Differential Sensitivity of 5′UTR-NS5A Recombinants of Hepatitis C Virus Genotypes 1−6 to Protease and NS5A Inhibitors

Accepted Manuscript Differential Sensitivity of 5’UTR-NS5A Recombinants of Hepatitis C Virus Genotypes 1–6 to Protease and NS5A Inhibitors Yi-Ping Li, Santseharay Ramirez, Daryl Humes, Sanne B. Jensen, Judith M. Gottwein, Jens Bukh PII: DOI: Reference:

S0016-5085(13)01655-7 10.1053/j.gastro.2013.11.009 YGAST 58797

To appear in: Gastroenterology Accepted Date: 13 November 2013 Please cite this article as: Li Y-P, Ramirez S, Humes D, Jensen SB, Gottwein JM, Bukh J, Differential Sensitivity of 5’UTR-NS5A Recombinants of Hepatitis C Virus Genotypes 1–6 to Protease and NS5A Inhibitors, Gastroenterology (2013), doi: 10.1053/j.gastro.2013.11.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in Gastroenterology are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs.

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Differential Sensitivity of 5’UTR-NS5A Recombinants of Hepatitis C Virus Genotypes 1–6 to Protease and NS5A Inhibitors

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Short Title: Novel HCV culture systems and DAA sensitivity

Authors: Yi-Ping Li, Santseharay Ramirez, Daryl Humes, Sanne B. Jensen, Judith M. Gottwein,

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and Jens Bukh*

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Affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

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Funding: This study was supported by research grants from the Lundbeck Foundation (Y.-P.L., J.G.M., and J.B.), the Danish Cancer Society (J.M.G. and J.B.), the Novo Nordisk Foundation (Y.P.L., J.M.G., and J.B.), the A. P. Møller og Hustru Chastine Mc-KinneyMøllers Fondation (J.B.),

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and the Danish Council for Independent Research–Medical Sciences (Y.-P.L., S.R., and J.B.). S.R. and D.G.H. are recipients of individual postdoctoral stipends from the Danish Council for

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Independent Research–Medical Sciences.

Abbreviations: HCV, hepatitis C virus; DAA, direct-acting antivirals; ORF, open reading frame; UTR, untranslated region; FFU/mL, Focus Forming Units per milliliter; IU/mL, international units per milliliter; PI, protease inhibitor; NS, nonstructural proteins.

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Corresponding author (*): Jens Bukh; Mailing address: Department of Infectious Diseases, Hvidovre Hospital, Kettegaard Alle 30, DK-2650 Hvidovre, Denmark. Phone: +4538626380. Fax:

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+4536474979. E-mail: [email protected].

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Disclosures: The authors disclose no conflicts.

Author contributions: Study concept and design: YPL, SR, JMG, and JB; acquisition of data: YPL,

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SR, DH, SBJ; analysis and interpretation of data: YPL, SR, DH, SBJ, JMG, and JB; drafting of the

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manuscript: YPL, SR, DH, JG, and JB; study supervision: YPL, JG, and JB.

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Abstract Background & Aims: Hepatitis C virus (HCV) therapy will benefit from the preclinical evaluation of direct-acting antiviral (DAA) agents in infectious culture systems that test the effects on different

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virus genotypes. We developed HCV recombinants comprising the 5’UTR-NS5A (5–5A) from genotypes 1–6 and 2a(JFH1) NS5B-3’UTR, and tested the effects of NS3 protease and NS5A inhibitors on these recombinants.

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Methods: The HCV 5–5A recombinants with previously identified mutations in the NS3- helicase (F1464L), NS4A (A1672S), and NS5B (D2929G) were adapted and improved, by incorporating

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additional recovered mutations that increase their propagation in Huh7.5 cells. Concentration– response profiles were determined for each DAA agent in replicate infected Huh7.5 cells. Results: Developed efficient 1a(H77), 1a(TN), 3a(S52), 4a(ED43), 5a(SA13), and 6a(HK6a) 5–5A recombinants did not require mutations after viral passage in the NS3 protease or NS5A domain-I regions targeted by the drugs. They were inhibited in a concentration-dependent manner by the NS3

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protease inhibitors telaprevir, boceprevir, asunaprevir, simeprevir, vaniprevir, faldaprevir, and MK5172 and by the NS5A inhibitor daclatasvir. The 1a(TN) 5–5A and JFH1-independent full-length viruses had similar levels of sensitivity to the DAA agents, validating the 5–5A recombinants as

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surrogates for full-length viruses in DAA testing. Compared to the 1a(TN) full-length virus, the 3a(S52) 5-5A recombinant was highly resistant to all protease inhibitors, whereas the 4a(ED43)

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recombinant was highly resistant to telaprevir and boceprevir, but most sensitive to other protease inhibitors. Compared to other protease inhibitors, MK-5172 had exceptional potency against all HCV genotypes. The NS5A inhibitor daclatasvir had the highest potency observed, but with genotype-dependent activity. Conclusions: The mutations F1464L, A1672S, and D2929G permitted the development of efficient HCV recombinants comprising genotype-specific 5’UTR-NS5A (5–5A), which include the natural NS3 protease and NS5A domain-I drug targets. The robust replication of adapted 5–5A 3

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recombinants allowed for direct comparison of NS3 protease and NS5A inhibitors against HCV strains of genotypes 1–6.

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KEYWORDS: drug resistance, cultures, vaccine, PI

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Introduction Over 130 million people are chronically infected with hepatitis C virus (HCV), which increases the

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risk of developing liver cirrhosis and end-stage liver diseases. HCV, belonging to the family Flaviviridae, has a ~9.6-kb positive single-stranded RNA genome consisting of one open reading frame (ORF) flanked by 5’ and 3’ untranslated regions (UTRs). The ORF is translated and processed into structural (Core, E1, and E2) and nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A,

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and NS5B) proteins. The NS3/NS4A-protease (NS3P), which is essential for viral polyprotein processing, the NS5A protein, which is important for replication and assembly, and the NS5B viral

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RNA polymerase are all major targets for direct acting antivirals (DAAs).1

Six epidemiologically important HCV genotypes, comprising numerous subtypes (a, b, etc.) exist. The differences within genotypes, subtypes, and isolates/strains at the nucleotide (nt) and amino acid (aa) level are approximately 30%, 20%, and 2–10%, respectively.1 The identity of the infecting

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HCV genotype affects the outcome of interferon-alpha/ribavirin (IFN-α/RBV)-based treatment.2 Genotypes 1, 2, and 3 are the most prevalent worldwide, and account for ~80% of global HCV infections.3 Genotype 3 accounts for the majority of infections in India, Pakistan and Brazil, and

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>20% of infections in many European and Asian countries, Canada, Israel, and Australia. Genotypes 4, 5, and 6 represent ~20% of worldwide HCV infections.4 Genotype 4 is prevalent in

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the Middle East and in many African countries, and is spreading to Western countries. Genotype 5 represents ~40% of the cases in South Africa, and is emerging in Europe. Finally, genotype 6 is found predominantly in South East Asia.4 In 2011, the NS3/NS4A protease inhibitors (PIs) telaprevir and boceprevir were licensed to complement the IFN-α/RBV therapy of genotype 1-infected HCV patients. Additional DAAs targeting the NS3P, NS5A, and NS5B polymerase have reached phase II or III clinical trials, and

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the first studies combining different DAAs highlighted their potential for use in IFN-free treatment regimens.5 However, DAAs have been studied primarily using HCV genotype 1 replicons and by performing clinical trials with genotype 1-infected patients, thus there is limited knowledge

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regarding DAA efficacy against the other major genotypes.

JFH1(genotype 2a)-independent full-length HCV infectious culture systems only exist for

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genotypes 1a, 2a, and 2b strains.6 We previously developed JFH1-based recombinants comprising 5’UTR-NS2, NS3P/NS4A, NS5A, or 5’UTR-NS3-protease plus NS4A-NS5A of other HCV

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genotype strains.7-10 Such systems have permitted genotype-specific studies of novel antivirals. However, several of these chimeric in vitro systems required mutations in the targeted HCV proteins, which we have found could influence their sensitivity to DAAs.6 Also, systems for key viral enzymes are missing for HCV genotypes 3-6.

We previously identified the LSG mutations F1464L (NS3-helicase), A1672S (NS4A), and

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D2929G (NS5B), which permitted the development of full-length HCV genotype 1 and 2 infectious culture systems11,12 [aa (and nt) positions according to genotype 1a strain H77; GenBank Accession No. AF009606]. In this study, we used these unique mutations to develop genotype(isolate) 1a(TN

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and H77), 3a(S52), 4a(ED43), 5a(SA13), and 6a(HK6a)-specific 5’UTR-NS5A (5-5A) prototype in vitro infectious clones (Figure 1A), and demonstrated genotype(isolate)-dependent responses to

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DAAs targeting the HCV NS3P and NS5A.

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Materials and Methods Construction of HCV 5’UTR-NS5A (5-5A) recombinants.

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To construct genotype(isolate)-specific recombinants (Figure 1A), the 5’UTR-NS5A sequences of genotype(isolate) 1a(H77), 3a(S52), and 4a(ED43) were cloned from pCV-H77C (GenBank Accession No. AF011751),13 pS52 (GU814263),14 and pED43(GU814265),14 respectively. For

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5a(SA13) and 6a(HK6a), previously reported 5’UTR-NS2,7 NS3P,8 and NS5A9 were used; the consensus sequences of the NS3-helicase and NS4B were obtained by analysis of clones of RT-

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PCR products amplified from plasma pools of experimentally infected chimpanzees.15 The 5’UTRNS5A sequences of 5a(SA13) and 6a(HK6a) are deposited in GenBank [5a(SA13), KF589888; 6a(HK6a), KF589889]. The JFH1 NS5B-3’UTR sequence, in junction with isolate-specific NS5A, was cloned from previously described plasmids.9 The JFH1-based genotype(isolate) 1a(H77), 3a(S52), 4a(ED43), 5a(SA13), and 6a(HK6a) 5’UTR-NS5A recombinants were generated, with

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LSG (F1464L/A1672S/D2979G)12 and other mutations identified in this study, by standard cloning procedures. Final plasmid preparations were sequenced covering the T7 promoter and the entire

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HCV genome (Macrogen).

Analysis of HCV recombinants in Huh7.5 cells.

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Transfection and infection of Huh7.5 cells was done as previously described12. Briefly, cells were seeded in 6-well plates ~24 hours before transfection or infection. For transfection, XbaI-linearized HCV plasmid was in vitro-transcribed into RNA using T7 RNA Polymerase (Promega). The RNA transcripts were transfected into the cells using Lipofectamine 2000 (Invitrogen) diluted in OptiMEM (Invitrogen). The transfected cultures were incubated with Opti-MEM medium overnight. For infection, cells were incubated with 1 ml HCV-containing culture supernatant overnight. The transfected or infected cells were sub-cultured every 2-3 days. HCV infected cells were detected by 7

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immunostaining using primary monoclonal anti-Core antibody C7-50 (Enzo Life Sciences) and/or anti-NS5A antibody 9E10 as described.12 When cultures reached peak infection (≥80% culture cells

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infected), supernatant was collected, filtered (0.45 µm) and stored at -80°C. HCV infectivity titers were determined by Focus-Forming Unit (FFU) assays, using a combination of C7-50 and 9E10 antibodies.12 The number of FFUs was determined by manual count with light microscopy, or by automated count with an ImmunoSpot Series 5 UV Analyzer with customized

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software (CTL Europe GmbH).9,14 In automated counting, the mean count of 6 negative control wells (≤15 FFUs/well) was subtracted from the count of each experimental wells (≤200 FFUs/well).

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The cutoff of detection was the mean of negative wells plus 3 standard deviations plus 3. The method for sequence analysis of the ORF of recovered viruses has been described previously.12 DAA treatment of HCV recombinant viruses.

DAAs were purchased from Acme Bioscience (Palo Alto, CA) and dissolved in dimethyl sulfoxide

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(Sigma). High throughput treatment assays have been previously established.8-10 Briefly, Huh7.5 cells plated on poly-D-lysine-coated 96-well plates (Nunc) were infected with HCV stocks and treated with drugs 24 hours post infection. Triplicate infections were treated with each drug

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concentration. Seventy-two hours post- infection, HCV Core/NS5A positive cells in each well were immunostained and counted using the ImmunoSpot Series 5 UV Analyzer. Counts from treated

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wells were related to the mean of counts from 6 infected, non-treated control wells. Concentrationresponse curves and EC50 values were calculated in GraphPad Prism 5 using previously described methods.8,10 Cytotoxicity assays were performed as previously described10 to ensure that the selected dose ranges of the DAAs were not cytotoxic.

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Results HCV genotype 2a-derived LSG mutations facilitated the development of 1a, 3a, 4a, 5a, and 6a

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specific 5’UTR-NS5A (5-5A) culture systems. We previously demonstrated that the LSG mutations permitted the development of J6cc (genotype 2a), J8cc (2b), and TNcc (1a) full-length infectious culture systems.6,11,12 Here, we initially tested whether LSG and additional TNcc-adaptive mutations could adapt another in vivo infectious full-

H77C

containing

LSG

or

LSG

plus

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length genotype 1a clone, H77C,13 for replication in cell culture. Huh7.5 cells transfected with the

TNcc-adaptive

mutations

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A1226G/Q1773H/N1927T/Y2981F/F2994S11 remained negative in HCV-antigen immunostaining assays.

We next tested a H77C 5-5A recombinant, designated 1a(H77)5-5A, as well as variants with either LSG or LSG/A1226G/Q1773H11 mutations that were previously found to efficiently adapt a

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1a(TN)5-5A recombinant (Figure 1A and Table 1). After two RNA transfections of Huh7.5 cells, 1a(H77)5-5A cultures were HCV negative for 29 and 31 days, respectively. In contrast, a 1a(H77)55A_LSG culture had HCV positive cells from day 4, indicating that LSG could initiate replication

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of 1a(H77)5-5A; after 36 days of follow-up, however, the virus did not spread. 1a(H77)55A_LSG/A1226G/Q1773H (KF134007) had ~5% HCV-antigen positive cells at day 1, and reached

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peak infection at day 8. After passage to naïve Huh7.5 cells, culture supernatant reached HCV infectivity titers of 104.1 FFU/ml. Sequence analysis of the ORF of first-passage virus revealed that the engineered mutations were maintained, and although putative adaptive mutations were observed in the NS3-helicase, no mutations were found in the NS3-protease (spanning nts 3420-3977 and aas 1027-1212) (Supplementary Table 1). Thus, although the LSG mutations could apparently not adapt H77C, they facilitated the development of 5-5A recombinant viruses for genotype 1a, strains TN and H77. 9

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Next, we used the LSG mutations to develop 5-5A recombinants for other genotype isolates. We previously demonstrated that 5’UTR-NS3-protease and NS4A-NS5A sequences of 3a(S52),10 5’UTR-NS2 and NS5A of 4a(ED43),7,9 and 5’UTR-NS2, NS3P, and NS5A of 5a(SA13) and

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6a(HK6a)7-9 were functional as part of JFH1-based recombinants. However, it remained unknown whether the NS3-helicase domain (spanning nts 3978-5312 and aas 1213-1657) encoded by these strains could support viral replication in cell culture. Here, we tested 3a(S52), 4a(ED43), 5a(SA13),

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and 6a(HK6a) specific 5-5A recombinants containing the LSG mutations (Figure 1A). After RNA transfection of Huh7.5 cells (Table 1), HCV positive cells emerged in 3a(S52) and 4a(ED43) 5-

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5A_LSG cultures at day 4, and the cultures reached peak infection at days 41 and 50, respectively. The 5a(SA13)5-5A_LSG culture showed 1-5% HCV positive cells at day 1 and reached peak infection at days 7 and 8 in two transfections, whereas the 6a(HK6a)5-5A_LSG culture had HCV positive cells at day 1 and reached peak infection at day 64. First-passage supernatants had peak infectivity titers of 104.2, 103.9, 104.7, and 103.5 FFU/ml for the 3a(S52), 4a(ED43), 5a(SA13), and

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6a(HK6a) 5-5A_LSG cultures, respectively. Sequence analysis of the amplified ORF of 3a(S52) (Supplementary Table 2), 4a(ED43) (Supplementary Table 3), 5a(SA13) (Supplementary Table 4), and 6a(HK6a) (Supplementary Table 5) 5-5A_LSG cultures revealed that the LSG mutations were

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maintained in all passaged viruses, except for a partial reversion (50/50 quasispecies) of G at aa 2979 in the 4a(ED43)5-5A_LSG virus; other mutations were observed in these viruses. Thus, we

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generated HCV genotype 3a, 4a, 5a, and 6a specific 5’UTR-NS5A recombinant viruses by using the LSG mutations, showing for the first time that the NS3-helicase of HCV genotypes 3-6 were functional for the complete viral life cycle in Huh7.5 cells.

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Development of efficient HCV recombinants with 5’UTR-NS5A of genotype 3a, 4a, 5a, and 6a prototype isolates.

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Although LSG could initiate replication of 5-5A recombinants (Table 1), the 3a(S52), 4a(ED43), and 6a(HK6a) 5-5A_LSG viruses showed impaired viral spread and low infectivity titers, while the 5a(SA13)5-5A_LSG recombinant had efficient viral spread, but relatively low infectivity titers. We further optimized these 5-5A recombinants by introducing mutations identified in the passaged

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viruses. Given the existence of genotype 1a full-length H77-S16 and efficient 1a(TN) 5-5A (Table 1) and full-length11 cultures, we did not further optimize 1a(H77) 5-5A recombinant, which had

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efficient viral spread, with >104 FFU/mL after first-passage. The final genotype 3-6 5-5A recombinant viruses did not have mutations in the NS3-protease and NS5A-domain-I (spanning nts 6258-6896 and aas 1973-2185) sequences, thus making them beneficial for studies of protease and NS5A inhibitors.

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Efficient 3a(S52) 5’UTR-NS5A recombinant. Based on ORF sequencing data of first-passage 3a(S52)5-5A_LSG, we tested H1819Q or D871G/H1819R in 3a(S52)5-5A_LSG (Supplementary Table 2); the D871G/H1819R recombinant spread more efficiently in transfected cultures (Table 1).

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After first-passage, all engineered mutations were maintained, and no common mutation was found in these two viruses (Supplementary Table 2). We performed a second-passage for the

which

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D871G/H1819R virus and identified two additional coding changes (Supplementary Table 2), were

engineered

into

the

genome.

In

two

transfections,

3a(S52)5-

5A_LSG/D871G/V1612E/H1819R/V2417A (KF134008) cultures had HCV positive cells at day 1, and reached peak infection at day 7 and 11, with peak titers of 104.0 and 103.6 FFU/ml, respectively. In first- and second-passage, this virus spread efficiently and reached peak infection within 7-9 days. Sequence analysis of respective second-passage viruses (104.1 and 104.3 FFU/ml) revealed no changes in the NS3-protease and NS5A-domain-I sequences (Supplementary Table 2). 11

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Efficient 4a(ED43) 5’UTR-NS5A recombinant. Based on ORF sequence analysis of first- and second-passage 4a(ED43)5-5A_LSG viruses, we tested selected combinations of mutations in a 4a(ED43)5-5A_LS genome (Supplementary Table 3). In two independent transfection cultures,

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4a(ED43)5-5A_LS/R781W/A1309P/A1786V (KF134009) was the most efficient recombinant, resulting in ~1% HCV positive cells at day 1, with spread to ≥80% infected cells at day 7, and with peak infectivity titers of 103.8 and 103.2 FFU/ml (Figure 1B and Table 1). Two second-passage

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viruses from independent transfections reached 103.2 and 103.1 FFU/ml, respectively (Table 1). The

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ORF sequence analysis of one of them revealed no additional mutations (Supplementary Table 3). Efficient 5a(SA13) 5’UTR-NS5A recombinant. We introduced two mutations identified from the ORF of second-passage 5a(SA13)5-5A_LSG virus pool (105.1 FFU/ml) (Supplementary Table 4) to generate 5a(SA13)5-5A_LSG/S294G/C1551F (KF134010) (Figure 1B and Table 1). In two independent transfections, the cultures showed 5-10% HCV positive cells at day 1, reached peak

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infection within 3 days, and produced peak infectivity titers of 104.5 FFU/ml (Table 1). ORF sequence analysis of one of the second-passage viruses (104.9 FFU/ml) revealed that no additional changes were required (Supplementary Table 4).

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Efficient 6a(HK6a) 5’UTR-NS5A recombinant. We introduced five mutations identified in firstand second passage 6a(HK6a)5-5A_LSG viruses (Supplementary Table 5) to generate 6a(HK6a)5-

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5A_LSG/T387I/S872P/V1550L/L1790M/S2218P (KF134011) (Figure 1B and Table 1). In two independent transfections, the cultures showed 1-5% HCV positive cells at day 1 and reached ≥80% infection at days 7 and 9, with peak infectivity titers of 103.4 and 103.3 FFU/ml, respectively. Sequence analysis of one second-passage virus (103.8 FFU/ml) revealed that all engineered mutations were maintained, and additional mutations were found in E1, NS2, and NS4B (Supplementary Table 5). 12

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Efficacy of NS3/NS4A protease inhibitors and an NS5A inhibitor against HCV recombinants with genotype 1-6 specific 5’UTR-NS5A. We generated virus stocks from first-passage 1a(H77 and TN) and second-passage 3a(S52),

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4a(ED43), 5a(SA13), and 6a(HK6a) 5-5A viruses (Table 1) for PI treatment assays. No mutations were detected in the NS3-protease of these virus stocks (Supplementary Tables 1-5). We tested virus sensitivity to seven PIs, including the licensed telaprevir (VX-950)17 and boceprevir

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(SCH503034),18 and others in clinical trials: asunaprevir (BMS-650032),19 simeprevir (TMC435350),20 vaniprevir (MK-7009),21 faldaprevir (BI201335),22 and MK-5172.23 For

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comparison, we also tested the previously developed 1a(TN) full-length11 (designated TN_FL) and JFH1-based NS3P recombinants.8 Finally, the 2a recombinant J6/JFH124 was included in each experiment. All recombinant viruses were inhibited by the different PIs in a concentrationdependent manner (Figure 2). The median effective concentration (EC50) was calculated from the concentration-response curves (Table 2). For a given inhibitor, fold-difference in EC50 relative to

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the TN_FL virus for each genotype recombinant was used to describe whether a recombinant was more sensitive (fold-difference <1) or resistant (fold-difference >1) than the TN_FL virus.

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For each 5-5A recombinant, differences in sensitivity to telaprevir and boceprevir were relatively small (Figure 2A and Table 2). Compared to the TN_FL virus, 3a(S52) and 4a(ED43) viruses were

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~13 and ~20-fold more resistant to telaprevir and boceprevir, respectively. 1a(H77), 5a(SA13), and J6/JFH1 viruses were ~3-fold more resistant to telaprevir and ~6-fold more resistant to boceprevir. 1a(TN) and 6a(HK6a) viruses showed ~1 to 3-fold differences compared to the TN_FL virus. Compared to TN_FL, 3a(S52)5-5A was ~33-fold more resistant to asunaprevir, while other recombinants had <2.5-fold differences. For simeprevir, 3a(S52)5-5A was ~85-fold more resistant than TN_FL, J6/JFH1 and 5a(SA13)5-5A viruses had ~3-4-fold resistance, whereas the 1a(H77) 13

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and 4a(ED43) 5-5A viruses were ~3- and 5-fold more sensitive, respectively. For vaniprevir, 3a(S52)5-5A and J6/JFH1 were ~119- and 6-fold more resistant, while the other 5-5A recombinants had <2-fold differences compared to TN_FL viruses. For faldaprevir, 3a(S52)5-5A and J6/JFH1

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were ~76- and ~4-fold more resistant than TN_FL, 4a(ED43)5-5A was ~10-fold more sensitive, while the other 5-5A viruses had <3-fold differences. For the 3a(S52)5-5A virus, all the EC50s values ranged only from 1215 to 2476 nM, indicating similar susceptibility of the 3a virus to all PIs

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tested.

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All recombinant viruses showed the highest sensitivity to MK-5172 as compared to the other PIs, indicating improved potency and broad activity of MK-5172 against HCV genotypes 1-6. However, 3a(S52)5-5A was ~22-fold more resistant than the TN_FL virus, while the remaining 5-5A viruses were only ~1 to 4-fold more resistant than TN_FL.

Overall, 3a(S52)5-5A was the most resistant virus to the PIs tested. Resistance of 3a(S52)5-5A to

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telaprevir, boceprevir, asunaprevir, simeprevir, vaniprevir, and faldaprevir was >60-fold more than that to MK-5172. Although the 4a(ED43)5-5A virus was relatively resistant to telaprevir and boceprevir, with EC50s similar to those of the 3a(S52)5-5A virus, it was the most sensitive virus to

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asunaprevir, simeprevir, vaniprevir, faldaprevir, and MK-5172.

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For NS3P recombinant viruses, 3a(S52) was the most resistant virus to all PIs tested compared to 5a(SA13) and 6a(HK6a) NS3P viruses (Figure 2B and Table 2), consistent with our previous observations for selected PIs.8 The NS3P and 5-5A viruses of respective isolates had a less than 4fold difference in EC50 for each PI tested. The 5-5A recombinant viruses did not appear to require adaptive mutations in NS5A-domain-I (Supplementary Tables 1-5), thus making them suitable for testing of NS5A inhibitors. The NS5Adomain-I directed inhibitor daclatasvir (BMS-790052)25 had the highest potency against all HCV 14

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genotypes (Figure 3), being ~30 to 100-fold more efficient than the most potent PI, MK-5172 (Tables 2 and 3). Among the different viruses, 3a(S52)5-5A was the most resistant, being ~6-fold more resistant than TN_FL, while 4a(ED43)5-5A was the most sensitive virus. The other genotypes

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showed ≤3-fold difference in sensitivity compared to TN_FL. The EC50s against 5-5A recombinants

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were similar to the reported values for the respective isolate NS5A recombinants9 (Table 3).

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Discussion In this study, we developed efficient infectious culture systems comprising 5’UTR-NS5A (5-5A)

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for HCV genotypes 3-6, representing 4 of the 6 major variants of this globally important human pathogen. Development of the 5-5A viruses described herein was aided by the previously identified LSG mutations.12 All recombinant viruses maintained the original patient NS3-protease and NS5Adomain-I sequences, thus the 5-5A viruses provide a panel of viruses for testing NS3-protease and

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NS5A inhibitors against the diverse HCV genotypes. Moreover, this is the first study to include an

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infectious HCV cell culture recombinant expressing the genotype 4 NS3P. We demonstrated concentration-dependent inhibition and genotype-specific activity profiles for HCV PIs for genotype 1-6 recombinant viruses in the context of complete viral life cycle. The 3a(S52)5-5A virus was most resistant to all PIs, while the 4a(ED43)5-5A was highly resistant to telaprevir and boceprevir, but was most sensitive to asunaprevir, simeprevir, vaniprevir, faldaprevir, and MK-5172.

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MK-5172 had the highest efficacy against all genotype recombinant viruses. In addition to their critical role in the development of full-length 1a, 2a, and 2b infectious culture systems,6,11,12 here the LSG mutations were found to initiate replication of 1a(H77), 3a(S52),

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4a(ED43), 5a(SA13), and 6a(HK6a) 5-5A recombinants, further demonstrating their cross-genotype effects. The JFH1 NS5B polymerase and 3’UTR were previously found to have unique

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characteristics favourable for HCV RNA replication.26,27 Combination of JFH1 NS5B-3’UTR with the LSG mutations could be advantageous for the initial replication of 5-5A recombinants, thus allowing for the acquisition of additional mutations for efficient virus production (Supplementary Tables 1-5). Mutations identified from the LSG-adapted TN 5-5A recombinant viruses have proven to be critical for the development of TNcc.11 Thus, in future studies it will be of great relevance to

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explore the potential of the LSG and other mutations identified in this study, in developing 5-5A or full-length culture systems of additional strains of HCV.

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The 5-5A culture systems for 3a(S52), 4a(ED43), 5a(SA13), and 6a(HK6a) also demonstrated for the first time that the NS3-helicase of these isolates was functional in Huh7.5 cells. The NS3helicase is critical for HCV RNA replication28 and could be a target of antivirals. Within the NS3helicase, all 5-5A recombinants contain the F1464L mutation.12 Additionally, the adapted

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4a(ED43)5-5A encodes the A1309P change in the helicase, a mutation that was identified

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previously in an ED43 replicon.29 Although recombinant-specific mutations in NS3-helicase were needed, no additional changes were required in this region after viral passage (Supplementary Tables 1-5), indicating genetic stability of the NS3-helicase sequence in the developed 5-5A recombinants. To date, only a few helicase inhibitors have been reported to decrease HCV RNA replication efficiency in cells, and none has been reported in clinical trials. The 5-5A culture

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systems will permit future screening or testing of the effect of helicase inhibitors in the context of complete viral life cycle for the major HCV genotypes. The 4a(ED43), 5a(SA13), and 6a(HK6a) 5-5A recombinants are the first infectious culture systems

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comprising NS4B (spanning nts 5475-6257 and aas 1712-1972) of genotypes 4, 5, and 6, and they represent a valuable tool for the development of inhibitors of NS4B, which has been suggested as a

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potential target for DAAs.30

Our 5-5A recombinants maintained wild-type HCV sequences in the NS3-protease and NS5Adomain-I (the target of daclatasvir25) (Supplementary Tables 1-5). These properties permitted assessment of genotype-specific profiles for either NS3-protease or NS5A inhibitors, which would most likely reflect relevant clinical differences in drug sensitivity without the possibility of being misled by mutations in the drug targets, as adaptive mutations could affect drug sensitivity in vitro.6 17

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Thus the 5-5A infectious culture systems could also be suitable models for future studies of combination treatment of protease and NS5A inhibitors.10

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For each PI, similar EC50s were observed for 1a(TN) 5-5A and full-length viruses (Table 2)6, and also for respective 5-5A and NS3P recombinants of 3a(S52), 5a(SA13), and 6a(HK6a) (Table 2). For telaprevir, boceprevir, simeprevir, and vaniprevir, the EC50s for NS3P viruses were also similar to those previously reported.8 The EC50s of asunaprevir and daclatasvir against 1a(TN), 1a(H77),

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and 3a(S52) 5-5A recombinants were similar to those previously observed for respective isolate 5’UTR-NS3-protease plus NS4A-NS5A.10 The EC50s of daclatasvir against 5-5A viruses were

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similar to those reported for NS5A recombinants of respective isolate.9 These similarities in EC50 values in various treatment studies validate in vitro antiviral studies using these different culture systems and suggest that JFH1 sequences and adaptive mutations engineered may not interfere with antiviral treatment studies. With the inclusion of a large portion of the genome sequence of

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respective isolates, the 5-5A recombinants may effectively recapitulate the infection cycle of the given genotype(isolate), thus representing a valid model for testing antivirals targeting 5’UTRNS5A regions and for studying viral escape and resistance in a genotype-specific manner.

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Clinical studies exploring IFN-free regimens with DAAs tested in this study, such as combination of asunaprevir and daclatasvir,31 have demonstrated that these drugs can improve sustained

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virological response (SVR) rates in genotype 1-infected patients. Given the limited knowledge on DAAs against genotype 3-6 infections, our experimental data on the efficacy of PIs against 5-5A infectious culture systems of genotypes 3a, 4a, 5a, and 6a may contribute to treatment guidelines for genotypes 3-6-infected patients. MK-5172 had activity against genotype 1 and 2 replicons and a high barrier to resistance.23 In this study, MK-5172 was tested for the first time against genotype 36 viruses, and notably was the most efficient PI against all viral genotypes (Figure 2 and Table 2). 18

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Compared to other recombinants, 3a(S52) 5-5A and NS3P viruses were the most resistant to the tested DAAs (Figure 2B, and Tables 2 and 3), in agreement with previous studies testing only NS3P recombinants for selected PIs.8 The high resistance of the genotype 3a recombinants to DAAs may

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reflect viral characteristics in clinical settings as genotype 3-infected patients have limited benefit from telaprevir monotherapy.32 Here we showed that MK-5172 and daclatasvir were 60 to 2250fold more efficient in inhibiting the 3a(S52)5-5A virus than the second most efficient PI, boceprevir

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(Tables 2 and 3). These results may contribute to the future design of treatment for genotype 3a-

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infected patients.

Limited clinical trials have been performed for genotypes 4, 5, and 6, although these genotypes represent ~20% of the global hepatitis C burden. Small-scale studies have shown that in long-term IFN-α/RBV treatment, genotype 5 and 6-infected patients could achieve SVR rates similar to those infected with genotypes 2 and 3, whereas SVR rates for chronic genotype 4-infected patients were

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<50%.4 Therapy with IFN-α/RBV plus DAA, or DAA combinations alone, may improve treatment outcome for these patients. However, the efficacy of DAAs against genotype 4a infectious viruses could not previously be assessed, as genotype 4a NS3P infectious cultures were not developed.33

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Thus, the 4a(ED43)5-5A recombinant developed here permits for the first time the evaluation of the efficacy of PIs against this important genotype in a high-throughput manner. The 4a(ED43)5-5A

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recombinant was highly resistant to telaprevir and boceprevir, in line with observations for a replicon of the same strain,29 but was the most sensitive virus to the other PIs and daclatasvir (Tables 2 and 3). These findings may contribute to future clinical DAAs guidelines for genotype 4infected patients.

In summary, we have developed efficient genotype-specific 5’UTR-NS5A infectious culture systems for HCV genotypes 3, 4, 5, and 6, and demonstrated concentration-dependent and 19

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genotype-specific viral responses to PIs and NS5A inhibitors. These efficient 5’UTR-NS5A culture systems have great potential for further use in functional and treatment studies that will directly contribute to HCV basic research and to the future development and assessment of DAAs, thus

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facilitating personalized IFN-free HCV therapy.

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Acknowledgments We thank T.B. Jensen for providing SA13 NS3-helicase and NS4B sequences, L.S. Mikkelsen for

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technical assistance, A.L. Sørensen and L. Ghanem for laboratory support, J.O. Nielsen, B. Ørskov Lindhardt, and O. Andersen for valuable support (all from Hvidovre Hospital), and C.M. Rice (Rockefeller University, NY) and T. Wakita (National Institute of Infectious Diseases, Tokyo) for

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providing reagents.

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Figure 1. Characteristics of HCV genotypes 3a, 4a, 5a, and 6a specific 5’UTR-NS5A (5-5A) recombinants in transfected Huh.7.5 cells. (A) Schematic diagram of J65’UTR-NS2/JFH17 and HCV 5-5A recombinants. LSG mutations (F1464L/A1672S/D2979G) are indicated. (B) RNA transcripts

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of 5-5A recombinants with indicated mutations were transfected into Huh7.5 cells, and the percentage of HCV Core and/or NS5A positive cells was estimated (left y-axis; lines). HCV infectivity titers in culture supernatants at peak infection were determined (mean of triplicate

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infections ± SEM, right y-axis; bar). J65’UTR-NS2/JFH17 was control. Duplicate transfection experiments performed for these recombinants yielded similar results. See Table 1 for details on

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transfection and second-passage viruses.

Figure 2. HCV genotype 1-6-specific 5-5A recombinants showed differential sensitivity to protease inhibitors, similar to NS3P recombinants of respective genotypes. Huh7.5 cells in 96well plates were infected with the 5-5A (A) and NS3P8 (B) viruses, and treated with seven PIs

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(Materials and Methods). Experimental control 2a(J6/JFH1) were included in each treatment. Control 1a(TN)_FL (full-length)11 was incorporated in A. Values are means of triplicates in the experiment ± SEM. EC50s for each drug against the different genotype viruses are shown in Table 2.

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Figure 3. HCV genotype 1-6-specific 5-5A recombinants showed differential sensitivity to NS5A inhibitor daclatasvir. Cultures infected with 5-5A recombinant viruses were treated with

3.

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daclatasvir. See legend of Figure 2 and Materials and Methods for details. EC50s are shown in Table

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Table 1. Characteristics of the HCV genotype-specific 5’UTR-NS5A (5-5A) recombinants in Huh7.5 cells. a, all genotype(isolate) 5-5A recombinants contained NS5B-3’UTR from JFH1 (Figure 1A). The wild-

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type sequence of genotype(isolate) 5’UTR-NS5A fused to JFH1 NS5B-3’UTR are deposited in GenBank [1a(TN), KF589882; 1a(H77), KF589883; 3a(S52), KF589884; 4a(ED43), KF589885; 5a(SA13), KF589886; 6a(HK6a), KF589887]. The final adapted recombinants showed efficient virus

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spread in transfection cultures (Figure 1B; see Results for GenBank accession numbers). Sequence analysis of the passage-recovered viruses is shown in Supplementary Tables 1-5.

F1464L/A1672S/D2929G.12

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b, aa positions are according to genotype 1a strain H77 (AF009606). LSG,

c, representative peak HCV infectivity titers determined on filtered cell-culture supernatant collected on the indicated day post-transfection or post-infection; HCV RNA titers in passaged viruses were from the day with peak FFU titer.

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d, GenBank accession number KF134006.

e, first-passage viruses collected from day 3, 5, and 7 (4.9 log10 FFU/ml) for 1a(TN) and from day 16 and 18 (4.1 log10 FFU/ml) for 1a(H77) 5-5A viruses were used for treatment assays (Figure 2A).

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f, HCV positive cells emerged at day 4 post-transfection.

and 3).

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g, virus pool collected at the days indicated, subsequently used for drug treatment assays (Figure 2A

h, in a separate second-passage, virus pool from days 14, 16 and 18 had infectivity titer of 3.9 log10 FFU/ml and RNA titer of 8.3 log10 IU/ml. n.d., not determined.

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Table 2. The EC50s of protease inhibitors against JFH1-based HCV recombinant viruses of different genotypes compared to TN (genotype 1a) full-length viruses. EC50 was calculated from the concentration-response curves in the treatment experiments (Figure 2).

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1a(TN) full-length11 and NS3P recombinants8 developed previously were included for comparison. J6/JFH124 was included as control in each experiment. Fold differences from 1a(TN) full-length (TN_FL) virus for each inhibitor was the EC50 of the respective recombinant relative to the EC50 of

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TN_FL virus. In the development of these PIs, the EC50s reported against genotype 1b(Con1) replicons for telaprevir (400 nM),17 boceprevir (200 nM),18 faldaprevir (7 nM)22, and MK-5172 (2

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nM)23 have a <3-fold difference from that found here for the 1a(TN and H77) 5-5A viruses, while the EC50s for asunaprevir (1.2 nM)19 were ~25-50-fold lower. For simeprevir (8 nM)20 and vaniprevir (5 nM),21 the EC50s against 1b(Con1) replicons were closer to 1a(H77) than 1a(TN) 5-5A viruses.

a, J6/JFH1 was control in each treatment; no mutations in the ORF of viruses used.

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b, from the same treatment experiment for respective drugs. Table 3. The EC50s of NS5A inhibitor daclatasvir against JFH1-based HCV recombinant

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viruses of different genotypes compared to 1a(TN) full-length viruses. See Table 2 legend for details. The reported EC50 for genotype 1b (Con1) replicon was 0.009 nM.25

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a, the previously reported EC50 values against 1a(TN) full-length6 and NS5A recombinants9 were included for comparison.

b, J6/JFH1 was control in each experiment.

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References

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1. Bartenschlager R, Lohmann V, Penin F. The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection. Nat Rev Microbiol 2013;11:482-496. 2. Zeuzem S. Interferon-based therapy for chronic hepatitis C: current and future perspectives. Nat Clin Pract Gastroenterol Hepatol 2008;5:610-622. 3. World Health Organization. Global distribution of HCV genotypes. Web site: http://www who int/vaccine_research/documents/ViralCancer7 pdf 2009;, Accessed 2013.

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4. Antaki N, Craxi A, Kamal S et al. The neglected hepatitis C virus genotypes 4, 5 and 6: an international consensus report. Liver Int 2010;30:342-355.

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5. Sarrazin C, Hezode C, Zeuzem S et al. Antiviral strategies in hepatitis C virus infection. J Hepatol 2012;56 Suppl 1:S88-100. 6. Ramirez S, Li YP, Jensen SB et al. Highly efficient infectious cell culture of three HCV genotype 2b strains and sensitivity to lead protease, NS5A, and polymerase inhibitors. Hepatology 2013;doi: 10.1002/hep.26660. [Epub ahead of print]. 7. Li YP, Gottwein JM, Scheel TK et al. MicroRNA-122 antagonism against hepatitis C virus genotype 1-6 and reduced efficacy by host RNA insertion or mutations in the HCV 5'UTR. Proc Natl Acad Sci U S A 2011;108:4991-4996.

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8. Gottwein JM, Scheel TK, Jensen TB et al. Differential Efficacy of Protease Inhibitors Against HCV Genotypes 2a, 3a, 5a, and 6a NS3/4A Protease Recombinant Viruses. Gastroenterology 2011;141:1067-1079. 9. Scheel TK, Gottwein JM, Mikkelsen LS et al. Recombinant HCV Variants with NS5A from Genotypes 1-7 Have Different Sensitivities to an NS5A Inhibitor but Not Interferon-alpha. Gastroenterology 2011;140:1032-1042.

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10. Gottwein JM, Jensen SB, Li YP et al. Combination Treatment with Hepatitis C Virus Protease and NS5A Inhibitors Is Effective against Recombinant Genotype 1a, 2a, and 3a Viruses. Antimicrob Agents Chemother 2013;57:1291-1303.

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11. Li YP, Ramirez S, Jensen SB et al. Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system. Proc Natl Acad Sci U S A 2012;109:19757-19762. 12. Li YP, Ramirez S, Gottwein JM et al. Robust full-length hepatitis C virus genotype 2a and 2b infectious cultures using mutations identified by a systematic approach applicable to patient strains. Proc Natl Acad Sci U S A 2012;109:E1101-E1110. 13. Yanagi M, Purcell RH, Emerson SU et al. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci U S A 1997;94:8738-8743. 14. Gottwein JM, Scheel TK, Callendret B et al. Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies. J Virol 2010;84:5277-5293. 25

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15. Bukh J, Meuleman P, Tellier R et al. Challenge pools of hepatitis C virus genotypes 1-6 prototype strains: replication fitness and pathogenicity in chimpanzees and human liver-chimeric mouse models. J Infect Dis 2010;201:1381-1389.

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16. Yi M, Villanueva RA, Thomas DL et al. Production of infectious genotype 1a hepatitis C virus (Hutchinson strain) in cultured human hepatoma cells. Proc Natl Acad Sci U S A 2006;103:23102315. 17. Lin C, Lin K, Luong YP et al. In vitro resistance studies of hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061: structural analysis indicates different resistance mechanisms. J Biol Chem 2004;279:17508-17514.

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18. Tong X, Bogen S, Chase R et al. Characterization of resistance mutations against HCV ketoamide protease inhibitors. Antiviral Res 2008;77:177-185.

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19. McPhee F, Sheaffer AK, Friborg J et al. Preclinical Profile and Characterization of the Hepatitis C Virus NS3 Protease Inhibitor Asunaprevir (BMS-650032). Antimicrob Agents Chemother 2012;56:5387-5396. 20. Lin TI, Lenz O, Fanning G et al. In vitro activity and preclinical profile of TMC435350, a potent hepatitis C virus protease inhibitor. Antimicrob Agents Chemother 2009;53:1377-1385. 21. Liverton NJ, Carroll SS, Dimuzio J et al. MK-7009, a potent and selective inhibitor of hepatitis C virus NS3/4A protease. Antimicrob Agents Chemother 2010;54:305-311. 22. White PW, Llinas-Brunet M., Amad M. et al. Preclinical characterization of non covalent HCV NS3/4A protease inhibitor BI201335. J Hepatol 2010;52:S302.

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23. Summa V, Ludmerer SW, McCauley JA et al. MK-5172, a selective inhibitor of hepatitis C virus NS3/4a protease with broad activity across genotypes and resistant variants. Antimicrob Agents Chemother 2012;56:4161-4167. 24. Lindenbach BD, Evans MJ, Syder AJ et al. Complete replication of hepatitis C virus in cell culture. Science 2005;309:623-626.

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25. Gao M, Nettles RE, Belema M et al. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature 2010;465:96-100.

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26. Schmitt M, Scrima N, Radujkovic D et al. A comprehensive structure-function comparison of hepatitis C virus strain JFH1 and J6 polymerases reveals a key residue stimulating replication in cell culture across genotypes. J Virol 2011;85:2565-2581. 27. Murayama A, Weng L, Date T et al. RNA polymerase activity and specific RNA structure are required for efficient HCV replication in cultured cells. PLoS Pathog 2010;6:e1000885. 28. Murayama A, Date T, Morikawa K et al. The NS3 helicase and NS5B-to-3'X regions are important for efficient hepatitis C virus strain JFH-1 replication in Huh7 cells. J Virol 2007;81:8030-8040. 29. Peng B, Yu M, Xu S et al. Development of robust hepatitis C virus genotype 4 subgenomic replicons. Gastroenterology 2013;144:59-61. 30. Dvory-Sobol H, Pang PS, Glenn JS. The Future of HCV Therapy: NS4B as an Antiviral Target. Viruses 2010;2:2481-2492. 26

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31. Suzuki Y, Ikeda K, Suzuki F et al. Dual oral therapy with daclatasvir and asunaprevir for patients with HCV genotype 1b infection and limited treatment options. J Hepatol 2013;58:655-662. 32. Schaefer EA, Chung RT. Anti-hepatitis C virus drugs in development. Gastroenterology 2012;142:1340-1350.

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33. Imhof I, Simmonds P. Development of an intergenotypic hepatitis C virus (HCV) cell culture method to assess antiviral susceptibilities and resistance development of HCV NS3 protease genes from HCV genotypes 1 to 6. J Virol 2010;84:4597-4610.

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Author names in bold designate shared co-first authorship.

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Table 1

Day with

Genotype(isolate)

1a(H77) 3a(S52)

4a(ED43)

cells

LSG/A1226G/Q1773H

4

LSG/A1226G/Q1773H

e

8

log10(FFU/ml)

(day) c

(day) c

3.9 (8)

4.8 (7)

2.4 (13) f

LSG

41

LSG/D871G/H1819R

13 f

LSG/H1819R

22

LSG/D871G/V1612E/H1819R/V2417A, exp. 1

11

LSG/D871G/V1612E/H1819R/V2417A, exp. 2

7 f

LSG

LSG/S294G/C1551F, exp. 2

8.1

8.6 7.7

3.7 (8)

8.9

3.5 (25)

n.d.

n.d.

3.6 (15)

4.1 (14)

8.5

4.0 (11)

4.3 (12)

7.2 g,h

3.9 (11,13)

7.8h

7

3.8 (7)

3.2 (15)

8.1

7

3.2 (7)

3.1 (15)

8.2

8

3.2 (13)

4.8 (5)

n.d.

7

2.8 (11)

5.1 (7,9,12)

3

4.5 (6)

4.9 (9)

3

4.5 (6)

4.9 (9)

g

8.1 7.9 8.1

g

64

2.5 (70)

3.8 (14,18)

7.4

LSG/T387I/S872P/V1550L/L1790M/S2218P, exp. 1

9

3.3 (9)

3.8 (22)

8.4

LSG/T387I/S872P/V1550L/L1790M/S2218P, exp. 2

7

3.4 (7)

3.6 (26)

8.3

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LSG

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LSG, exp. 2

4.5 (5,7,10)

g

log10(IU/ml)c

3.3 (13)

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LSG, exp. 1

LSG/S294G/C1551F, exp. 1

2.5 (44)

4.3 (11)

Peak

2.4 (52)

50

LS/R781W/A1309P/A1786V, exp. 2

6a(HK6a)

log10(FFU/ml)

infected d,e

LS/R781W/A1309P/A1786V, exp. 1

5a(SA13)

Peak

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Mutations engineered

recombinanta 1a(TN)

Peak

≥80%

b

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Second-passage

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Table 2

Genotype(isolate)specific HCV

Boceprevir (SCH503034)

Asunaprevir (BMS-650032)

Simeprevir (TMC435350)

2a(J6/JFH1)

EC50 (nM)

Fold to TN_FL

EC50 (nM)

Fold to TN_FL

EC50 (nM)

Fold to TN_FL

EC50 (nM)

Fold to TN_FL

EC50 (nM)

Fold to TN_FL

EC50 (nM)

Fold to TN_FL

149

-

65

-

64

-

29

16

-

20

b

644

4.3

399

6.1

159

265

1.8

203

3.1

64

456

3.1

425

6.5

31

JFH1-based HCV 5'UTR-NS5A 1a(TN) 1a(H77)

b

2.7

b

88

-

0.9

-

5.5

b

87

4.4

1.6

1.8

1.0

45

1.6

21

1.3

22

1.1

3

3.3

0.5

10

0.3

15

0.9

7

0.4

3

3.3

b

493

3.3

405

6.2

3a(S52)

2000

13.4

1215

18.7

4a(ED43)

1949

13.1

1387

21.3

5a(SA13)

539

3.6

403

6.2

6a(HK6a)

124

0.8

141

2.2

2a(J6/JFH1)a

608

4.1

3a(S52)

2745

18.4

5a(SA13)

695

4.7

6a(HK6a)

412

2.8

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-

77

2a(J6/JFH1)

NS3/NS4A protease

b

2.5

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a

MK-5172

Fold to TN_FL

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a

Faldaprevir (BI201335)

EC50 (nM)

Full-length HCV 1a(TN)

Vaniprevir (MK-7009)

Phase II trials

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Telaprevir (VX-950)

Phase III trials

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Licensed for clinical use

b

159

2.5

91

3.1

88

5.5

87

4.4

3.3

3.7

2143

33.5

2476

85.4

1900

118.8

1512

75.6

20

22.2

37

0.6

5

0.2

10

0.6

2

0.1

1

1.1

82

1.3

109

3.8

26

1.6

11

0.6

3

3.3

55

0.9

56

1.9

17

1.1

21

1.1

2

2.2

7.7

67

1.0

77b

2.7

64

4.0

54

2.7

2.9

3.2

1417

21.8

3721

58.1

2366

81.6

1599

99.9

1832

91.6

59

65.6

875

13.5

79

1.2

127

4.4

32

2.0

14

0.7

5

5.6

435

6.7

96

1.5

78

2.7

36

2.3

8

0.4

7

7.8

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Table 3 EC50 (nM)

Fold to TN_FL

0.03 0.09

3.0

5'UTR-NS5A 1a(TN) 1a(H77) 2a(J6/JFH1)b 3a(S52) 4a(ED43) 5a(SA13) 6a(HK6a)

0.06 0.05 0.09 0.54 0.02 0.03 0.07

2.0 1.7 3.0 18.0 0.7 1.0 2.3

NS5Aa 1a(TN) 1a(H77) 2a(J6/JFH1)b 3a(S52) 4a(ED43) 5a(SA13) 6a(HK6a)

0.04 0.03 0.10 0.91 0.02 0.04 0.03

1.3 1.0 3.3 30.3 0.7 1.3 1.0

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Genotype(isolate)-specific HCV Full-length HCV 1a(TN)a 2a(J6/JFH1)b

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JFH1-based HCV

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Supplementary Table 1. Sequence analysis of the 1a(H77) and 1a(TN) 5’UTR-NS5A recombinant viruses.

6279 6279 A . . a/G

9277 9277 A G G G

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5355 5355 G T T T

5660 5660 A C C C

1672 1672 A-S

1773 1773 Q-H

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NS4A NS4B NS5A NS5B

2979 2979 D-G

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HCV Passage (day) E2 p7 NS2 NS3 NS3 NS3 Nucleotide position Recombinant specific 2494 2584 3006 4018 4443 4731 H77 reference (AF009606) 2494 2584 3006 4018 4443 4731 Recombinant nucleotide A T A C T T 1a(TN)_LSG/A1226G/Q1773H 1st (3, 5, 7)a . t/C A/G G . C 1a(H77)_LSG/A1226G/Q1773H 1st (10) . . . G T/C C 1st (16, 18)a A/G . . G T/C C Amino acid position Recombinant specific 718 748 889 1226 1368 1464 H77 reference (AF009606) 718 748 889 1226 1368 1464 Amino acid change T-C L-S T-A A-G S-P F-L

1980 1980 I-V

One milliliter of transfection-derived virus was passaged to naïve Huh7.5 cells (~4x105 cells), and filtered culture supernatant collected after peak infection (≥80% culture cells infected) was subjected to ORF sequence analysis. Primers used for RT-PCR haved been described

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previously.1-3 Nucleotide and amino acid positions of the specific recombinant with mutations are listed; the corresponding position of genotype 1a strain H77 (GenBank Accession No. AF009606) is given. Shading indicates the engineered mutations; the J6-derived LSG

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(F1464L, A1672S, and D2979G) mutations4 are shown in dark shading and the passaged recombinant-derived mutations are shown in light shading. Coding mutations identified in direct sequencing are listed; two capital letters separated by a slash indicates a nucleotide

AC C

quasispecies (50/50), while a capital letter separated from a lowercase letter indicates a dominant/minor ratio. Dots indicate identity with original sequence. Peak viral infectivity titers and associated RNA titers of the passage viruses are shown in Table 1.

1

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a, viruses collected from first-passage culture supernatants at the indicated days were pooled and used for antiviral treatment (Figures 2A

AC C

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SC

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and 3).

2

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Supplementary Table 2. Sequence analysis of the passage-recovered 3a(S52) 5’UTR-NS5A recombinant viruses.

Continued Table HCV Nucleotide position Recombinant specific H77 reference (AF009606) Recombinant nucleotide 3a(S52) 5'UTR-NS5A recombinant a +LSG

E2

E2

E2

NS2

NS2

NS3

NS3

NS3

NS3 NS4A NS4A

385 387 A

1366 1372 1583 1589 1936 1937 2921 2969 4535 4747 5191 5192 5347 5371 1368 1374 1585 1591 1935 1936 2905 2953 4519 4731 5175 5176 5331 5355 A T A A A A C A T T G T G G

. A/G . . . . .

. T/g . . T/g A/G A/G . . . . . . T/G . . T/g/c . . T/g .

A/c A/C . . . . .

. . . . A/G . A/g

. A/G . . . . .

C/A C/a . . . . .

A/G . G G . G G

16 16 I-V

343 345 415 343 345 415 T-A F-V/L N-S

417 417 N-T

533 532 N-D

533 532 N-S

861 855 A-D

877 1399 1470 1618 1618 1670 1678 871 1393 1464 1612 1612 1664 1672 D-G F-S F-L V-M V-E G-R A-S

. T/C . . . . .

C C C C C C C

G/a G/A . . . . .

. T/a . T/A . A A

. . G/A . . . .

T T T T T T T

NS4B NS4B NS4B NS4B NS4B NS4B NS5A NS5A NS5A NS5A NS5A NS5A NS5A NS5B NS5B

. A/G . . . . .

5663 5647 T

+LSG/H1819R +LSG/D871G/V1612E/H1819R/V2417A, exp. 1 +LSG/D871G/V1612E/H1819R/V2417A, exp. 2 Amino acid position

5730 5714 G

5813 5797 A

5814 5798 T

6269 6253 C

6740 6724 T

6926 6910 T

7183 7167 A

7597 7569 A

7601 7573 A

7607 7579 A

7619 7591 T

8653 8625 G

9305 9277 A

. G/A . . . . .

A/Gc . G G G G G

T/Ac T/A . . . . .

. . T . . . .

. T/C . . . . .

. . C . . . .

A/G A/G . . . . .

. . a/G . . . .

A/g a/G . . . . .

. . . A/t A/G . .

T/c T/c . T/C . C C

. g/A . . . . .

G G G G G G G

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5567 5551 A

T/C T/C . . . C C

AC C

+LSG/D871G/H1819R

E2

SC

+LSG/H1819R +LSG/D871G/V1612E/H1819R/V2417A, exp. 1 +LSG/D871G/V1612E/H1819R/V2417A, exp. 2 Amino acid position Recombinant specific H77 reference (AF009606) Amino acid change

E1

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+LSG/D871G/H1819R

1st (15) 2nd (5,7,10)b 1st (17) 2nd (14) 1st (14) 2nd (14) 2nd (14)

E1

RI PT

Passage (day) Core

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HCV Nucleotide position Recombinant specific H77 reference (AF009606) Recombinant nucleotide 3a(S52) 5'UTR-NS5A recombinant a +LSG

3

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Recombinant specific H77 reference (AF009606) Amino acid change

1743 1737 Q-R

1775 1769 V-A

1797 1791 M-I

1825 1819 H-R

1825 1819 H-Q

1977 1971 P-L

2134 2128 V-A

2196 2190 L-P

2282 2276 K-E

2420 2410 S-G

2421 2423 2427 2411 2413 2417 D-G E-V/G V-A

2772 2762 A-T

2989 2979 D-G

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For details see legend of Supplementary Table 1. One milliliter of transfection- or first passage-derived virus was passaged to naïve Huh7.5 cells (~4x105 cells). Primers used for RT-PCR have been described previously.1,5 Peak viral infectivity titers and associated RNA titers of

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the passage viruses are shown in Table 1.

a, in first-passage 3a(S52)5-5A_LSG, six 50/50 quasispecies mutations were identified, coding for changes at five aa positions, A855A/D

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and D871D/G (NS2), V1769V/A and H1819H/R/Q (NS4B), and K2276K/E (NS5A). Clonal analysis of PCR products covering NS2 and NS4B mutations revealed that the D871G/H1819R combination appeared in 3 of 8 clones, while A855D/V1769A, A855D/H1819R, A855D/H1819Q, H1819Q, and wild-type were each found in 1 clone. We thus engineered D871G/H1819R and H1819R into 3a(S52)55A_LSG. The 3a(S52)5-5A_LSG/D871G/H1819R culture showed 1% HCV positive cells on day 1 post-transfection and reached peak

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infection at day 13 with an infectivity titer of 103.3 FFU/ml. In contrast, 3a(S52)5-5A_LSG/H1819R was HCV negative until day 4 and had delayed spread (Table 1). Two 50/50 quasispecies changes V1612V/E (NS3-helicase) and V2417V/A (NS5A-domain-III) identified in

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second-passage 3a(S52)5-5A_LSG/D871G/H1819R virus were introduced back to the genome to make the final recombinant 3a(S52)5-

AC C

5A_LSG/D871G/V1612E/H1819R/V2417A (KF134008) (see Results). The mutations identified here are different from those reported in the 3a(S52)6 and 3a(S310)7 replicon systems.

b, a virus stock made from second-passage supernatants collected at days 5, 7, and 10 was used for antiviral treatment (Figures 2A and 3). c, cloning analysis of PCR products (8 clones) showed that these mutations did not co-exist. 4

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Supplementary Table 3. Sequence analysis of the 4a(ED43) 5’UTR-NS5A recombinant viruses. NS2

NS2

NS3

2681 2682 C

2819 2820 A

2946 2947 C

4265 4266 G

C/T C/T C/T T

A/g A/g a/G .

C/T C/T C/T .

C C C C

781 781 R-W

For details see legends of Supplementary Table 1 and 2.

827 827 T-A

869 869 P-L

NS3

1309 1309 A-P

NS4A

RI PT

1st (13) 2nd (11, 13)c 2nd (14, 16, 18)d 2nd (13)

p7

NS4B

NS4B

NS5A

NS5A

NS5B

4730 4731 T

5354 5355 G

5697 5698 C

5729 5730 G

6431 6432 T

7134 7144 T

9267 9277 A

C C C C

T T T T

T T T T

G/a G/A G/A .

T/c T/C . .

T/C T/C T/C .

A/G . . .

1464 1464 F-L

1672 1672 A-S

1786 1786 A-V

1797 1797 V-I

2031 2031 C-R

2265 2268 V-A

2976 2979 D-G

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+LS/R781W/A1309P/A1786V, exp. 1 Amino acid position Recombinant specific H77 reference (AF009606) Amino acid change

Passage (day)

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HCV Nucleotide position Recombinant specific H77 reference (AF009606) Recombinant nucleotide 4a(ED43) 5’UTR-NS5A recombinanta +LSG

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a, In ORF sequence analysis of one first-passage and two independent second-passage 4a(ED43)5-5A_LSG viruses, the “G” of LSG had reverted to 50/50 quasispecies in first-passage virus (nt A9267A/G in dark shading) and to wild-type sequence only in two second-passage

EP

viruses. Two complete changes (A1309P and V1797I) and four 50/50 quasispecies aa changes (R781R/W, P869P/L, V1797V/I, and

AC C

V2268V/A) were found in at least two of these three viruses. Clonal analysis of PCR products spanning the p7 and NS2 mutations, amplified from the first-passage 4a(ED43)_LSG, showed that R781W, P869L, and wild-type were found in 7 clones, 2 clones, and 1 clone, respectively. In clonal analysis of NS4B and NS5A mutations, V1797I/V2268A, V1797I, V2268A, and wild-type were found in 4 clones, 2 clones, 3 clones, and 3 clones, respectively. We thus engineered three sets of mutations, A1309P/A1786V (two complete changes in 5

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sequenced viruses; A1309A/P was also identified in an ED43 replicon6), R781W/A1309P/A1786V, and P869L/A1309P/A1786V, into a

RI PT

4a(ED43)5-5A_LS genome. The 4a(ED43)5-5A_LS/A1309P/A1786V and 4a(ED43)5-5A_LS/P869L/A1309P/A1786V cultures were HCV positive but spread to only 50% of culture cells at day 16. In contrast, 4a(ED43)5-5A_LS/R781W/A1309P/A1786V (KF134009) reached ≥80% at day 7 in two independent transfections (Figure 1B and Table 1). We also tested these three sets of mutations in the

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5A_LS/R781W/A1309P/A1786V was most efficient recombinant (see Results).

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original 4a(ED43)5-5A_LSG recombinant; all showed only ≤10% HCV positive cells after 30 days of follow-up. Thus, the 4a(ED43)5-

c, a virus stock made from a second-passage supernatants collected at days 11 and 13 (Table 1) was used for antiviral treatment (Figures 2A and 3).

AC C

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d, a virus stock made from a separate second-passage experiment; supernatants were collected at days 14, 16 and 18 (Table 1 legend).

6

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Supplementary Table 4. Sequence analysis of the 5a(SA13) 5’UTR-NS5A recombinant viruses.

+LSG/S294G/C1551F, exp. 1 Amino acid position Recombinant specific H77 reference (AF009606) Amino acid change

NS2

NS3

NS3

NS4A

NS5B

1219 1221 A

2828 2827 T

4732 4731 T

4994 4993 G

5356 5355 G

9284 9277 A

A/g A/g A/g a/G G

T/c T/C . . .

C C C C C

. . G/t T T

T T T T T

G G G G G

294 294 S-G

830 829 F-S

1465 1464 F-L

1552 1551 C-F

1673 1672 A-S

2982 2979 D-G

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1st (15) 2nd (5) 1st (10) 2nd (7, 9 ,12)b 2nd (5)

E1

SC

+LSG, exp. 2

Passage (day)

M AN U

HCV Nucleotide position Recombinant specific H77 reference (AF009606) Recombinant nucleotide 5a(SA13) 5’UTR-NS5A recombinanta +LSG, exp. 1

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For details see legend of Supplementary Table 1 and 2. Primers used for RT-PCR were previously described 8-10. a, 5a(SA13)5-5A_LSG viruses from two independent transfections (Table 1) were first- and second-passaged and ORFs of recovered

EP

viruses were sequenced.

b, a virus stock (105.1 FFU/ml) made from second-passage supernatants collected at days 7, 9, and 12 was used for antiviral treatment

AC C

(Figures 2A and 3). A dominant aa change S294G and a complete aa change C1551F (NS3-helicase) found in this virus were engineered back to the 5a(SA13)5-5A_LSG to make 5a(SA13)5-5A_LSG/S294G/C1551F (KF134010), which was the most efficient and stable 5a(SA13) 5’UTR-NS5A recombinant (see Results).

7

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Supplementary Table 5. Sequence analysis of the 6a(HK6a) 5’UTR-NS5A recombinant viruses. Passage (day)

E1

E2

NS2

NS2

NS3

NS3 NS4A NS4B NS4B NS5A NS5B

RI PT

HCV Nucleotide position Recombinant specific H77 reference (AF009606) Recombinant nucleotide 6a(HK6a) 5’UTR-NS5A recombinant +LSG

1389 1502 2974 3139 4750 5008 5374 5503 5728 7012 9305 1385 1501 2955 3120 4731 4989 5355 5484 5709 6993 9277 A C T A T G G A T T A c/T T T

C C C

. . a/G

349 348 I-M

387 387 T-I

878 872 S-P

933 927 R-G

C C C

SC

. . G

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+LSG/T387I/S872P/V1550L/L1790M/S2218P, exp. 1 Amino acid position Recombinant specific H77 reference (AF009606) Amino acid change

1st (42) 2nd (14, 18)a 2nd (22)

T T T

T T T

. . T

A A A

C C C

G G G

1470 1556 1678 1721 1796 2224 2988 1464 1550 1672 1715 1790 2218 2979 F-L V-L A-S I-F L-M S-P D-G

For details see legends of Supplementary Table 1 and 2. One milliliter of transfection- or first passage-derived virus was passaged to naïve 8,10,11

. Peak viral infectivity titers and associated

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Huh7.5 cells (~4x105 cells). Primers used for RT-PCR have been described previously RNA titers of the passage viruses are shown in Table 1.

a, a virus stock made from second-passage supernatants collected at days 14 and 18 was used for antiviral treatment (Figures 2A and 3).

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Five mutations found in this virus stock, as well as the first-passage virus, coding for aa change T387I, S872P, V1550L (NS3-helicase),

AC C

L1790M, and S2218P (NS5A-low-complexity-sequence-I) were introduced into 6a(HK6a)5-5A_LSG to make 6a(HK6a)55A_LSG/T387I/S872P/V1550L/L1790M/S2218P (KF134011), which had efficient growth in vitro (see Results).

8

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References

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1. Gottwein JM, Scheel TK, Hoegh AM et al. Robust hepatitis C genotype 3a cell culture releasing adapted intergenotypic 3a/2a (S52/JFH1) viruses. Gastroenterology 2007;133:1614-1626.

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2. Sakai A, Takikawa S, Thimme R et al. In vivo study of the HC-TN strain of hepatitis C virus recovered from a patient with fulminant hepatitis: RNA transcripts of a molecular clone (pHC-TN) are infectious in chimpanzees but not in Huh7.5 cells. J Virol 2007;81:7208-7219. 3. Yanagi M, Purcell RH, Emerson SU et al. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci U S A 1997;94:8738-8743. 4. Li YP, Ramirez S, Jensen SB et al. Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture system. Proc Natl Acad Sci U S A 2012;109:19757-19762.

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5. Gottwein JM, Scheel TK, Callendret B et al. Novel infectious cDNA clones of hepatitis C virus genotype 3a (strain S52) and 4a (strain ED43): genetic analyses and in vivo pathogenesis studies. J Virol 2010;84:5277-5293. 6. Saeed M, Scheel TK, Gottwein JM et al. Efficient replication of genotype 3a and 4a hepatitis C virus replicons in human hepatoma cells. Antimicrob Agents Chemother 2012;56:5365-5373.

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7. Saeed M, Gondeau C, Hmwe S et al. Replication of hepatitis C virus genotype 3a in cultured cells. Gastroenterology 2013;144:56-58.

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8. Gottwein JM, Scheel TK, Jensen TB et al. Differential Efficacy of Protease Inhibitors Against HCV Genotypes 2a, 3a, 5a, and 6a NS3/4A Protease Recombinant Viruses. Gastroenterology 2011;141:1067-1079. 9. Jensen TB, Gottwein JM, Scheel TK et al. Highly efficient JFH1-based cell-culture system for hepatitis C virus genotype 5a: failure of homologous neutralizing-antibody treatment to control infection. J Infect Dis 2008;198:1756-1765. 10. Scheel TK, Gottwein JM, Mikkelsen LS et al. Recombinant HCV Variants with NS5A from Genotypes 1-7 Have Different Sensitivities to an NS5A Inhibitor but Not Interferon-alpha. Gastroenterology 2011;140:1032-1042. 9

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11. Gottwein JM, Scheel TK, Jensen TB et al. Development and characterization of hepatitis C virus genotype 1-7 cell culture systems: role of CD81 and scavenger receptor class B type I and effect of antiviral drugs. Hepatology 2009;49:364-377.

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A

J65’UTR-NS2/JFH1

5’UTR C

p7

E1

E2

NS2

NS3

NS4A NS4B

2a(J6)

2a(JFH1)

D2979G

J65'UTR-NS2/JFH1 Genotype(isolate)-specific 5'UTR-NS5A recombinant with mutations 4a(ED43)_LS/R871W/ A1309P/A1786V

5a(SA13)_LSG/S294G/ C1551F

6a(HK6a)_LSG/T387I/S872P/ V1550L/L1790M/S2218P

100 5

80

4

60

3

40

2

20

1 0

0 1

3

5

7

9

11 13 15

1

3

5

7

10 12 14

1

3

6

8

11

1

3

5

7

9

F F U /m l( lo g 1 0 ) ( b a r s )

p o s itiv e c e lls ( lin e s )

3a(S52)_LSG/D871G/V1612E/ H1819R/V2417A % H C V a n tig e n

NS5B

A1672S F1464L

Genotype(isolate) specific 5’UTR-NS5A recombinant with LSG mutations

B

3'UTR NS5A

12 14

Day post transfection Figure 1. Characteristics of HCV genotypes 3a, 4a, 5a, and 6a specific 5’UTR-NS5A (5-5A) recombinants in transfected Huh.7.5 cells. (A) Schematic diagram of J65’UTR-NS2/JFH17 and HCV 5-5A recombinants. LSG mutations (F1464L/A1672S/D2979G) are indicated. (B) RNA transcripts of 5-5A recombinants with indicated mutations were transfected into Huh7.5 cells, and the percentage of HCV Core and/or NS5A positive cells was estimated (left y-axis; lines). HCV infectivity titers in culture supernatants at peak infection were 22

determined (mean of triplicate infections ± SEM, right y-axis; bar). J65’UTR-NS2/JFH17 was control. Duplicate transfection experiments performed for these recombinants yielded similar results. See Table 1 for details on transfection and second-passage viruses.

23

C o n tro ls :

r e la tiv e to n o n tre a te d c o n tro l

1 a (T N )_ F L

2 a ( J 6 /J F H 1 )

5 'U T R - N S 5 A r e c o m b in a n ts : 1 a (H 7 7 ) 1 a (T N ) 3 a (S 5 2 )

4 a (E D 4 3 )

5 a (S A 1 3 )

6 a (H K 6 a )

120

120

120

120

100

100

100

100

80

80

80

80

60

60

60

60

40

40

40

40

20

20

20

20

r e la tiv e to n o n tre a te d c o n tro l

% H C V a n tig e n p o s itiv e c e lls

% H C V a n tig e n p o s itiv e c e lls

A

0 0 1 2 3 4 0 1 2 3 4 B o c e p r e v ir (S C H 5 0 3 0 3 4 ) ( lo g 1 0 n M ) A s u n a p re v ir (B M S -6 5 0 0 3 2 ) (lo g 1 0 n M ) 0

0 0 1 2 3 4 T e la p re v ir (V X -9 5 0 ) (lo g 1 0 n M )

120

120

120

100

100

100

80

80

80

60

60

60

40

40

40

20

20

20

0

0

0

0 1 2 3 4 V a n ip re v ir (M K - 7 0 0 9 ) (lo g 1 0 n M )

C o n tro l:

B % H C V a n tig e n p o s itiv e c e lls

r e la tiv e to n o n tre a te d c o n tro l

% H C V a n tig e n p o s itiv e c e lls

r e la tiv e to n o n tre a te d c o n tro l

2 a ( J 6 /J F H 1 )

0

0 1 2 3 4 F a ld a p r e v ir ( B I2 0 1 3 3 5 ) ( lo g 1 0 n M )

1 2 3 M K - 5 1 7 2 ( lo g 1 0 n M )

0 0

1

2

3

4

S im e p re v ir (T M C 4 3 5 3 5 0 ) ( lo g 1 0 n M )

4

N S 3 /N S 4 A - p ro te a s e re c o m b in a n ts : 3 a (S 5 2 )

5 a (S A 1 3 )

6 a (H K 6 a )

120

120

120

120

100

100

100

100

80

80

80

80

60

60

60

60

40

40

40

40

20

20

20

20

0

0

0

0

0 1 2 3 4 T e la p re v ir (V X -9 5 0 ) (lo g 1 0 n M )

0 1 2 3 4 0 1 2 3 4 B o c e p r e v ir (S C H 5 0 3 0 3 4 ) ( lo g 1 0 n M ) A s u n a p re v ir (B M S -6 5 0 0 3 2 ) (lo g 1 0 n M )

120

120

120

100

100

100

80

80

80

60

60

60

40

40

40

20

20

20

0

0

0

0 1 2 3 4 V a n ip re v ir (M K - 7 0 0 9 ) (lo g 1 0 n M )

0 1 2 3 4 F a ld a p r e v ir ( B I2 0 1 3 3 5 ) ( lo g 1 0 n M )

0

0 1 2 3 4 S im e p re v ir (T M C 4 3 5 3 5 0 ) ( lo g 1 0 n M )

1 2 3 4 M K - 5 1 7 2 ( lo g 1 0 n M )

Figure 2. HCV genotype 1-6-specific 5-5A recombinants showed differential sensitivity to protease inhibitors, similar to NS3P recombinants of respective genotypes. Huh7.5 cells in 96well plates were infected with the 5-5A (A) and NS3P8 (B) viruses, and treated with seven PIs (Materials and Methods). Experimental control 2a(J6/JFH1) were included in each treatment. Control 1a(TN)_FL (full-length)11 was incorporated in A. Values are means of triplicates in the experiment ± SEM. EC50s for each drug against the different genotype viruses are shown in Table 2. 24

C o n tro ls :

1 a (T N )_ F L

2 a ( J 6 /J F H 1 )

re la tiv e to n o n tre a te d c o n tro l

% H C V a n tig e n p o s itiv e c e lls

5 'U T R - N S 5 A r e c o m b in a n ts : 1 a (T N ) 1 a (H 7 7 ) 4 a (E D 4 3 )

5 a (S A 1 3 )

3 a (S 5 2 ) 6 a (H K 6 a )

120 100 80 60 40 20 0 -3

-2

-1

0

1

2

D a c la ta s v ir (B M S -7 9 0 0 5 2 ) (lo g 1 0 n M )

Figure 3. HCV genotype 1-6-specific 5-5A recombinants showed differential sensitivity to NS5A inhibitor daclatasvir. Cultures infected with 5-5A recombinant viruses were treated with daclatasvir. See legend of Figure 2 and Materials and Methods for details. EC50s are shown in Table 3.

25