Chronic hepatitis C virus (HCV) infection remains a. Development of Robust Hepatitis C Virus Genotype 4 Subgenomic Replicons

GASTROENTEROLOGY 2013;144:59 61 Development of Robust Hepatitis C Virus Genotype 4 Subgenomic Replicons BETTY PENG, 1 MEI YU, 1 SIMIN XU, 1 YU JEN LEE, 1 YANG TIAN, 1 HUILING YANG, 1 KATIE CHAN, 1 HONGMEI MO, 1 JOHN MCHUTCHISON, 2 WILLIAM DELANEY IV, 1 and GUOFENG CHENG 1 BRIEF REPORTS 1 Biology Department and 2 Clinical Research, Gilead Sciences, Foster City, California See Covering the Cover synopsis on page 1; see editorial on page 13. Despite the prevalence of hepatitis C virus genotype 4, no replicon system is available for study of the genotype. To facilitate discovery and development of reagents against this virus, we synthesized and transcribed a genotype 4a subgenomic replicon and transfected Huh7-Lunet cells with it, which yielded very few colonies. However, when we used a new Huh- 7 derived cell line, colony formation increased 70- fold. We identified multiple adaptive mutations in the virus s nonstructural 3 or 4A proteins that allowed the cells to maintain stable, genotype 4a luciferase-encoding replicons. Several classes of hepatitis C virus inhibitors had different antiviral effects on genotypes 4a vs 1b. The genotype 4a replicon system we created will aid in the development of treatment regimens for all genotypes of hepatitis C virus. Keywords: Pan-Genotypic; Q34R; Cured Huh-7 Cell Line; GS-5885; GS-7977. Chronic hepatitis C virus (HCV) infection remains a significant global health burden, with an estimated 170 million people infected worldwide. Although genotype (GT) 1 is generally predominant worldwide, GT4 is prevalent in the Middle East and many African countries, and is being observed increasingly in central and northern Europe. 1 3 GT4 represents 93% of HCV infections in Egypt, and 5% 15% of infections in several European countries. 1 Despite its prevalence, the current standard of care has limited efficacy or is not approved (eg, telaprevir or boceprevir) for GT4 patients. 4 Clearly, there is a need for additional therapies for GT4 patients. However, no GT4 replicons have been available to facilitate antiviral discovery. HCV replicons are self-replicating HCV RNA sequences that have served as essential tools for molecular virology studies and drug discovery. 5,6 To date, replicons have only been established for genotypes 1a, 1b, and 2a. 5,7,8 Here we report on the development of the first GT4a replicon that efficiently replicates in vitro. To establish a subgenomic GT4a replicon, the consensus sequence of the GT4a ED43 strain, based on the ED43 infectious clone (GenBank accession # GU814266), 9 was constructed as described previously by Lohmann et al. 5 In addition, the nonstructural protein (NS) 5A mutation S232I (or S2204I), previously shown to enhance GT1 replication, was incorporated (Figure 1A). 8 In vitro transcribed replicon RNA was electroporated into Huh-7 Lunet, 51C, and 1C cells and selected with Geneticin (G418). In Lunet cells, stable replicon colonies were selected at very low frequency, 0.05 colony-forming unit (CFU)/ g RNA. In 51C cells, 10 stable colonies were selected at an improved frequency of 0.3 CFU/ g RNA. Strikingly, in 1C, a cell line generated by curing a GT1a replicon clone derived from 51C cells and highly permissive to GT1a replicon replication, GT4a replicon colonies were readily selected and colony-formation efficiency was improved 70-fold compared with Lunet cells. NS5A mutation S232I was important to the selection of stable replicon colonies because no colony survived in the absence of S232I in 1C cells (Figure 1B). Twelve GT4a replicon colonies isolated from 1C cells were expanded, and the remaining colonies were pooled together. High levels of HCV replicon RNA were detected in all selected replicon cells (Supplementary Figure 1). In consistency, Western blot analyses readily detected NS5A protein in all replicon cells and an immunohistochemical analysis of the GT4a replicon pool indicated strong staining for NS5A in the perinuclear region (Supplementary Figure 1). In addition, 1 g total cellular RNA of the GT4a replicon pool yielded hundreds of colonies in Lunet cells (Supplementary Figure 2). Together, these results confirmed that the selected colonies harbored replicating GT4a replicons. These replicons were subsequently sequenced and multiple amino acid changes were identified (Supplementary Table 1). NS3 helicase mutations of T343K or T343R were observed in 4 clones, A200E in 2 clones, and T511R in 1 clone together with L179P in NS5A. The remaining 5 clones had mutations in NS4A protein. Four had either Q34K or Q34R and the final clone had E52V. To investigate whether emerging mutations enhanced replication, NS4A Q34R mutation was introduced into the GT4a neomycin (neo) construct and validated to enhance colony-formation efficiency almost 3000-fold (Figure 1B). All emerging singlet mutations were then individually introduced into a GT4a Renilla luciferase-neo construct to facilitate the quantification of replicon replication (Figure 1C). Abbreviations used in this paper: GT, genotype; HCV, hepatitis C virus; NS3, nonstructural protein 3; neo, neomycin. 2013 by the AGA Institute 0016-5085/$36.00 http://dx.doi.org/10.1053/j.gastro.2012.09.033

60 PENG ET AL GASTROENTEROLOGY Vol. 144, No. 1 BRIEF REPORTS Figure 1. NS3 and NS4A mutations were selected to enhance GT4a replicon replication. (A) GT4a ED43 strain replicons encode a neomycin phosphotransferase gene (neo) or a Renilla luciferase (Rluc)-neo fusion reporter., NS5A S232I mutation. (B) Ten-microgram GT4a replicon RNA as indicated was in vitro transcribed and electroporated into 1C cells. Forty-eight hours after seeding, medium was replaced with complete Dulbecco s modified Eagle medium supplemented with 0.5 mg/ml G418 and refreshed twice per week. (C) Individual mutations were introduced into the ED43 Rluc-neo construct by site-directed mutagenesis, respectively. All replicon RNAs were transfected into 1C cells and 10,000 transfected cells were plated into wells in a 96-well plate without G418. At indicated times, cells were analyzed for luciferase activity. Dotted line indicates the background relative light units (RLU) value. Data represent mean of 2 independent experiments with error bars. Replicons encoding any emerging mutation actively replicated as evidenced by significant increases in luciferase signal within 24 72 h of transfection. In contrast, the parental replicons without the mutations did not show any signal increase, similar to the replication-defective polymerase GND mutant. Interestingly, lack of NS5A mutation S232I abolished the replicon RNA replication, suggesting it is critical for the GT4a replicon adaptation. The adaptive functions of these emerging mutations were also confirmed in the replicon colony formation assays (Supplementary Figure 3 and Supplementary Table 2). In addition, all adaptive replicons showed enhanced RNA replication in Lunet cells, although at a level lower than in 1C cells (Supplementary Figure 4 and Supplementary Table 2). These results suggest that all emerging mutations are GT4a adaptive mutations with T343R in NS3 helicase, and Q34R in NS4A being the most effective, which are comparable to adapted GT1a Renilla luciferase-neo replicon, but weaker than GT1b replicon. We next sought to establish cell lines stably replicating luciferase-encoding replicons for antiviral assays. Although the parental Renilla luciferase-neo replicon failed to yield any colonies after G418 selection, a Q34R mutant replicon significantly improved colony-formation efficiency (Supplementary Figure 2). A stable cell line (clone- 23) was selected for an antiviral study of 6 different classes of HCV inhibitors. EC 50 values against both GT1b and GT4a replicons were successfully generated using a 384- well antiviral assay measuring luciferase activity (Table 1).

January 2013 GENERATION OF GENOTYPE 4A HCV REPLICONS 61 Table 1. Antiviral Activities of HCV Inhibitors Against GT1b and GT4a Replicons Inhibitor classes Compounds GT1b RLucNeo EC 50 (nm) GT4a RlucNeo EC 50 (nm) GT4a/1b EC 50 ratio NS3 protease Telaprevir 831 450 3356 1374 4.0 Boceprevir 513 203 1080 355 2.1 GS-9451 8.2 3.2 11 3.0 1.3 ITMN-191 0.94 0.39 1.6 0.6 1.7 TMC-435 4.1 2.2 3.8 1.5 0.9 NS4A ACH-806 38 9.6 365 50 9.6 NS5A GS-5885 0.002 0.001 0.12 0.04 60 BMS-790052 0.009 0.002 0.003 0.001 0.3 NS5B Nuc GS-7977 0.045 0.010 0.048 0.016 1.1 2-CMeA 162 87 123 15 0.8 NS5B non-nuc GS-9669 2.2 0.1 389 137 177 MK-3281 247 35 4340 971 18 GS-9190 5.5 0.2 49 32 8.9 Filibuvir 236 94 4444 19 A-837093 8.4 2.7 4444 529 HCV-796 4.0 0.9 21 11 5.3 Host target CsA 163 16 118 34 0.7 Interferon alfa (IU/mL) 1.3 0.4 1.5 0.2 1.2 BRIEF REPORTS NOTE. Data represent a mean of at least 2 independent experiments with standard deviations. CsA, cyclosporine A; Rluc, Renilla luciferase. Of the 6 classes, non-nucleoside NS5B polymerase inhibitors exhibited the greatest deviation in activity between genotypes. Many inhibitors did not have measurable activity against GT4a ( 4.44 M). Most NS3 protease inhibitors had similar potency, but telaprevir (VX-950) demonstrated approximately 4-fold less susceptibility to GT4a compared with GT1b. This result is in agreement with clinical trial data showing that telaprevir had significantly less efficacy in GT4 HCV-infected patients compared with GT1. 11 Inhibitors targeting the NS5B active site and host factors had similar potency against both GT1b and GT4a, consistent with previous pan-genotypic results. 4,12 Both NS5A inhibitors remained potent against GT4a. Overall, these results demonstrate the utility of this novel GT4a replicon cell line for antiviral drug discovery. In summary, we report the isolation of the first GT4a replicon that efficiently replicates in vitro. Using a novel permissive cell line, stable GT4a colonies were readily identified, confirming that cellular factors play a critical role in HCV replication in cell culture. 13 The 1C permissive cell line might facilitate efforts to establish replicons from other genotypes. Multiple adaptive mutations in NS3 or NS4A were demonstrated to enhance GT4a replicon replication. The localization of these mutations suggest that they may be involved in protein protein interactions, such as modulating NS3/4A protease activity. 14 Although the article was under revision, a functional GT4a replicon was independently reported with a diverse set of adaptive mutations. 15 These adaptive mutations permitted the establishment of cells stably replicating luciferase-encoding GT4a replicons. These novel cell lines have allowed us to efficiently profile the antiviral activity of multiple classes of HCV inhibitors, with results consistent with previous clinical observations. 4,12 Overall, this novel GT4a replicon will serve as a valuable tool for antiviral drug discovery and development. In conjunction with existing replicons, it will aid the development of pan-genotypic HCV regimens for the treatment of HCV worldwide. Supplementary Material Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at www.gastrojournal.org, and at http:// dx.doi:10.1053/j.gastro.2012.09.033. References 1. Cornberg M, et al. Liver Int 2011;31(Suppl 2):30 60. 2. Sievert W, et al. Liver Int 2011;31(Suppl 2):61 80. 3. Lavanchy D, et al. Clin Microbiol Infect 2011;17:107 115. 4. Ghany MG, et al. Hepatology 2009;49:1335 1374. 5. Lohmann V, et al. Science 1999;285:110 113. 6. Bartenschlager R, et al. J Hepatol 2005;43:210 216. 7. Kato T, et al. Gastroenterology 2003;125:1808 1817. 8. Blight KJ, et al. J Virol 2003;77:3181 3190. 9. Gottwein JM, et al. J Virol 2010;84:5277 5293. 10. Robinson M, et al. AAC 2010;54:3099 3106. 11. Benhamou Y, et al. the 61st AASLD 2010. 12. Hassanein T, et al. HEPDart 2011. 13. Sumpter R, et al. J Virol 2005;79:2689 2699. 14. Yao N, et al. Structure 1999;7:1353 1363. 15. Saeed M, et al. AAC 2012; Epub ahead of print. Received February 2, 2012. Accepted September 17, 2012. Reprint requests Address requests for reprints to: Guofeng Cheng, PhD, Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94404. e-mail: Guofeng.cheng@gilead.com; fax: (650) 522-5890. Acknowledgments We are grateful to Amy Corsa for her suggestions on designing the replicon constructs. Conflicts of interest The authors disclose the following: All of the authors are stockholders and employees of Gilead Sciences. This article contains information describing the in vitro activity of Gilead compounds currently in development as treatments for HCV.

61.e1 PENG ET AL GASTROENTEROLOGY Vol. 144, No. 1 Supplementary Materials and Methods Cell Culture The following HCV permissive cell lines were used during these studies: Huh7-Lunet, 51C, and 1C. Huh7- Lunet was obtained from ReBLikon GmbH (Mainz, Germany). 1 The derivation of 51C cells, and stable genotype 1a H77 and genotype 1b Con-1 Renilla luciferase (Rluc)- neo replicon cells were described previously. 2 1C cells were derived by curing a GS-5885 resistant genotype 1a replicon clone derived from 51C cells. The 1C cells have been intensively validated for a complete cure and no surviving colonies have been observed after G418 treatment. All cell lines were propagated as described previously. 2 Replicon cell lines were selected and maintained with 0.5 mg/ml G418 (Geneticin; Invitrogen, Carlsbad, CA). Construction of Plasmids Encoding Genotype 4a HCV Subgenomic Replicons A plasmid (pgt4aed43sg) encoding a subgenomic genotype 4a replicon based on the ED43 infectious clone (GenBank accession # GU814266) 3 was prepared by DNA synthesis and cloning (Genescript, Piscataway, NJ). The synthesized replicon incorporated the following elements from 5= to 3= (Figure 1): (1) the ED43 5=UTR, extending to the first 48 nucleotides of core, (2) a linker 5=-GGC GCG CCA-3= that introduces the AscI restriction site (underlined), (3) the neo gene, (4) a linker 5 -GGC CGG CCG CGG CCG CAA-3 that introduces FseI and Not I restriction sites (underlined), (5) the encephalomyocarditis virus (EMCV) IRES, (6) a linker 5=-ACG CGT ATG-3= that introduces the MluI restriction site (underlined) and an ATG start codon, (7) the NS3 NS5B polyprotein region of ED43 including an NS5A adaptive mutation (S232I), and (8) the 3=UTR of ED43. Construction of Plasmids Encoding Genotype 4a HCV Rluc Subgenomic Replicons The plasmid (pgt4aed43rlucsg) encoding a subgenomic replicon that incorporated the humanized Rluc reporter gene was generated as follows: The pgt4aed43sg plasmid described here was cut using AscI and MluI restriction enzymes (to remove the neo gene) and gel purified using a commercial kit (Qiagen, Valencia, CA). A gene fragment encoding the humanized Rluc gene fused with the neo gene along with the EMCV region from pjfh1 (Toray, Japan), were polymerase chain reaction (PCR) amplified by using Accuprime super mix I (Invitrogen) with the following primers from the phrlucneosg2a plasmid described here: 2aRlucNeoAscI- For: 5=-AAC ACC AAC GGC GCG CCA ATG GCT TCC AAG GTG TAC-3= (AscI site is introduced by the primer and is underlined), 2aEMCVIRESMluIRev: 5=-TGG GCA TAA GCA GTG ATG GGA GCC ATA CGC GTA TCG 3= (MluI site underlined). The subsequent PCR fragment was cut with AscI and MluI and gel purified using a commercial kit (Qiagen). The vector and insert pieces were ligated using LigaFast Rapid DNA Ligation System per manufacturer s protocol (Promega, Madison, WI). The resulting vector, pgt4aed43rlucsg was sequenced to confirm the correct orientation and sequence of the hrluc- Neo. The phrlucneosg2a plasmid was constructed by replacing the Luc-Neo fragment in the plasmid plucneosg2a with the hrluc-neo gene amplified from the plasmid hrluc-neo Flexi(R) (Promega) as described previously. 2,4 RNA Transcription and Transfection and Selection of Stable Replicon Cell Lines RNA transcription and transfection was performed as described previously. 2 Forty-eight hours after plating the transfected cells, medium was replaced with complete Dulbecco s modified Eagle medium supplemented with 0.5 mg/ml G418, which was refreshed twice per week. Cell clones were isolated after approximately 3 weeks of G418 selection, expanded, and cryopreserved at early passages. Genotypic Analysis and Quantification of HCV RNA HCV RNA isolation, reverse transcription (RT)- PCR, and sequencing were performed by TACGen (Hayward, CA). Otherwise HCV replicon cellular RNA was extracted and purified using an RNeasy kit (Qiagen) according to manufacturer s protocol. RT-PCR was performed using the SuperScript III first-strand synthesis system (Invitrogen), followed by amplification of PCR products using the high-fidelity PCR polymerase (Invitrogen). PCR products were sequenced by TACGen (Hayward). HCV RNA copy numbers were determined using HCV RNA real-time RT-PCR assay supplied by EraGen Biosciences (Madison, WI). HCV RNA amounts were normalized by glyceraldehyde-3-phosphate dehydrogenase copy numbers that were determined by following the glyceraldehyde-3-phosphate dehydrogenase real-time RT- PCR assay protocol recommended by the manufacturer (Qiagen). Replicon Antiviral Assays Replicon cells were seeded in 384-well plates at a density of 2000 cells/well in 90 L Dulbecco s modified Eagle medium culture medium excluding G418. Cells were treated with 3-fold serial drug dilutions with 10 different concentrations, and replicon antiviral activities were determined by assaying luciferase activity as markers for replicon levels as described previously. 2 Antiviral Compounds VX-950 (telaprevir), boceprevir, ITMN-191, and 2-C-methyl adenosine (2-CMeA) were purchased from Acme Bioscience (Belmont, CA). Interferon alfa and cyclosporine A were purchased from Sigma-Aldrich (St

January 2013 GENERATION OF GENOTYPE 4A HCV REPLICONS 61.e2 Louis, MO). An Abbott palm site I benzothiadiazine NS5B polymerase inhibitor (A-837093) was synthesized by ChemALong Laboratories (Lemont, IL). The Wyeth HCV NS5B site palm site II inhibitor HCV-796 was synthesized by Curragh Chemistries (Cleveland, OH). Gilead compounds GS-5885, GS-9190, GS-9451, GS-9669, and GS-7977; Achillion NS4A inhibitor ACH-806, Tibotec protease inhibitor TMC-435; Merck NS5B thumb site I inhibitor MK-3281; Pfizer NS5B thumb site II inhibitor Filibuvir; and the Bristol-Myers Squibb NS5A inhibitor (BMS-790052) were synthesized by Gilead Sciences. Supplementary References 1. Friebe P, et al. J Virol 2005;79:380 392. 2. Robinson M, et al. Antimicrob Agents Chemother 2010;54:3099 3106. 3. Gottwein JM, et al. J Virol 2010;84:5277 5293. 4. Cheng G, et al. Antimicrob Agents Chemother 2011;55:2197 2205.

61.e3 PENG ET AL GASTROENTEROLOGY Vol. 144, No. 1 A 10 8 B HCV RNA Copies per µg Cellular RNA 64kd 50kd 10 7 10 6 10 5 GT4a-1 GT4a-2 GT4a-3 GT4a-4 GT4a-5 GT4a-6 GT4a-7 GT4a-8 GT4a-9 GT4a-10 GT4a-11 GT4a-12 GT4a Pool GT1a H77 Replicon Clones Selected GT4a clones 1 2 3 4 5 6 7 8 9 10 11 12 pool GT1b Bip NS5A C GT4a replicon cells 1C cells Supplementary Figure 1. (A) Intracellular HCV replicon RNA expression level. Selected genotype 4a replicon cell lines were measured for their intracellular HCV replicon RNA level by using HCV-specific real-time RT quantitative PCR (RT-QPCR), normalized by cellular glyceraldehydes-3- phosphate dehydrogenase (GAPDH) level as described in the Supplementary Material and Methods. Genotype 1a H77 Rluc-neo stable replicon cells were included as a reference. (B) NS5A expression in selected replicon cells. The selected replicon cell lines were pelleted and completely lysed in sodium dodecyl sulfate loading buffer. The Western blot was analyzed with primary anti-ns5a antibody (clone 9E10; Apath, Brooklyn, NY), which was co-stained with anti-bip antibody as a loading control. The staining was analyzed by Odyssey Imaging (LI-COR, Lincoln, NE). (C) NS5A immunostaining of the selected replicon cells. The genotype 4a replicon cell pool was stained with the same primary anti-ns5a antibody as above (red) and Hoechst 33342 (blue, indicating nuclei). 1C cells were stained as a negative control.

January 2013 GENERATION OF GENOTYPE 4A HCV REPLICONS 61.e4 In vitro transcribed GT4a RNA 16 µg Total RNA from GT4a replicon clone 1 µg 16 µg Supplementary Figure 2. Efficient colony formation of stable GT4a replicon clones. Total cellular RNA was extracted from the GT4a replicon pool cell line and then electroporated into Huh-7 Lunet cells at the indicated amounts. The efficiency of colony formation was determined as described in the Supplementary Materials and Methods. In vitro transcribed parental GT4a replicon RNA was transfected in parallel as a control. Supplementary Table 1. Mutations Identified in Genotype 4a Stable Replicon Cell Lines Mutations No. of occurrence Nucleotide positions a Nucleotide mutations Replicon clone NS3 A200E 3 4017/2436 C to G #7, #8 and pool NS3 T343K 3 4446/2865 C to A #1, #3, and #11 NS3 T343R 1 4446/2865 C to G #6 NS3 T511R 1 4950/3369 C to G #10 NS5A L179P 6792/5211 T to C NS4A Q34K 3 5411/3830 C to A #2, #4 and pool NS4A Q34R 3 5412/3831 A to G #9, #12 and pool NS4A E52V 1 5466/3885 A to T #5 a The nucleotide positions were presented in both ED43 full-length virus genome/rlu-neo subgenomic replicon.

61.e5 PENG ET AL GASTROENTEROLOGY Vol. 144, No. 1 Supplementary Figure 3. Selected NS3 or NS4A mutations enhance GT4a Rluc-neo replicon colony formation. All GT4a replicon constructs were prepared by introducing indicated mutations into wild-type GT4a Rluc-neo replicon plasmid using site-directed mutagenesis. Ten micrograms each GT4a replicon RNA was in vitro transcribed and electroporated into 1C cells. Forty-eight hours after seeding in 150-cm dishes, medium was replaced with complete Dulbecco s modified Eagle medium supplemented with 0.5 mg/ml G418 and refreshed twice per week. After 3 weeks of G418 selection, cells were stained with 0.05% crystal violet. GT1a H77 Rluc-neo replicon was included as a reference. The colony-formation efficiency (CFU/ g RNA) was determined and the data are summarized in Supplementary Table 2. Supplementary Table 2. Stable Colony Formation and Transient Replication Efficiency of HCV Replicons in 1C and Lunet Cells Replicons CFU/ g RNA in 1C cells CFU/ g RNA in lunet Replication in 1C (144hvs4h) a Replication in lunet (144hvs4h) a GT4aNeo wt 0.05 0.05 ND ND GT4aNeo wt NS5A S232I 3.4 0.05 ND ND GT4aNeo wt NS5A S232I NS4A Q34R 9950 1651 ND ND GT4aRlucNeo wt NS5A S232I 0.05 ND 0.02 b 0.1 GT4aRlucNeo wt NS4A Q34R 0.41 ND 0.06 b 0.2 GT4aRlucNeo wt NS5A S232I NS4A Q34R 980 179 0.63 1.4 GT4aRlucNeo wt NS5A S232I NS4A Q34K 121 ND 0.37 0.97 GT4aRlucNeo wt NS5A S232I NS4A E52V 21 ND 0.22 0.28 GT4aRlucNeo wt NS5A S232I NS3 A200E 43 ND 0.1 0.31 GT4aRlucNeo wt NS5A S232I NS3 T343R 299 ND 0.79 0.9 GT4aRlucNeo wt NS5A S232I NS3 T343K 39 ND 0.08 0.61 GT4aRlucNeo wt NS5A S232I NS5B GND 0.05 0.05 0.04 b 0.23 GT1aRlucNeo H77 NS5A S232I NS3 P470L 1103 15.5 0.4 0.21 GT1bRlucNeo Con-1 NS3 E176G, T254I NS4B K135T 10,000 3500 c 6.3 2.3 ND, not determined; Rluc, Renilla luciferase; wt, wild-type. a The values represent the ratio of Rluc signal of 144 h (day 6) to 4 h post transfection. b The value could be overestimated because their Rluc signals at day 6 post transfection were essentially at the background level. c The value was derived from previous studies, not in parallel experiments.

January 2013 GENERATION OF GENOTYPE 4A HCV REPLICONS 61.e6 Supplementary Figure 4. 1C cells are more permissive to GT4a replicon replication than Lunet cells. GT4a replicon RNA was in vitro transcribed and electroporated into 1C and Lunet cells. Forty-eight hours after seeding, medium was replaced with complete Dulbecco s modified Eagle medium supplemented with 0.5 mg/ml G418 and refreshed twice per week. After 3 weeks of G418 selection, cells were stained with 0.05% crystal violet. Colony-formation efficiency (CFU/ g RNA) was determined and the data are summarized in Supplementary Table 2.