh-Lunet cells. Among the different 1a cured cell lines, permissivity, as measured by colony formation, ranged from approximately - fold (1a H- and 1a H-) to 0-fold of that observed with Huh-Lunet (Table 1). Two Cured replicon cell clones, 1C (Fig. A) and C, exhibited the highest degree of permissiveness to genotype 1a replication and were used in subsequent studies. To determine if the cured cell lines retained permissivity to replicons encoding other HCV genotypes, we transfected genotype 1a, 1b and a luciferase-encoding replicons into C Downloaded from on January, 01 by guest 1

15 and Huh-Lunet cells (Fig. B). Three days post electroporation, the luciferase-labeled 1a replicon replicated at much higher levels in C cells (>0 fold) compared to Huh-Lunet cells. The genotype 1b replicon replicated at similar levels in C and Huh-Lunet cells (~-fold greater levels in C). C cells were less permissive to genotype a replicons (>-fold) compared to Huh-Lunet cells, although this replicon was readily quantifiable in both cell types. Overall, these transient transfection assays indicate that C cells support robust replication of genotypes 1a, 1b, and a. However, in comparison to Huh-Lunet cells, C cells were selectively more permissive to genotype 1a rather than globally more permissive to all three genotypes. Generation of Cell Lines Stably Replicating Luciferase-encoding Genotype 1a replicons. To establish stable 1a luciferase replicon cell lines, we electroporated in vitro-transcribed RNA for reporter and parental replicons (Figure 1) into Huh-Lunet, 1C, or C cells. Remarkably, all 1C and C cells transfected with the 1a H and 1a SF luciferase replicons yielded a significant number of G1-resistant colonies while Huh-Lunet cells yielded none (consistent with our initial experiments, data not shown). To assess replication levels in these new genotype 1a stable replicon cell lines, luciferase activity was measured (Figure ). All stable 1a replicon luciferase cell lines showed robust luciferase expression and luciferase signal-to-background ratios in excess of 0 were observed after miniaturization into -well format (Table ). Z- factors, which measure assay quality (), were calculated for each luciferase reporter replicon using the NS inhibitor BILN-01 as a positive control or an equivalent volume of DMSO as a negative control (Table ). Over three independent assays, both hrluc and hgluc 1a replicon Downloaded from on January, 01 by guest 1

16 cell lines produced Z-factors >0., supporting the potential use of these cells in high-throughput screening applications Drug Screening and Antiviral Activity. To validate these cell lines for antiviral activity assays, we assessed the response of 1a replicons expressing either hgluc or hrluc using a panel of six known HCV replication inhibitors in a - well assay format. Compounds were chosen to represent distinct mechanistic classes that target different nonstructural genes, such as NS protease inhibitors (BILN-01 and VX-0) and nucleoside ( C-methyl adenosine [ CMA]) or non-nucleoside (HCV-, A-) NSB polymerase inhibitors, and host antiviral cytokines (IFN-α) (,, 1, ). Compounds were also selected to exhibit a broad range of antiviral potency; HCV-, for example, is approximately 0-fold more potent than CMA in previous replicon assays. Antiviral activity, as measured by EC 0 values, was consistent for each compound among the various 1a luciferase replicon cell lines (including four 1a H luciferase replicon cell lines and three 1a SF luciferase replicon cell lines (Figure ). The potencies observed in this experiment for both 1a H and 1a SF replicons were consistent with EC 0 values we have previously generated in the 1a H- non-reporter replicon cell line (data not shown). These results confirm the phenotypic similarity of 1a replicon cell lines with luciferase reporters (hgluc or hrluc) from different isolates (H or SF) in distinct cured cell lines (1C or C). Downloaded from on January, 01 by guest 1

17 We next compared the potency of compounds between genotype 1a and genotype 1b replicons in a -well assay format. In addition to BILN-01, HCV-, CMA, and VX- 0, we also tested a monomeric NSA inhibitor as this class of compounds has been reported to have significant differences in potency between genotypes 1a and 1b (1). EC 0 values for each compound in each genotype are summarized in table. CMA, VX-0, and HCV- had similar potency against both genotypes (<.1-fold differences) (Table ). The protease inhibitor, BILN-01, was slightly less potent against the genotype 1a replicon (~.-fold). However, the monomeric NSA inhibitor was over 0 fold less potent against genotype 1a compared to genotype 1b. Genotypic Analysis of the New Stable 1a Replicons. In addition to the cellular background, adaptive mutations in replicon sequence are known to contribute to increased replication efficiency of replicon RNA. To investigate whether adaptive mutations were responsible for the enhanced replication levels of our genotype 1a luciferase replicons, we sequenced the nonstructural regions of replicon RNAs extracted from these cell lines and compared them to baseline replicon sequences. We first sequenced the coding region of the non-luciferase replicon RNA extracted from the five 1a H non-reporter cell lines (derived from Huh-Lunet cells) to determine if any mutations arose during selection (Table ). For each replicon cell line, we isolated total RNA and sequenced the entire replicon coding regions. Baseline adaptive mutations in the parental 1a H-neo replicon included the NSA mutation, S0I and the NS mutation P1L. In all Downloaded from on January, 01 by guest 1

18 five of the 1a H replicon cell lines these baseline adaptive mutations were maintained. However, additional HCV mutations were also observed in all five cell lines. One of these cell lines (1a H-) contained the previously reported NSA adaptive mutation K11R (). The other clones contained mutations that have not been previously reported (S1C, A1S, M1I, I1T, and IT), but which were not consistently selected across multiple clones. A similar analysis was then performed on the luciferase-encoding replicon cell lines. As with the non-luciferase replicon cell lines, baseline adaptive mutations encoded in the parental H replicon were maintained in each clone after selection (Table ). Similarly, adaptive mutations in the parental SF replicon (S0I in NSA, EK and DY in NS) were maintained in each clonal cell line after selection. In some, but not all of the luciferase-encoding replicon cell lines additional mutations were identified. The known adaptive mutation K11R was identified in the 1a-H- cell line and also appeared in the 1a 1C-H-Gluc clone which was derived from the H 1a strain. The NSB mutation E1G was observed in two 1a H luciferase clones and the NSB mutation E0G was identified in one H luciferase clone. However, the 1a H Rluc polyclonal pooled cell line did not show any consensus mutations beyond the parental adaptive mutations. Examination of the 1a SF luciferase replicons identified two mutations present in one clone each: I1T in NS and LS in NSB. The third SF replicon cell line did not contain any mutations. Overall, these analyses did not identify any specific mutations that were required for genotype 1a replication (beyond the parental adaptive mutations) in Huh-Lunet, 1C, or C cell lines. Downloaded from on January, 01 by guest 1

19 Discussion While the widely-used Huh- cell line, and particularly, the derivative cell line cured of the genotype 1b replicon known as Huh-Lunet, support robust replication of genotype 1b reporter replicons, these cell lines do not appear to support the generation of stable genotype 1a reporter replicon cell lines. Indeed, we were unable to generate any genotype 1a stable replicon colonies when using replicon RNAs encoding a luciferase reporter gene. In contrast, but consistent with what others have reported, we were readily able to establish genotype 1a replicon cell lines using replicons without a luciferase reporter. To facilitate the generation of luciferase-encoding genotype 1a replicons, we cured cell lines stably replicating a genotype 1a replicon without a reporter gene, using a combination of HCV inhibitors. The resulting cured Huh cell lines were remarkably more permissive to genotype 1a replicons with or without reporter genes; in fact, two out of five cured cell lines had permissivity to non-reporter genotype 1a replicons enhanced greater than 0-fold compared to Huh-Lunet cells during colony formation assays. We also tested the ability of the C cured cell line to support replication of genotype 1b and genotype a replicons. We found that this cell line supported robust replication of all three genotypes (1a, 1b and a). However, in contrast to genotype 1a which replicated 0-fold better in C compared to Huh-Lunet, genotype 1b showed little enhancement (approximately -fold) while genotype a replicated better in Huh-Lunet cells. These results suggest the presence of cellular factors that are able to influence HCV replication in a genotype-specific manner since permissivity was not globally enhanced for all genotypes. Downloaded from on January, 01 by guest 1

20 After establishing these 1a reporter replicons, we next validated their utility in antiviral assays. We found that under standard antiviral screening conditions (e.g. three day drug treatment of replicon cells in well plates) replicon cell lines encoding either the Renilla or Gaussia luciferases had signal-to-noise ratios of >0 with robust Z factors (>0.). Furthermore, multiple luciferase cell lines including those based on the H and SF 1a HCV strains were demonstrated to have antiviral susceptibilities for multiple drug classes similar to those observed in non luciferase-encoding cell lines. As genotype 1b and a luciferase replicons have been previously described () (1), these new genotype 1a replicons will allow side-by-side direct comparison of antiviral potency among the three genotypes in a high throughput reporter format. Furthermore, these 1a replicons encode either Renilla or Gaussia luciferase which utilize coelenterazine-based substrates whereas typical genotype 1b or genotype b replicons utilize Firefly luciferase (which utilizes luciferin-based substrates); this opens the possibility to conveniently multiplex these new 1a replicons with 1b or a replicons using commercially available luminescence assay kits designed for this purpose. With the compounds that we tested, we did not note any significant difference in antiviral sensitivity between the H and SF strains. However, these two 1a strains have a high level of overall homology (.1% identity at the protein level). The NS protease domain and NSB polymerase have particularly high homology at.% and.% identity, respectively. NSA and NSB, which are emerging targets for drug development, have.% and.% homology, respectively. Overall, having two independent genotype 1a strains to validate inhibitor activity will be valuable for drug discovery efforts, especially given the natural heterogeneity of HCV Downloaded from on January, 01 by guest 0

21 and the significant impact polymorphisms have already been described to have on some HCV drug classes. We did, in contrast, confirm that a monomeric NSA inhibitor had dramatically less potency against genotype 1a compared to genotype 1b (>0-fold less). Such potency information is valuable for assessing potential variability in the clinic, but might also yield valuable insight to inhibitor mechanism of action or aid molecular target identification for new drug classes. Sequencing the 1a replicons stably transfected into Huh-Lunet or 1a cured cell lines to explore the potential evolution of additional genotype 1a adaptive mutations did not reveal any high frequency mutations in conjunction with the pre-existing adaptive mutations. We observed the previously reported NSA adaptive mutation, K11R, and the nearby I1T mutation twice each out of 1 sequenced 1a replicon cell lines. I1, whose role in replication has not yet been characterized, is highly conserved amongst genotype 1a patient isolates. Positions (NSB), 1 (NS), and 1 (NS) are also highly conserved among genotype 1a patient isolates, and mutations at these residues have an uncertain impact on HCV replication. It should also be noted that some clonal and polyclonal cell lines selected during these studies had no amino acid changes from the parental replicon sequences. Together, these observations suggest that factors specific to the cured cell lines, and not additional adaptive mutations in replicon sequence, are the greatest contributor to the enhanced replication phenotype of genotype 1a replicons in these cells. In summary, the new luciferase replicon cell lines described in this report will enable convenient high-throughput screening and characterization of antivirals against genotype 1a Downloaded from on January, 01 by guest 1

22 1 HCV. The approach of creating cell lines with enhanced permissivity was crucial to the generation of the new 1a reporter replicon cell lines, and did not rely on identifying or preengineered adaptive mutations beyond those already commonly described. Given the high prevalence of chronic genotype 1a infection, we envision that both the cured cell lines and luciferase encoding 1a replicons described here will facilitate efforts to develop new therapies for HCV. Indeed, we have recently taken advantage of the enhanced permissivity of 1a cured cell lines by using them to phenotype chimeric replicons encoding target genes from clinical isolates (data not shown). In this context, these cell lines are particularly valuable since such chimeric replicons often have significantly reduced replication fitness compared to laboratory HCV strains. Finally, these cell lines could be used, along with other permissive cell lines (e.g. Huh-Lunet, Huh-.) to study cellular factors and pathways that govern HCV replication in a pan-genotype or genotype/subtype specific manner. Downloaded from on January, 01 by guest

23 Acknowledgements We the following members of Gilead Sciences: Matthew Paulson for assistance with luciferase optimization, James Nugteren and Brian Stephens for compound management assistance, Johnny Lee for assistance with replicon assays, Victor Chen and Mark Kenney for informatics support, Hongmei Mo for helpful conversations, and Weidong Zhong for critical review of this manuscript. We also thank Helen Lee and Dr. Robert Lanford (Southwest Foundation for Biomedical Research) for helpful discussions. Downloaded from on January, 01 by guest

24 References Bartenschlager, R., and V. Lohmann Replication of hepatitis C virus. J Gen Virol 1:-.. Beaulieu, P. L. 00. Non-nucleoside inhibitors of the HCV NSB polymerase: progress in the discovery and development of novel agents for the treatment of HCV infections. Curr Opin Investig Drugs :1-.. Blight, K. J., A. A. Kolykhalov, and C. M. Rice Efficient initiation of HCV RNA replication in cell culture. Science 0:1-.. Blight, K. J., J. A. McKeating, J. Marcotrigiano, and C. M. Rice. 00. Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture. J Virol :-0.. Blight, K. J., J. A. McKeating, and C. M. Rice. 00. Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication. J Virol : De Francesco, R., and A. Carfi. 00. Advances in the development of new therapeutic agents targeting the NS-A serine protease or the NSB RNA-dependent RNA polymerase of the hepatitis C virus. Adv Drug Deliv Rev :1-.. Di Bisceglie, A. M. 1. Hepatitis C. Lancet 1:1-.. Feinstone, S. M., H. J. Alter, H. P. Dienes, Y. Shimizu, H. Popper, D. Blackmore, D. Sly, W. T. London, and R. H. Purcell. 11. Non-A, non-b hepatitis in chimpanzees and marmosets. J Infect Dis 1:-.. Friebe, P., V. Lohmann, N. Krieger, and R. Bartenschlager Sequences in the ' nontranslated region of hepatitis C virus required for RNA replication. J Virol :-. Downloaded from on January, 01 by guest

25 Fried, M. W., M. L. Shiffman, K. R. Reddy, C. Smith, G. Marinos, F. L. Goncales, Jr., D. Haussinger, M. Diago, G. Carosi, D. Dhumeaux, A. Craxi, A. Lin, J. Hoffman, and J. Yu. 00. Peginterferon Alfa-a plus Ribavirin for Chronic Hepatitis C Virus Infection. N Engl J Med :-.. Guo, J. T., V. V. Bichko, and C. Seeger Effect of alpha interferon on the hepatitis C virus replicon. J Virol : Hao, W., K. J. Herlihy, N. J. Zhang, S. A. Fuhrman, C. Doan, A. K. Patick, and R. Duggal. 00. Development of a novel dicistronic reporter-selectable hepatitis C virus replicon suitable for high-throughput inhibitor screening. Antimicrob Agents Chemother 1:-. 1. Huang, Z., M. G. Murray, and J. A. Secrist, rd. 00. Recent development of therapeutics for chronic HCV infection. Antiviral Res 1: Kato, T., T. Date, M. Miyamoto, A. Furusaka, K. Tokushige, M. Mizokami, and T. Wakita. 00. Efficient replication of the genotype a hepatitis C virus subgenomic replicon. Gastroenterology 1: Kim, C. S., J. H. Jung, T. Wakita, S. K. Yoon, and S. K. Jang. 00. Monitoring the antiviral effect of alpha interferon on individual cells. J Virol 1: Krieger, N., V. Lohmann, and R. Bartenschlager Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol : Lanford, R. E., H. Lee, D. Chavez, B. Guerra, and K. M. Brasky Infectious cdna clone of the hepatitis C virus genotype 1 prototype sequence. J Gen Virol :11-. Downloaded from on January, 01 by guest

26 Lemm, J. A., D. O'Boyle, nd, M. Liu, P. T. Nower, R. Colonno, M. S. Deshpande, L. B. Snyder, S. W. Martin, D. R. St Laurent, M. H. Serrano-Wu, J. L. Romine, N. A. Meanwell, and M. Gao. Identification of hepatitis C virus NSA inhibitors. J Virol : Lohmann, V., S. Hoffmann, U. Herian, F. Penin, and R. Bartenschlager. 00. Viral and cellular determinants of hepatitis C virus RNA replication in cell culture. J Virol : Lohmann, V., F. Korner, A. Dobierzewska, and R. Bartenschlager Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J Virol : Lohmann, V., F. Korner, J. Koch, U. Herian, L. Theilmann, and R. Bartenschlager. 1. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science :1-.. Manns, M. P., J. G. McHutchison, S. C. Gordon, V. K. Rustgi, M. Shiffman, R. Reindollar, Z. D. Goodman, K. Koury, M. Ling, and J. K. Albrecht Peginterferon alfa-b plus ribavirin compared with interferon alfa-b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet :-.. Matthews, J. C., K. Hori, and M. J. Cormier. 1. Purification and properties of Renilla reniformis luciferase. Biochemistry 1:-1.. Murray, E. M., J. A. Grobler, E. J. Markel, M. F. Pagnoni, G. Paonessa, A. J. Simon, and O. A. Flores. 00. Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh cells. J Virol :-. Downloaded from on January, 01 by guest

27 Paulson, M. S., H. Yang, I. H. Shih, J. Y. Feng, E. M. Mabery, M. F. Robinson, W. Zhong, and W. E. t. Delaney. 00. Comparison of HCV NS protease and NSB polymerase inhibitor activity in 1a, 1b and a replicons and a infectious virus. Antiviral Res :1-.. Reed, K. E., and C. M. Rice Overview of hepatitis C virus genome structure, polyprotein processing, and protein properties. Curr Top Microbiol Immunol :-.. Robertson, B., G. Myers, C. Howard, T. Brettin, J. Bukh, B. Gaschen, T. Gojobori, G. Maertens, M. Mizokami, O. Nainan, S. Netesov, K. Nishioka, T. Shin i, P. Simmonds, D. Smith, L. Stuyver, and A. Weiner. 1. Classification, nomenclature, and database development for hepatitis C virus (HCV) and related viruses: proposals for standardization. International Committee on Virus Taxonomy. Arch Virol 1:-0.. Rodger, A. J., S. Roberts, A. Lanigan, S. Bowden, T. Brown, and N. Crofts Assessment of long-term outcomes of community-acquired hepatitis C infection in a cohort with sera stored from 11 to 1. Hepatology :-.. Simmonds, P. 00. Genetic diversity and evolution of hepatitis C virus--1 years on. J Gen Virol : Simmonds, P., A. Alberti, H. J. Alter, F. Bonino, D. W. Bradley, C. Brechot, J. T. Brouwer, S. W. Chan, K. Chayama, D. S. Chen, and et al. 1. A proposed system for the nomenclature of hepatitis C viral genotypes. Hepatology 1:-. 1. Sudo, K., K. Yamaji, K. Kawamura, T. Nishijima, N. Kojima, K. Aibe, K. Shimotohno, and Y. Shimizu. 00. High-throughput screening of low molecular weight NS-NSA protease inhibitors using a fluorescence resonance energy transfer substrate. Antivir Chem Chemother 1:-. Downloaded from on January, 01 by guest

28 Tannous, B. A., D. E. Kim, J. L. Fernandez, R. Weissleder, and X. O. Breakefield. 00. Codon-optimized Gaussia luciferase cdna for mammalian gene expression in culture and in vivo. Mol Ther :-.. Tong, X., Z. Guo, J. Wright-Minogue, E. Xia, A. Prongay, V. Madison, P. Qiu, S. Venkatraman, F. Velazquez, F. G. Njoroge, and B. A. Malcolm. 00. Impact of naturally occurring variants of HCV protease on the binding of different classes of protease inhibitors. Biochemistry :1-1.. Wasley, A., and M. J. Alter Epidemiology of hepatitis C: geographic differences and temporal trends. Semin Liver Dis 0:1-1.. Yang, H., and W. E. Delaney IV 00. A Novel Fluorescence-based Protease Assay Using the Endogenous NS/A Protease Activity Present in the Total Cell Lysates of HCV Replicon Cells. Journal of Clinical Virology :S.. Yi, M., F. Bodola, and S. M. Lemon. 00. Subgenomic hepatitis C virus replicons inducing expression of a secreted enzymatic reporter protein. Virology 0:1-.. Yi, M., and S. M. Lemon. 00. Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh cells. J Virol :0-1.. Zein, N. N Clinical significance of hepatitis C virus genotypes. Clin Microbiol Rev 1:-.. Zhang, J. H., T. D. Chung, and K. R. Oldenburg. 1. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen :-. Downloaded from on January, 01 by guest

29 Figures Figure 1. Design of novel 1a reporter replicons. HCV replicons used to generate novel genotype 1a stable replicon cell lines. A neomycin (Neo) encoding H 1a strain replicon (A) An H 1a strain replicon encoding a Gaussia luciferase (Gluc)-neomycin fusion reporter (B). An H 1a strain replicon encoding a Renilla luciferase (Rluc)-neomycin fusion reporter (C). An SF 1a strain replicon encoding a Renilla luciferaseneomycin fusion reporter (D). indicates HCV core sequence. indicates SF sequence. L indicates the P1L adaptive mutation. I indicates the S0I adaptive mutation. K indicates the EK adaptive mutation. Y indicates the DY adaptive mutation. The and non-translated regions (NTR), and Encephalomyocarditis virus (EMCV) IRES are indicated. Figure. Replication levels in eleven unique clonal cell lines harboring a genotype 1a replicon without a luciferase reporter. After transfection with the 1a H-neo replicon, individual G1-resistant colonies were isolated, expanded, and tested in an NS protease activity assay (as a marker for HCV replication levels). For comparative purposes, the genotype 1b luciferase replicon cell line, Huh-Luc, was also tested in the assay. Figure. Cured cell lines are highly permissive to genotype 1a replication but also support replication of genotype 1b and a. RNA encoding the genotype 1a replicon ph/sg-neo(l+i) was electroporated into the Huh- Lunet (A, top left) or the 1C Cured cell line (A, top right). After three weeks of selection in Downloaded from on January, 01 by guest

30 G1, resistant colonies, which harbor the genotype 1a replicon, were visualized with crystal violet. The 1C cell line (A, top right) gave rise to a significantly greater number of stable replicon clones compared to the Huh-Lunet cell line (A, top left). Confirming that 1C cells are cured of the HCV replicon, electroporation without RNA (Mock) resulted in fully G1- sensitive cells for both cell lines (A, bottom). Genotype 1a (ph/sg-glucneo(l+i)), 1b (pfkrep PI-luc/.1), and a replicons (plucneoa) were transfected into either Cured (white bars) or Lunet cells (black bars), and luciferase levels -days post transfection were measured (B). To control for transfection efficiency, data were normalized to luciferase levels at -hours post transfection. For each genotype, replication in Lunet cells is displayed as a percent of replication in Cured cells. Figure. Robust luciferase expression in 1a-luciferase cell clones. Stable cell lines replicating either the 1a H-Rluc-neo, 1a H-Gluc-neo, or the 1a-SF-Rluc-neo replicon were assayed for Luciferase activity. In the nomenclature of the cell lines, 1C/C refers to the cured cell line that the cells were derived from, H/SF refers to the strain of HCV the replicons were constructed with, Gluc/Rluc refer to the reporter gene (Gaussia luciferase/renilla luciferase) and the terminal number indicates the clone number. Figure. Antiviral response is consistent across multiple genotype 1a replicon cell lines. Novel genotype 1a luciferase-encoding replicon cell lines were seeded in well plates and treated with the indicated HCV inhibitors for three days. After drug treatment, the dose-response for each antiviral was quantified by measuring luciferase activity in duplicate and EC 0 values were calculated by non-linear regression. EC 0 values are plotted on the Y-axis, the antiviral Downloaded from on January, 01 by guest 0

31 compounds on the X-axis, and the cell lines are indicated by the shape and fill of the data points EC 0 values are plotted on the Y-axis, the antiviral compounds on the X-axis, and the cell lines are indicated by the shape and fill of the data points ( 1A 1C-H-Gluc1, 1a 1C-H Gluc, 1a 1C-H-Gluc, 1a C-H-RlucP, 1a C-SF-Rluc1, 1a C-SF- Rluc, 1a C-SF-Rluc). Each data-point represents the arithmetic mean of two independent experiments. Downloaded from on January, 01 by guest 1

32 Table 1. Novel cured cell lines are more permissive to genotype 1a replicon than Huh- Lunet cells Relative Ability to Form Colonies After Cell Line Parental Cell Line Genotype 1a Replicon Transfection and Selection* C 1a H- ++ 1C 1a H C 1a H C 1a H- +++ C 1a H- ++ Huh-Lunet Huh + * The + symbols indicates relative number of colonies formed. Downloaded from on January, 01 by guest

33 Table. Genotype 1a reporter replicons in cured cells produce robust luciferase signal Replicon (Cell Line) Signal-to-noise ratio a Z factor b Firefly luciferase 1b (Huh-luc) Renilla luciferase 1a (1a C-SF-Rluc) Gaussia 1a (1a 1C-H-Gluc1) a Signal-to-noise ratio was calculated by dividing the mean signal from negative DMSO treated wells by the mean signal from positive BILN-01 treated wells. b Z factor was calculated using positive (BILN-01 treated) and negative (DMSO treated) control wells from triplicate experiments Downloaded from on January, 01 by guest

34 Table : EC 0 Values of Direct Acting HCV Antivirals Against Genotype 1b and 1a replicons Compound Genotype 1b EC 0 (nm) Genotype 1a EC 0 (nm) Fold Difference CMA 1 ± 1 ± 1.1 BILN-01 ± 1 1 ±. VX-0 1 ± 0 1 ± 1 0. HCV- ± ± 1.0 BMS-Am. ± 0. >, ± N/A >1 Downloaded from on January, 01 by guest

35 Table. Mutations identified in stable genotype 1a replicon cell lines Cell Line Parental Cell Line HCV Strain Baseline Adaptive Mutations Additional Mutations 1a H- Huh-Lunet H P1L, S0I S1C (NS), I1T (NSA) 1a H-1 Huh-Lunet H P1L, S0I A1S (NS) 1a H- Huh-Lunet H P1L, S0I K11R (NSA)* 1a H- Huh-Lunet H P1L, S0I I1T (NSA) 1a H- Huh-Lunet H P1L, S0I M1I (NS), IT (NSA) 1a 1C-H-Gluc1 1C H P1L, S0I E1G (NSB) 1a 1C-H-Gluc 1C H P1L, S0I K11R (NSA)* 1a 1C-H-Gluc 1C H P1L, S0I E1G (NSB), E0G (NSB) 1a C-H-RlucP C H P1L, S0I 1a C-SF-Rluc1 C SF EK, DY, S0I I1T (NS) 1a C-SF-Rluc C SF EK, DY, S0I 1a C-SF-Rluc C SF EK, DY, S0I LS (NSB) *K11R is a previously-reported adaptive mutation () Downloaded from on January, 01 by guest

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