Efficacy of NS5A Inhibitors Against Hepatitis C Virus Genotypes 1–7 and Escape Variants

Accepted Manuscript Efficacy of NS5A Inhibitors Against Hepatitis C Virus Genotypes 1–7 and Escape Variants Judith M. Gottwein, Long V. Pham, Lotte S. Mikkelsen, Lubna Ghanem, Santseharay Ramirez, Troels K.H. Scheel, Thomas H.R. Carlsen, Jens Bukh PII: DOI: Reference:

S0016-5085(17)36720-3 10.1053/j.gastro.2017.12.015 YGAST 61591

To appear in: Gastroenterology Accepted Date: 18 December 2017 Please cite this article as: Gottwein JM, Pham LV, Mikkelsen LS, Ghanem L, Ramirez S, Scheel TKH, Carlsen THR, Bukh J, Efficacy of NS5A Inhibitors Against Hepatitis C Virus Genotypes 1–7 and Escape Variants, Gastroenterology (2018), doi: 10.1053/j.gastro.2017.12.015. 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.

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Hepatitis C virus NS5A inhibitors pibrentasvir and velpatasvir show pangenotypic efficacy

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HCV genotype Size~EC50 1 5 2 6 3 7 4

Pibrentasvir showed highest efficacy against hepatitis C virus genotype 1-7 escape variants Sequencing of genotype 1-7 NS5A domain I

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ACCEPTED MANUSCRIPT Efficacy of NS5A Inhibitors Against Hepatitis C Virus Genotypes 1–7 and Escape Variants

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Short title: HCV Genotype 1-7 NS5A Inhibitor Resistance

Authors: Judith M. Gottwein*, Long V. Pham, Lotte S. Mikkelsen, Lubna Ghanem, Santseharay

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Ramirez, Troels K. H. Scheel, Thomas H. R. Carlsen# and Jens Bukh*

Affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and

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Clinical Research Centre, Hvidovre Hospital and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark

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Current address (#): Department of Virology, Novo Nordisk A/S, Denmark

Grant Support: This work was supported by grants from Region H Foundation (J.M.G., S.R., J.B.), The Lundbeck Foundation (J.M.G, S.R., J.B.), The Novo Nordisk Foundation (J.M.G., J.B.),

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and The Danish Council for Independent Research (DFF), Medical Sciences (J.M.G., S.R., T.K.H.S., J.B.). J.B. is the 2014 recipient of an advanced top researcher grant from DFF and the

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2015 recipient of the Novo Nordisk Prize.

Abbreviations: HCV, hepatitis C virus; DAA, direct-acting antiviral; aa, amino acid; FFU; focus forming unit; NS5A, nonstructural protein 5A; RAS, resistance-associated substitution; SEM, standard error of the mean.

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ACCEPTED MANUSCRIPT Correspondence (*): Judith Gottwein, M.D. ([email protected]) and Jens Bukh, M.D. ([email protected]); Mailing address: Department of Infectious Diseases #144, Hvidovre Hospital,

Disclosures: The authors disclose no conflicts.

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Kettegaard Alle 30, DK-2650 Hvidovre, Denmark.

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Author Contributions: study concept and design (J.M.G, T.K.H.S, J.B.), acquisition of data (J.M.G., L.V.P., L.S.M., L.G., S.R., T.C.), analysis and interpretation of data (J.M.G., L.V.P., S.R.,

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J.B.), drafting of the manuscript (J.M.G., J.B.), and study supervision (J.M.G., J.B.).

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Abstract Background & Aims: Inhibitors of the hepatitis C virus (HCV) NS5A protein are a key component of effective treatment regimens, but the genetic heterogeneity of HCV has limited the efficacy of

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these agents and mutations lead to resistance. We directly compared the efficacy of all clinically relevant NS5A inhibitors against HCV genotype 1–7 prototype isolates and resistant escape variants, and investigated the effects of pre-existing resistance-associated substitutions (RAS) on

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HCV escape from treatment.

Methods: We measured the efficacy of different concentrations of daclatasvir, ledipasvir,

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ombitasvir, elbasvir, ruzasvir, velpatasvir, and pibrentasvir in cultured cells infected with HCV recombinants expressing genotype 1–7 NS5A proteins with or without RAS. We engineered HCV variants that included RAS identified in escape experiments, using recombinants with or without T/Y93H and daclatasvir, or that contained RAS previously reported from patients.

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Results: NS5A inhibitors had varying levels of efficacy against original and resistant viruses. Only velpatasvir and pibrentasvir had uniform high activity against all HCV genotypes tested. RAS hotspots in NS5A were found at amino acids 28, 30, 31, and 93. Engineered escape variants had

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high levels of fitness. Pibrentasvir had the highest level of efficacy against variants; viruses with RAS at amino acids 28, 30, or 31 had no apparent resistance to pibrentasvir, and HCV with RAS at

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amino acid 93 had a low level of resistance to this drug. However, specific combinations of RAS and deletion of amino acid 32 led to significant resistance to pibrentasvir. For the remaining NS5A inhibitors tested, RAS at amino acids 28 and 93 led to high levels of resistance. Among these inhibitors, velpatasvir was more effective against variants with RAS at amino acid 30 and some variants with RAS at amino acid 31 than the other agents. Variants with the pre-existing RAS T/Y93H acquired additional NS5A changes during escape experiments, resulting in HCV variants with specific combinations of RAS, showing high fitness and high resistance.

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ACCEPTED MANUSCRIPT Conclusions: We performed a comprehensive comparison of the efficacy of the 7 clinically relevant inhibitors of HCV NS5A and identified variants associated with resistance to each agent.

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These findings could improve treatment of patients with HCV infection.

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KEY WORDS: liver disease; direct acting antiviral; DAA; drug resistance

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Introduction Almost all novel combination treatments against hepatitis C virus (HCV) include direct acting antivirals (DAA) targeting domain I of HCV NS5A,1-4 a protein with an essential role for viral

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replication and assembly.5-7 Since 2014, six NS5A-inhibitors, daclatasvir, ledipasvir, ombitasvir, elbasvir, velpatasvir, and pibrentasvir were approved for clinical use, while ruzasvir has been in advanced stages of clinical development. While approval was initially limited to treatment of HCV

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genotype 1 infected patients, it was subsequently expanded to treatment of patients infected with other major HCV genotypes.1-4,8,9 HCV is classified within the Flaviviridae family and has seven

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highly divergent major genotypes, further subdivided into subtypes.8-10 Up to 200 million individuals are estimated to be chronically infected with HCV and have an increased risk for liver cirrhosis and liver cancer, resulting in up to 750.000 deaths per year.11 Despite high success rates of DAA-based treatment regimens, a small percentage of

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patients experiences treatment failure.12,13 HCV resistance is an important cause of treatment failure and the majority of these patients harbor HCV resistant variants with resistance-associated substitutions (RAS) in the drug protein targets.12 Recent evidence suggests that NS5A RAS persist

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long-term in patients following treatment failure,12,14 facilitating spread of resistant variants in populations.15 In addition, NS5A RAS are found as naturally occurring polymorphisms in a

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relatively high percentage of treatment naïve patients.12,14 Clinical studies demonstrated that for various DAA-based treatment regimens pre-existing RAS result in lower cure rates.12,14,16 However, the virological correlates of treatment escape of HCV variants with pre-existing RAS have not been elucidated.

Given their frequency and impact on the success of DAA-based treatments, it is of great importance to identify and characterize NS5A RAS for the seven major HCV genotypes and important subtypes.8,9 In addition, it is essential to compare the efficacy of relevant NS5A-

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ACCEPTED MANUSCRIPT inhibitors against isolates of different genotypes and resistant variants. In vitro studies allow systematic head-to-head comparisons of DAA efficacy for a large number of DAA and HCV variants, and are required to determine the degree of resistance conferred by putative RAS

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identified in vitro or in patients. Such studies have mostly been done for HCV genotype 1, while data for other genotypes are limited.12,14,17-28 Even though NS5A inhibitors were shown to act on different steps of the viral life cycle, the majority of reported in vitro studies used replicon systems, only mimicking viral replication.17-26,28-30 In contrast, infectious culture systems reflect the entire

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viral life cycle,31 and data on efficacy of and resistance to NS5A-inhibitors obtained in these

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systems have proven to be in agreement with clinical data.20,27,32-39 However, limited data on NS5Ainhibitor treatment escape have been obtained in such systems.27,33,38

We aimed at providing an independent head-to-head comparison of the efficacy of all currently licensed HCV NS5A-inhibitors against the major HCV genotypes and important subtypes,

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as well as resistant escape variants, in infectious culture systems. We first determined the efficacy of daclatasvir, ledipasvir, ombitasvir, elbasvir, ruzasvir, velpatasvir and pibrentasvir against HCV recombinants with genotype 1-7 specific NS5A. To generate a panel of relevant resistant variants,

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we also used these genotype 1-7 recombinants to induce viral escape from treatment with the firstin-class NS5A-inhibitor daclatasvir.20 Engineered escape variants were characterized regarding

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fitness and resistance to NS5A-inhibitors. At last, we aimed at investigating the influence of preexisting RAS at NS5A position 93 on resistance development.

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Material and Methods HCV recombinant viruses. J6/JFH140, in this study designated 2a(JFH1), and J6/JFH1-based recombinants with genotype(isolate) 1a(H77), 1a(TN), 1a(DH6), 1a(HCV1), 1a(J1),

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1b(J4), 2a(J6), 3a(S52), 4a(ED43), 5a(SA13), 6a(HK6a) and 7a(QC69) specific NS5A and derived T/Y93H mutants and their respective GenBank IDs were reported previously.32 3a(S52) was further adapted by C2419R (aa positions in this paragraph are absolute H77 (GenBank ID AF009606)

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reference numbers, whereas in the following NS5A substitutions are indicated relative to H77 NS5A).41 For 2b(J8) (GenBank ID MG406988), the pJ8CF NS5A sequence34 was cloned into

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J6/JFH1. For 1b(DH1) (GenBank ID MG406987),42 the NS5A consensus sequence was determined by analysis of clones of reverse-transcription polymerase-chain-reaction amplicons and cloned into J6/JFH1 with adaptive substitutions R867H/C1185S, previously identified for 1b(J4).32 1b(DH1) and 2b(J8) yielded peak infectivity titers of 3.2-3.9 log10 focus forming units (FFU)/ml in viral

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passage cultures. Recombinants with NS5A mutations were engineered by standard cloning procedures. The HCV sequence of final plasmid preparations was confirmed (Macrogen, Seoul). Huh7.5 cell culture experiments. Huh7.5 cells were cultured as described.43 For

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induction of viral escape, 3.5x105 cells were plated per well of a 6-well plate. Cultures were infected with HCV containing culture supernatants. Every 2-3 days, cultures were split, treated with

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specified fold-EC50 concentrations of daclatasvir (BMS-790052), and immunostaining for HCV antigen was carried out. Transfections with lipofectamine 2000 (Invitrogen, Carlsbad) and viral passages to naïve cells for generation of virus stocks were done as described.44 Percentage of HCVantigen positive culture cells was determined by fluorescence microscopy following immunostaining with primary anti-Core antibody B2 (Anogen, Yes Biotech Laboratories, Mississauga).43 Culture supernatant infectivity titers were determined following infection of triplicate cultures with serially diluted supernatants, staining with a mix of primary anti-Core C7-50

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ACCEPTED MANUSCRIPT (1:450) (Enzo Life Sciences, Farmingdale and AbCam, Cambridge) and anti-NS3 H23 (1:1000) antibodies (Abcam), and automated FFU counting.32,45,46 Viral fitness of HCV NS5A variants compared to original recombinants of the same isolate was estimated by calculation of titer

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differences (log10 FFU/ml). In most instances where the original virus yielded peak titers earlier than the variant, the titer differences were calculated as (infectivity titer of the variant on the same day as the original virus achieved peak titer minus peak infectivity titer of the original virus). In

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instances where the variant achieved peak titer earlier than the original virus, the titer differences were calculated as (peak infectivity titer of variant minus titer of the original virus on the same

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day). Recombinants of the original virus and variants were transfected in the same experiment. Supernatants derived at the peak of infection from transfection cultures were used to inoculate naïve Huh7.5 cells. For treatment assays, virus stocks were generated from supernatants collected at the peak of infection from resulting first viral passage cultures or subsequent second viral passage

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cultures, and evaluated for genetic stability by sequencing of NS5A domain I. Direct sequence analysis of NS5A of HCV from cell culture supernatant. Procedures for RNA extraction from culture supernatant, reverse-transcription polymerase-chain-

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reaction, and direct sequence analysis have been described.43 The complete NS5A domain I sequence was analyzed. Primers are specified in Supplemental Table 1. In selected cases with

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potentially combined substitutions, amplicons were TOPO TA cloned (Invitrogen) and individual clones were sequenced. Analysis was done with Sequencher (GeneCodes, Ann-Arbor). High-throughput treatment assays and statistical analysis. The procedures have

been described in detail previously.32,46 In brief, Huh7.5 cells, plated at 6x103 cells per well on poly-D-lysine-coated 96-well plates (Nunc) the previous day, were infected with supernatants from viral passage cultures. 24 hrs post infection, cultures were treated with serial dilutions of NS5Ainhibitors daclatasvir (BMS-790052), ledipasvir (GS-5885), ombitasvir (ABT-267), elbasvir (MK-

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ACCEPTED MANUSCRIPT 8742), ruzasvir (MK-8408), velpatasvir (GS-5816), and pibrentasvir (ABT-530) (Acme Bioscience, Palo Alto), dissolved in dimethyl sulfoxide. 72 hours post infection, immunostaining was carried out as for infectivity titration. Cell viability was monitored using the CellTiter 96 AQueous One

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Solution Cell Proliferation Assay (Promega). Single HCV-antigen positive cells were counted automatically. Counts from treated cultures were related to means of counts from infected, nontreated cultures. Following transformation of X values, sigmoidal concentration-response curves

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were fitted [Y=Top/(1+10[log10EC50-X]*HillSlope)] to obtain EC50 values using GraphPad Prism 6. For each HCV variant and inhibitor, at least two independent treatment experiments were carried out.

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Initial experiments were typically carried out with one to two replicates per concentration. However, unless otherwise indicated, final experiments were carried out with three replicates per concentration; of these final experiments one representative experiment was used to calculate fold resistance values.

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Authors. All authors had access to the study data and reviewed and approved the final manuscript.

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Results Efficacy of NS5A-inhibitors against the seven major HCV genotypes and important subtypes. We determined the efficacy of daclatasvir, ledipasvir, ombitasvir, elbasvir,

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ruzasvir, velpatasvir and pibrentasvir against HCV recombinants with NS5A of HCV genotype 1-7 prototype isolates (Figure 1 and Supplemental Table 2).47,48 Compared to daclatasvir, ledipasvir was less efficient against genotypes 2 to 7, while ombitasvir was more efficient against genotypes 2, 3

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and 4, but less efficient against genotype 6. Compared to daclatasvir, elbasvir and ruzasvir showed improved efficacy against genotype 2b and 3, and velpatasvir and pibrentasvir showed improved

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efficacy against genotype 2 and 3. Specific isolates showed differences of up to 5 orders of magnitude in sensitivity to different inhibitors. The greatest EC50 differences between the least and most sensitive isolate were observed for ledipasvir (~6 log), followed by daclatasvir, ombitasvir, elbasvir and ruzasvir (3-4 log). The smallest EC50 differences were observed for velpatasvir (66-

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fold) and pibrentasvir (8-fold), thus showing uniform pangenotypic efficacy. Universal escape of HCV genotypes 1-7 from daclatasvir and identification of NS5A RAS. To identify relevant resistant variants, we induced viral escape from the first approved

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NS5A-inhibitor, daclatasvir.20 Initiation of treatment with 4- to 256-fold EC50 of daclatasvir typically led to decreases in the percentages of HCV-antigen positive cells. Subsequently, for most

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cultures an increase in the number of HCV-antigen positive cells to at least 80% was observed. All cultures showed viral escape, except 2b(J8) treated with 256-fold EC50 (Supplemental Figure 1A). We determined the consensus sequence of NS5A domain I of genotype 1-7 viruses

recovered at the time of viral escape to identify putative RAS (Figure 2A, Supplemental Tables 317). Across isolates, substitutions at position 28, 30, 31 and 93 were most frequent, however with isolate specific selection patterns. The complexity of substitution patterns varied greatly. For certain

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ACCEPTED MANUSCRIPT isolates single substitutions were selected, while for other isolates multiple different substitutions were found. Fitness of engineered HCV escape variants. We engineered 74 genotype 1-7

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variants with putative NS5A RAS, focusing on genotype 2-7, for which least data was published. Most variants showed relatively high fitness, evaluated by comparison of their spread kinetics and infectivity titers to that of the respective original viruses following transfection (Figure 3). Certain

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aa changes such as F28S or Y93H had differential impact on the fitness of different isolates. All variants except 2a(J6)T94K could be passaged to naïve cells. For all 73 first-passage viruses the

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engineered substitution(s) persisted, not showing signs of reversion to the wild-type sequence, and additional substitutions, suggesting a requirement for compensation of RAS-induced fitness cost, were only acquired in 4 passaged viruses (Figure 3). Analyzing 12 double mutants, V8A compensated for fitness impairment induced by L30H or P58L in 4a(ED43). S24T increased fitness

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of 2b(J8)Y93H and T99A increased fitness of 3a(S52)Y93H (Figure 3). Resistance of engineered HCV escape variants to daclatasvir. Virus stocks of 73 variants were tested for sensitivity to daclatasvir (Figure 4 and Supplemental Figure 2). No

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resistance (≤ 3-fold change in EC50) was found for 24 variants, low resistance (4- to 79-fold change in EC50) for 14 variants, intermediate resistance (80- to 999-fold change in EC50) for 23 variants,

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and high resistance (≥1.000-fold change in EC50) for 12 variants. Substitutions at positions 8, 62, 69, and 74, and downstream of position 93, did not

confer resistance. Position 24 RAS showed no-to-low resistance. At positions 28 and 30 the degree of resistance greatly depended on the specific substitution; thus no-to-high resistance was found. All positions 31 RAS mediated intermediate resistance, while position 58 and 92 RAS mediated low resistance. Position 93 RAS mediated low-to-high resistance. Positions 28, 30, 31 and 93 RAS induced intermediate-to-high resistance for at least 2 viruses, thus representing hotspots for RAS.

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ACCEPTED MANUSCRIPT Eleven of the 12 double mutants tested showed intermediate-to-high resistance. Compared to the most resistant of the respective single mutants, 7a(QC69)K24R,S30G, 1b(J4)L28M,Y93N, 2a(J6)F28S,Y93H and 3a(S52)L31F,Y93H showed increased resistance.

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Resistance of daclatasvir resistant variants to newer NS5A-inhibitors. Variants with at least intermediate resistance to daclatasvir were tested for cross-resistance to ledipasvir, ombitasvir, elbasvir, ruzasvir, velpatasvir, and pibrentasvir (Figure 4; Supplemental Figure 2). The

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two genotype 2a viruses with position 28 RAS showed low-to-high resistance to all inhibitors except pibrentasvir, showing high efficacy. Pibrentasvir showed exceptional efficacy against

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position 30 and 31 variants with no apparent resistance. Also velpatasvir showed relatively high efficacy against position 30 and 31 variants, with no-to-low resistance. For the other inhibitors, Q30K/R conferred low-to-high resistance, while L/Q30H and L30S conferred no-to-intermediate resistance; position 31 RAS conferred low-to-high resistance, except L31M not conferring

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resistance to ombitasvir. Pibrentasvir showed the highest efficacy against position 93 variants with no-to-low resistance. For velpatasvir no-to-high resistance and for the other inhibitors low-to-high resistance was observed for position 93 variants. While pibrentasvir also showed the highest

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efficacy against the eleven tested double mutants, 2a(J6)F28S,Y93H and 3a(S52)L31F,Y93H showed intermediate resistance to pibrentasvir.

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Resistance of additional clinically relevant HCV resistant variants to NS5A inhibitors. Several clinically relevant RAS, not commonly selected during the escape experiments with daclatasvir, including L31V, Y93N, the combination of L31M and Y93H and a deletion at position 32 (∆32), were engineered in relevant recombinants.14,49 Here we found that the resulting variants had relatively low fitness requiring long-term adaptation in transfection and passage culture (data not shown). Against genotype 1-6 variants with V31, only pibrentasvir showed high efficacy, while all other inhibitors showed varying efficacy with no-to-high resistance (Figure 5,

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ACCEPTED MANUSCRIPT Supplemental Figure 3). In contrast, ∆32 conferred high resistance to all inhibitors including pibrentasvir (19.000- to 177.000-fold resistance to pibrentasvir). Pibrentasvir showed comparatively high efficacy against Y93N and L31M+Y93H variants, which conferred high resistance to most

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other inhibitors. Effect of pre-existing T/Y93H polymorphism on resistance development. H93 is naturally occurring in up to 16% of isolates.12,14 To study the effect of this key RAS on resistance

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development, genotype 1-6 recombinants engineered with H9332 were treated with 4- to 256-fold their EC50 of daclatasvir (Supplemental Figure 1B). For genotypes 2-6, identified substitutions

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selected during viral escape (Figure 2B, Supplemental Tables 18-25) were evaluated regarding their fitness (Figure 6) and sensitivity to daclatasvir (Figure 7).

Distinct selection patterns in T/Y93H escape variants. With a few exceptions (Figure 2B and Supplemental Tables 18 and 25) the engineered T/Y93H was maintained. For most

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recombinants, hotspots for acquisition of additional substitutions overlapped those previously observed, and included positions 30, 31 and 58 (Figure 2B). In contrast to the original recombinants, substitutions at position 31 were not selected in 2a(JFH1)Y93H and 6a(HK6a)T93H,

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and substitutions at position 24 were not selected in 2a(J6)Y93H (Figure 2). Also, T/Y93H recombinants selected substitutions at positions not observed to change for the respective original

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recombinants. This included almost all substitutions for 1a(H77) and 1b(J4), P29S for 2a(JFH1)Y93H and 2a(J6)Y93H, and Q30H and P32L for 5a(SA13)T93H. In several instances, different substitutions at the same position were selected for specific recombinants with versus without T/Y93H.

Increased fitness of T/Y93H escape variants. The 23 engineered genotype 2-6 T/Y93H double and triple mutants were all viable and showed high fitness (Figure 6). Fitness of the T/Y93H recombinants was improved by combination with L31V for 3a(S52) and R30H for

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ACCEPTED MANUSCRIPT 6a(HK6a), possibly explaining the preferential selection of these substitution in the T/Y93H versus the original recombinants. Also, fitness of recombinants with F28L, P29S, L30H, or P32L was increased by combination with T/Y93H. 2a(JFH1)P29S and 5a(SA13)P32L showed large fitness

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impairment, which might explain why these variants were not selected in the original recombinants. When comparing variants with different substitutions at the same positions selected based on original versus Y93H recombinants, such as 2a(J6)F28L,Y93H versus 2a(J6)F28S,Y93H and

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3a(S52)L31V,Y93H versus 3a(S52)L31F,Y93H, variants selected based on Y93H recombinants showed higher fitness. For triple mutants V15A increased fitness of 4a(ED43)L30P,Y93H.

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Increased resistance of T/Y93H-escape variants. T/Y93H escape variants showed higher resistance than the original escape variants, determined in proof-of-concept studies using daclatasvir (Figure 7). All double mutants had increased resistance compared to the most resistant of the respective single mutants. Even though T93H only induced low resistance when tested

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singly, it significantly increased resistance of the T93H double mutants. For triple mutants, L28I 6a(HK6a)R30H,T93H

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Impact of different substitutions at position Y93 on 1a(H77). To investigate the principal impact of different substitutions at position 93, we mutated H93 in 1a(H77) to all possible

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aa residues (Supplemental Figure 4). Interestingly, most recombinants had fitness comparable to that of the original 1a(H77). Most viable and genetically stable recombinants showed high resistance, determined in proof-of concept studies using daclatasvir. Variants with Y93G/N/R showed >10.000 fold resistance to daclatasvir. Thus, various substitutions at position 93 were permissive and resulted in significant resistance.

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ACCEPTED MANUSCRIPT Discussion In this comprehensive study, we provide an unprecedented comparison of the efficacy of seven relevant NS5A-inhibitors against a wide range of HCV variants using infectious culture

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systems. Pibrentasvir and velpatasvir, the most advanced NS5A-inhibitors, recently approved for treatment of all HCV genotypes showed uniform pangenotypic activity.3,4 To identify NS5A RAS, we induced viral escape in genotype 1-7 recombinants using daclatasvir. Engineered escape variants showed high fitness and persistence of engineered RAS, in specific cases depending on fitness

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compensating substitutions. Resistance testing revealed NS5A positions 28, 30, 31 and 93 as

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hotspots for RAS across genotypes. Pibrentasvir showed the highest efficacy against engineered resistant variants. At last, we showed that pre-existing RAS T/Y93H facilitated selection of resistant variants with increased fitness and resistance under treatment. This comprehensive analysis in the context of the complete viral life cycle was facilitated by infectious HCV JFH1-based cell culture systems with genotype 1-7 specific NS5A.32

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Of note, even though none of the cell culture adaptive substitutions were localized at positions known to influence resistance to NS5A-inhibitors, an influence on selection of RAS cannot be

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excluded. Further, compared to full-length viruses isolated from patients, the chimeric nature of the recombinants might influence the results in this study. Nevertheless, NS5A-inhibitors showed

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similar efficacy against more recently developed TNcc (1a), J6cc (2a) and J8cc (2b) full-length viruses as against the respective JFH1-based NS5A recombinant.34,35,37,38 Further, results in infectious culture systems showed good correlation with clinical data.20,27,32-39,46 Also in vitro results in this study correlated with findings in clinical trials regarding the efficacy of NS5Ainhibitors against different genotypes and against HCV variants with RAS pre-existing at initiation of treatment.1-4,12,14

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ACCEPTED MANUSCRIPT In addition, hotspots for resistance were identified across genotypes that were similar to those described in replicons and patients, even though these studies mostly focused on genotype 1.12,14 Also, RAS selected for 1a(TN), 1a(H77), 2a(JFH1), 2a(J6) and 3a(S52) were similar to those

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selected in the few prior escape studies using infectious systems expressing NS5A of the same isolates.27,33 Isolate specific differences regarding selection of RAS were associated with presence of naturally occurring polymorphisms. A number of isolates had polymorphisms at position 30, 31

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or 93 and did not select RAS at these positions.

Compared to protease inhibitor resistant variants, NS5A-inhibitor escape variants

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selected in this study showed high fitness and RAS persisted throughout transfection and viral passage.50,51 Clinical studies suggested short-term versus long-term persistence of protease inhibitor versus NS5A-inhibitor resistant variants.12,14 In future studies it would be interesting to study longterm persistence of the selected RAS throughout additional viral passages in cell culture. Interestingly, for specific RAS, previous data in genotype 1a/b replicons suggested a somewhat

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greater negative effect on fitness than observed in our infectious systems.17 In infectious systems minor effects on replication might be compensated by additional substitutions acting on different

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steps of the viral life cycle.51 Alternatively, there could be isolate specific differences, with the effect of substitutions on fitness depending on the genetic context.42 Such dependency might also

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explain why clinically relevant resistant variants, which were not selected in escape experiments but engineered here based on prior clinical findings, showed relatively low fitness. In comparison to protease inhibitors and sofosbuvir, NS5A-inhibitors showed a very

low barrier to resistance.38,51 This finding might be due to several factors: (I) High fitness of most resistant variants. (II) A low genetic barrier to resistance, since most RAS were induced by single nucleotide changes. (III) In many instances, a single aa substitution caused high resistance. (IV) A number of positions localizing to the first 93 aa of domain I of NS5A could serve as sites for RAS.

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ACCEPTED MANUSCRIPT (V) At many of these positions different substitutions were able to mediate resistance, as also observed in patients12,14. (VI) There were many possibilities for combinations of RAS with secondary resistance substitutions and fitness compensatory substitutions. Also in patients,

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combinations of multiple RAS have been found.12 Given these factors, it appeared that almost any given HCV variant, even naturally highly resistant 2a(J6) and 2b(J8) as well as recombinants with pre-existing Y93H could become more resistant under treatment.

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Pre-existing T/Y93H led to the selection of highly resistant escape variants, which might partly be due to higher absolute inhibitor concentrations applied in these experiments.

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Sequential selection of RAS in T/Y93H viruses apparently drove the selection of resistant variants with improved fitness compared to variants selected in escape experiments with original viruses. Thus, repeated unsuccessful treatment attempts could give rise to highly fit and resistant variants. Only pibrentasvir and velpatasvir showed uniform pangenotypic efficacy. Several

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NS5A-inhibitors and especially ledipasvir showed poor efficacy on genotype 2 and 3 isolates. Top resistance levels of NS5A-inhibitor resistant variants (up to several million-fold resistance) exceeded those we previously observed for protease inhibitor resistant variants (up to ~6000-fold

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resistance) and for the polymerase inhibitor sofosbuvir (~10-fold resistance).38,50,51 Even though pibrentasvir had the highest efficacy against resistant variants, position 93 RAS, especially in

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combination with position 28 and 31 RAS, and ∆32 conferred significant resistance also to this inhibitor. Position 28 and 93 and selected position 31 RAS conferred significant resistance to velpatasvir. As different isolates showed great differences in EC50, resistant variants of different isolates might, despite similar fold-resistance, show big differences in EC50, of relevance for treatment responses to a given inhibitor concentration in patients. However, in addition to the EC50 of the specific NS5A inhibitor, the clinical impact of RAS is expected to depend on the used DAAcombination. While overall cure rates of DAA-combination regimens are high even in individuals

17

ACCEPTED MANUSCRIPT with detectable RAS, pre-existing NS5A RAS were shown to decrease cure rates depending on the DAA-combinations and patient group.12,14,16 Given the large number of HCV infected individuals that will be treated with DAA, even a low percentage of treatment failures might result in the

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emergence and spread of HCV resistant and multi-resistant variants in populations and over time

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result in decreased efficacy of currently highly effective treatment regimens.

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ACCEPTED MANUSCRIPT Acknowledgements: We thank Anna-Louise Sørensen for laboratory assistance, Steen Ladelund for statistical advice, Bjarne Ø. Lindhardt, Ove Andersen and Jens Ole Nielsen for support (all at Copenhagen University Hospital, Hvidovre), Carsten Geisler (University of Copenhagen) for

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support, Charles Rice (Rockefeller University) for research material, and CTL Europe GmbH for

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customized software.

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ACCEPTED MANUSCRIPT 28. Liu R, Curry S, McMonagle P, et al. Susceptibilities of genotype 1a, 1b, and 3 hepatitis C virus variants to the NS5A inhibitor elbasvir. Antimicrob Agents Chemother 2015;59:69226929.

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29. McGivern DR, Masaki T, Williford S, et al. Kinetic analyses reveal potent and early blockade of hepatitis C virus assembly by NS5A inhibitors. Gastroenterology 2014;147:453-462.

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30. 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

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2012;56:5365-5373.

31. Thomas E, Liang TJ. Experimental models of hepatitis B and C - new insights and progress. Nat Rev Gastroenterol Hepatol 2016;13:362-374.

32. Scheel TK, Gottwein JM, Mikkelsen LS, et al. Recombinant HCV variants with NS5A

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from genotypes 1-7 have different sensitivities to an NS5A inhibitor but not interferonalpha. Gastroenterology 2011;140:1032-1042. 33. Gottwein JM, Jensen SB, Li YP, et al. Combination treatment with hepatitis C virus

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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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34. 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. 35. 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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ACCEPTED MANUSCRIPT 36. Li YP, Ramirez S, Humes D, et al. Differential sensitivity of 5'UTR-NS5A recombinants of hepatitis C virus genotypes 1-6 to protease and NS5A inhibitors. Gastroenterology 2014;146:812-821.

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37. Ramirez S, Li YP, Jensen SB, et al. Highly efficient infectious cell culture of three hepatitis C virus genotype 2b strains and sensitivity to lead protease, nonstructural protein 5A, and polymerase inhibitors. Hepatology 2014;59:395-407.

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38. Ramirez S, Mikkelsen LS, Gottwein JM, et al. Robust HCV Genotype 3a Infectious Cell Culture System Permits Identification of Escape Variants With Resistance to Sofosbuvir.

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Gastroenterology 2016;151:973-985.

39. Pham LV, Ramirez S, Carlsen THR, et al. Efficient Hepatitis C Virus Genotype 1b CoreNS5A Recombinants Permit Efficacy Testing of Protease and NS5A Inhibitors. Antimicrob Agents Chemother 2017;61.

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40. Lindenbach BD, Evans MJ, Syder AJ, et al. Complete replication of hepatitis C virus in cell culture. Science 2005;309:623-626.

41. Kuiken C, Combet C, Bukh J, et al. A comprehensive system for consistent numbering of

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HCV sequences, proteins and epitopes. Hepatology 2006;44:1355-1361. 42. Scheel TK, Gottwein JM, Carlsen TH, et al. Efficient culture adaptation of hepatitis C virus

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recombinants with genotype-specific core-NS2 by using previously identified mutations. J Virol 2011;85:2891-2906. 43. 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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ACCEPTED MANUSCRIPT 44. Gottwein JM, Jensen TB, Mathiesen CK, et al. Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A. J Virol 2011;85:8913-8928.

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45. 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.

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46. 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.

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Gastroenterology 2011;141:1067-1079.

47. 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 liverchimeric mouse models. J Infect Dis 2010;201:1381-1389.

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48. Murphy DG, Sablon E, Chamberland J, et al. Hepatitis C virus genotype 7, a new genotype originating from central Africa. J Clin Microbiol 2015;53:967-972. 49. Younossi ZM, Stepanova M, Feld J, et al. Sofosbuvir/velpatasvir improves patient-reported

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outcomes in HCV patients: Results from ASTRAL-1 placebo-controlled trial. J Hepatol 2016;65:33-39.

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50. Jensen SB, Serre SB, Humes DG, et al. Substitutions at NS3 Residue 155, 156, or 168 of Hepatitis C Virus Genotypes 2 to 6 Induce Complex Patterns of Protease Inhibitor Resistance. Antimicrob Agents Chemother 2015;59:7426-7436. 51. Serre SB, Jensen SB, Ghanem L, et al. Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle. Antimicrob Agents Chemother 2016;60:3563-3578.

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ACCEPTED MANUSCRIPT 52. Simmonds P, Bukh J, Combet C, et al. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes. Hepatology 2005;42:962-973.

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Author names in bold designate shared co-first authorship

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ACCEPTED MANUSCRIPT Figure legends Figure 1. Efficacy of NS5A-inhibitors against HCV genotypes 1-7. Original HCV recombinants expressing NS5A of the indicated genotypes(isolates) were subjected to treatment assays using

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NS5A-inhibitors daclatasvir, ledipasvir, ombitasvir, elbasvir, ruzasvir, velpatasvir, and pibrentasvir. Data are from representative experiments; data points represent means of triplicates±SEM; curves were fitted as described in Materials and Methods. For median EC50 values and 95% CI of

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replicate experiments see Supplemental Table 2, which also includes analysis of 3 additional genotype 1a isolates (DH6, HCV1 and J1).

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Figure 2. Putative resistance substitutions in NS5A domain I identified for HCV genotype 1-7 daclatasvir escape viruses without (A) and with (B) pre-existing T/Y93H. a

, Shown are aa positions in NS5A domain I, numbered relative to H77 (GenBank ID AF009606)

NS5A,52 at which substitutions for at least one of the recombinants subjected to treatment with

b

c

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different concentrations of daclatasvir was identified by direct sequence analysis. , Beneath, aa residues for H77 at these positions are specified.

, For each recombinant with genotype(isolate) specific NS5A a dot indicates that the aa residue at

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the specified position is conserved with H77. Non-conserved aa residues are indicated by a single letter without shading. Putative resistance substitutions are indicated by the original and the mutated

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residue with grey shading. The mutated residue is color coded in accordance with the highest foldEC50 concentration under which it was selected. *Substitutions not only detected in escape cultures but also in non-treated control cultures were not included in this Figure; as an exemption, R30H for 6a(HK6a)T93H was included. When direct sequencing indicated that several nucleotides of the same codon had changed, only aa substitutions confirmed by subclonal analysis are shown. Green shading indicates cell culture adaptive substitutions localizing to NS5A. Orange shading indicates engineered T/Y93H substitutions. Khaki brown shading indicates engineered substitutions that

27

ACCEPTED MANUSCRIPT changed under treatment. Detailed datasets for individual recombinants are shown in Supplemental Tables 3-25. Figure 3. Fitness of engineered HCV genotype 1-7 NS5A-inhibitor escape variants. , Titer differences (log10 FFU/ml) were calculated by relating supernatant infectivity titers of

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a

cultures transfected with HCV RNA transcripts of variants with indicated substitutions to that of original recombinants of the same genotype(isolate) as described in Materials and Methods. Double

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mutants with increased fitness, defined as an at least 0.5 log10 increase in the titer difference for the recombinant with combinations of substitutions as compared to the least fit of the recombinants

b

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with single substitutions, are indicated by red font.

, Direct sequence analysis of virus stocks revealed that the following variants had acquired

additional substitutions in NS5A domain I, estimated to be present in at least 50% of viral genomes: 1a(TN)Q30H: F161L; 1a(TN)Q30R: F161L/f; 7a(QC69)L31F: I158M/i; 2a(J6)F28S,Y93H: T24I. , variants were engineered previously;32 titer differences are based on experiments done in the

present study.

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c

Figure 4. Efficacy of NS5A-inhibitors against engineered HCV genotype 1-7 escape variants. a,

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Fold resistance values were calculated by relating EC50 of variants with indicated substitutions to that of original viruses for the indicated genotypes(isolates) for the NS5A-inhibitors daclatasvir (D),

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ledipasvir (L), ombitasvir (O), elbasvir (E), ruzasvir (R), velpatasvir (V) and pibrentasvir (P) and are shown in Supplemental Figure 2. Values are based on one representative experiment. The following 24 variants with ≤3-fold resistance to daclatasvir were not included here: 4a(ED43)V8A, 7a(Q69C)K24R, 7a(Q69C)K24T, 2b(J8)S24F, 2b(J8)S24T, 2a(J6)F28I, 2a(J6)F28L, 2a(J6)N62D, 1a(TN)N69H, 1a(H77)N105D,

1a(H77)N69T,

1a(TN)N69T,

5a(SA13)N105H,

2a(J6)I74T,

7a(QC69)T116M,

3a(S52)T99A,

2a(J6)Q123R,

2a(J6)P104S,

5a(SA13)Y129H,

7a(QC69)Q197L, 2a(J6)M200V, 2a(J6)M202V, 1b(J4)T204A, 1a(TN)H208Y. Double mutants

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ACCEPTED MANUSCRIPT with at least 4-fold increased resistance to daclatasvir, compared to the most resistant of the respective single mutants, are indicated by red font. Cases where the respective recombinant was not inhibited by the highest non-cytotoxic inhibitor concentration applied, are indicated by “>”. nd,

b

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not done. , Additional aa changes, estimated to be present in at least 50% of viral genomes by direct

sequence analysis of NS5A domain I, were recorded in the following virus stocks: 1a(TN)Q30H: F161L; 1a(TN)Q30R: F161L/f; 7a(Q69)L31F: I158M/i; 2a(J6)F28S,Y93H: T24I. , Previously reported data.32

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c

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Figure 5. Efficacy of NS5A-inhibitors against additional HCV variants with clinically relevant RAS. a, Fold resistance values were calculated by relating EC50 of variants with indicated substitutions to that of original viruses for the indicated genotypes(isolates) for the NS5A-inhibitors daclatasvir (D), ledipasvir (L), ombitasvir (O), elbasvir (E), ruzasvir (R), velpatasvir (V), and

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pibrentasvir (P) and are shown in Supplemental Figure 3. Values are based on one representative experiment with two replicates per concentration. Cases where the respective recombinant was not inhibited by the highest non-cytotoxic inhibitor concentration applied, are indicated by “>”.

c

, Data are reproduced from Figure 4 for comparison.

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b

, Direct sequence analysis of virus stocks revealed that the following variants used for the indicated

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treatments had acquired additional substitutions in NS5A domain I: 1a(TN)L31V: Y93C; 1a(TN)∆32: R6G, Y93H, T204I; 1b(J4)L31V: C165R; 1b(J4)∆32: K139M. Figure 6. Fitness of engineered HCV genotype 2-6 NS5A-inhibitor escape variants with preexisting T/Y93H. a

, Titer differences (log10 FFU/ml) were calculated by relating supernatant infectivity titers of

cultures transfected with HCV RNA transcripts of variants with indicated substitutions to that of original recombinants of the same genotype(isolate) as described in Materials and Methods.

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ACCEPTED MANUSCRIPT *Values were also presented in Figure 3 and are derived from the same experiment. Double / triple mutants with increased fitness, defined as an at least 0.5 log10 increase in the titer difference for the recombinant with combinations of substitutions as compared to the least fit of the recombinants

b

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with single / double substitutions, are indicated by red font. , Direct sequence analysis of NS5A domain I revealed that 2a(JFH1)P29S had acquired K26R and

K30G.

, Variants were engineered previously;32 titer differences are based on experiments done in the

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present study.

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Figure 7. Efficacy of daclatasvir against engineered HCV genotype 2-6 escape variants with pre-existing T/Y93H. Fold resistance values (y-axis, logarithmic scale) were calculated by relating EC50 of variants of the indicated genotype(isolate) with indicated substitutions (x-axis) to that of the original viruses without T/Y93H treated in the same experiment for daclatasvir based on one

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representative experiment. Striped columns indicate that the respective variant was not inhibited by the highest non-cytotoxic concentration of daclatasvir applied; a minimum fold resistance value was calculated using this concentration. Double and triple mutants were selected under treatment; single

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mutants are included for comparison. Direct sequence analysis of NS5A domain I revealed that 2a(JFH1)P29S had acquired K26R and K30G. *, double / triple mutants with at least 4-fold

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increased resistance to daclatasvir compared to the most resistant of the respective single / double mutants. Selected fold resistance values are also shown in Figure 4 and are derived from the same experiment. Fold resistance values for daclatasvir against 2a(J6)Y93H, 5a(SA13)T93H and 6a(HK6a)T93H were previously reported.32

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ACCEPTED MANUSCRIPT

1a(H77) 1a(TN) 1b(J4) 1b(DH1) 2a(JFH1) 2a(J6)

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2b(J8) 3a(S52) 4a(ED43)

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residual infectivity (percent)

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5a(SA13) 6a(HK6a) 7a(QC69)

ACCEPTED MANUSCRIPT H77 aa rel ref # a H77 aa

b

Genotype Isolate

6

8

24

25

28

30

31

43 46 54 56

58

62 64

69

93

94 99

R

I

K

A

M

Q

L

Y

V

H

R

H

E

T

N

74 81 85 87 92 I

R

S

T

A

Y

T

T

c

H77

●

●

●

●

●

●

Q-H/R

●

●

●

●

●

●

●

●

N-T

●

●

●

●

●

Y-C/H

●

●

1a

TN

●

●

●

●

●

●

Q-H/K/R

●

●

●

●

●

●

●

●

N-H/T

●

●

●

●

●

Y-C/H

●

●

1a

HCV1

●

●

●

●

●

●

Q-R

●

●

V-I

●

●

●

●

●

●

●

R-W

●

●

●

Y-H/N/S

●

●

1a

J1

●

●

●

●

T-A

M-T

H

●

●

●

H-D

●

●

●

●

●

●

●

S-G

●

●

Y-H

●

●

1a

DH6

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

T-A

●

Y-C

●

●

1b

J4

●

●

V

Q

S

L-M

R

●

●

●

Q

T

P

Q

A

●

●

S

H

●

●

Y-H/N

●

●

1b

DH1

●

R-G V

Q

S

L

Q-H

L-I

●

●

●

T

P

Q

●

2a

JFH1

●

●

V

T

S

F-S

K

L-M

●

●

T

●

P

N

S

2a

J6

●

●

V T-A/S S

F-I/L/S

K

M

●

●

T

●

P

2b

J8

●

Q

●

L

K

M

●

●

T

●

P

N

S

3a

S52

D-G

●

●

●

●

A

L-F

●

●

S

●

P

S

●

S-F/T S S

N-D S

ED43

●

●

●

L

L-H/S

M

●

E

●

T

P-L

D

A

5a

SA13

T R-G

●

Q

●

L

●

L-F/V

●

●

S

K

P

T

S

6a

HK6a

●

●

V

●

●

L-F

R-H

L-M

●

●

●

T T-A/N V

A

7a

QC69

●

●

V K-R/T

●

L

S-G

L-F Y-H E

●

S Y-H

●

●

Y-H

●

●

L

●

M

Q

●

C

Y-H

●

A

L

I-T

M

Q

●

C-S Y-H/N T-K V

M

●

L

Q

●

C-R

Y-H

●

V

●

L

A

H

●

E

Y-H

●

T-A

●

●

S

H

●

●

Y-H

●

V

●

●

S

H

●

●

T

●

V

●

●

S

H

●

●

T

●

P

L A-V

●

●

●

●

W

●

S

H

●

S

104 105 116 123 129 137 144 156 158 186 197 200 202 204 207 208 P

N

E

R

Y

N

I

V

L

S

c

1a

H77

●

N-D

●

●

●

●

●

●

●

●

1a

TN

●

●

●

Q

●

●

V

●

●

●

1a

HCV1

●

●

●

Q

●

●

V

●

●

●

1a

J1

●

●

●

●

●

●

V

●

●

●

1a

DH6

●

●

●

●

●

●

V

●

●

●

1b

J4

●

●

●

●

●

●

V

●

●

●

1b

DH1

●

●

●

●

●

N-D V

●

●

●

2a

JFH1

T

●

S

Q

●

●

L

●

I

2a

J6

P-S

●

S Q-R

●

●

L-V

●

I

2b

J8

●

●

S

Q

●

3a

S52

●

●

S

●

●

4a

ED43

●

●

●

●

●

5a

SA13

●

6a

HK6a

●

●

7a

QC69

H

C T-M

H77 aa rel ref # a

●

●

Y-H

●

●

●

●

T

M

T

S

●

●

●

●

●

●

●

●

●

●

●

H-Y

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

T

●

●

T-A

●

●

V

●

●

●

●

●

R

●

●

P

●

●

●

● ●

S-F D

D M-V M-V

●

TE D

N-H A

A

H

M AN U

b

Genotype Isolate

N K-E

●

SC

4a

H77 aa

W V-A

RI PT

1a

H77 aa rel ref # a

●

V

●

I

●

E

A

●

●

●

E

V

●

●

●

S

●

●

R

●

●

●

V

I

●

●

●

●

●

●

●

●

●

V

●

I

●

T

●

●

S

A

●

G

V

●

I

●

T

●

●

●

T

●

D

V V-A I-V

L

●

S-P

●

Q-L S

●

3

6

14 15 28 29

30

31

32 38

58

62 75 78 81 92 93 123 133 137

S

R

E

V

Q

L

P

H

E

V

R

R

A

Y

R

M

N

F

H77

●

●

●

J4

●

●

T

2a

JFH1

●

●

T

I

2a

J6

●

●

T

I

S52

D-G

●

S

●

4a

ED43

●

5a

SA13

T R-G T

6a

HK6a

●

H77 aa

b

Genotype Isolate 1a 1b

3a

c

P

●

●

●

●

●

●

●

●

●

R-S

●

H-R

●

M-V

●

F-L

●

●

L

●

R

L-I/V

●

●

P

Q

●

●

S

●

H

●

M-T

●

●

●

F-C P-S

K

●

●

●

P

N

T K-R M

C

H

Q

L

●

●

●

●

F-L P-S

K-N

M

●

●

P

M C-W H

Q

L

●

●

●

F D-N

●

P

S A-V R

A

E

H

●

A

E

F-Y

●

●

S

●

●

●

P-S

D

T

K

S

●

H R-K V

●

●

●

F

●

●

●

●

P

T

V

K

S

●

H

●

V

N-D

●

T

I-V

●

●

T-A

V

T

K

S

●

H-R

●

V

G

●

T

●

E-K

●

●

A

L-V

●

W T-A V-A L

●

L-H/P/S

M

●

●

T-A

A ●

L P-S

L-I

●

Q-H

L-F/I/V P-L

R-H*

putative RAS selected under treatment with: 4xEC50

16xEC50

64xEC50

256xEC50

cell culture adaptive substitution engineered substitution engineered and changed under treatment

●

●

S

149 166 182 197 209 212

M

AC C

B

3 S

EP

A

S-F H-D

N-D T

K

K

Y

A

I

E

●

●

●

●

●

T

●

●

D

●

●

●

●

N-D F-I ●

●

ACCEPTED MANUSCRIPT Titer Difference (log10 FFU/ml)a 1b(J4)

2a(J6)

2a(JFH1)

2b(J8)

3a(S52)

4a(ED43) 5a(SA13) 6a(HK6a) 7a(QC69) 0.4 -0.3 -0.5

-0.1 0.1

-0.3 -0.4 -1.2

RI PT

-0.2 -0.1

-0.1

-0.4

0.1 -0.5 0.1 b b

SC

-0.2 0.1 -0.1

0.0

-0.8

-0.8 -1.0

-0.4

0.0

0.0

M AN U

0.4

-0.6

0.0 0.1 0.0 -0.1

0.0 -0.1

0.0

-0.4 1.5 c

-0.3 -0.5 <-1.2

c

0.0

c

-0.7

TE D

0.0 -0.1

-0.7

c

0.7

c

0.1

0.3

-1.1 -0.7

0.1

0.1 -0.4

0.4 0.0

0.2

AC C

K24R K24T S24F S24T T24A T24S F28I F28L F28S L28F L28M L30H L30S Q30H 0.2 Q30K Q30R 0.1 R30H S30G L31F L31M L31V P58L T58A T58N N62D N69H N69T 0.3 I74T C92R C92S Y93C 0.3 Y93H -0.1 c Y93N T94K T99A P104S N105D 0.2 N105H T116M Q123R Y129H Q197L M200V M202V T204A H208Y Double mutants V8A,L30H V8A,P58L K24R,S30G K24R,L31F S24F,Y93H S24T,Y93H L28M,Y93N F28S,Y93H L31F,Y93H L31F,Y129H N62D,Y93H Y93H,T99A

1a(TN)

EP

1a(H77) Single mutants V8A

-0.3

0.4 0.2 -0.5 <-1.3 -0.5 -0.1

0.0 <-1.0

b

-1.0 -0.1 0.1 0.0

Titer difference

<-0.9

strong fitness impairment

(log10 FFU/ml)

≥-0.9 to ≤-0.5

moderate fitness impairment ≥+0.5

>-0.5 to <+0.5 comparable fitness increased fitness

b

ACCEPTED MANUSCRIPT Fold Resistance a 1a(H77) D L OE RV P Single mutants T24A T24S

1a(TN)

1b(J4)

D L OE RV P

2a(J6)

D L OE RV P

2a(JFH1)

D L OERV P

F28S L28F L28M

3a(S52)

D L OE RV P

D L OE RV P

4a(ED43) D L OE RV P

5a(SA13) D L OE RV P

6a(HK6a) D L OE RV P

7a(QC69) D L OE RV P

b b

b

b b

b b

b b

b

b b

SC

b b

RI PT

>

L30H L30S Q30H Q30K Q30R R30H S30G

M AN U

L31F L31M L31V P58L T58A T58N C92R C92S

Fold resistance compared to original recombinant ≤3 no resistance low resistance 4-79 80-999 intermediate resistance high resistance 1000 ≥

nd nd nd nd

c

EP

c

TE D

nd nd nd nd

AC C

Y93C Y93H c Y93N Double mutants V8A,L30H V8A,P58L K24R,S30G K24R,L31F S24F,Y93H S24T,Y93H L28M,Y93N F28S,Y93H L31F,Y93H L31F,Y129H N62D,Y93H Y93H,T99A

2b(J8)

D L OE RV P

>

> >

b >b >b >b b >b b

>

>

b b

b b

b

b b

Fold Resistance a 1a(H77)

1a(TN)

1b(J4) 2a(JFH1) 3a(S52) ACCEPTED MANUSCRIPT

D L OE RV P Single mutants

D L OE RV P

D L OE RV P

L31V M31V

c

c

Del 32

>c c

c

c

c

c

c

c

c >c c >c c

c

c

c

> c

c

c

c

c

D L OE RV P

4a(ED43)

D L OE RV P

D L OE RV P

5a(SA13)b D L OE RV P

c

Y93N Double mutants L31M,Y93H

AC C

EP

TE D

M AN U

SC

RI PT

Fold resistance compared to original recombinant ≤3 no resistance low resistance 4-79 80-999 intermediate resistance high resistance ≥1000

6a(HK6a) D L OE RV P

ACCEPTED MANUSCRIPT Titer Difference (log10 FFU/ml)a 2a(J6) 2a(JFH1) 3a(S52) Single mutants for comparison T14A V15A F28C 0.2 F28L * -0.4 L28I P29S <-0.9 b L30H L30P L30S Q30H R30H

Titer difference (log10 FFU/ml)

6a(HK6a) -0.5

0.2 * * -0.5 -0.4 0.4 0.1 -1.3

0.0

*

0.0

*

* *

* 0.3 0.2 -0.7

*c

*c

0.7

0.2 -0.4 0.0 -0.6 0.1

c

-1.2

*c

-0.5

-0.1

-0.1 0.4

-0.1

0.3 -0.2

0.1

-0.1

-0.1

0.0 0.2

0.1

-0.5

<-0.9 ≥-0.9 to ≤-0.5 >-0.5 to <+0.5 ≥+0.5

c

M AN U

0

TE D

*c

SC

-0.2

RI PT

-0.5 -0.2 0.1

0.1

AC C

T/Y93H triple mutants T14A,L30H,Y93H T14A,R30H,T93H T14A,P58S,Y93H V15A,L30P,Y93H L28I,R30H,T93H F28L,N62D,Y93H 0.2 F28L,C92W,Y93H -0.5 P29S,K78R,Y93H R30H,T58A,T93H

5a(SA13)

0.2 0.2

EP

L31F L31I L31V P32L P58S T58A N62D 0.0 K78R C92W -0.4 T93H Y93H -0.3 T/Y93H double mutants F28C,Y93H F28L,Y93H 0.1 P29S,Y93H L30H,Y93H L30P,Y93H L30S,Y93H Q30H,T93H R30H,T93H L31F,T93H L31I,T93H L31V,Y93H L31V,T93H P32L,T93H P58S,Y93H

4a(ED43)

strong fitness impairment moderate fitness impairment comparable fitness increased fitness

0.2

ACCEPTED MANUSCRIPT

100000 10000 1000

RI PT

Fold resistance

1000000

100 10

SC

1

M AN U

Fold resistance

100000 10000 1000 100 10

100

10

1

EP

1000

AC C

Fold resistance

10000

TE D

1

ACCEPTED MANUSCRIPT

RI PT

Supporting Documents

Supplemental Table 1. Primers for nested reverse-transcription polymerase chain reaction (RT-PCR) for direct sequence analysis

Primer direction

Primer name

Primer sequence

M AN U

RT-PCR step

SC

of NS5A domain I of HCV JFH1-based genotype 1-7 NS5A recombinants.

Reverse

9470R(24)_JFH1

5'-CTATGGAGTGTACCTAGTGTGTGC-3'

1st round PCR

Forward Reverse

JF5951 JR8368

5'-AAGATCATGTCTGGCGAGAAG-3' 5'-CCTGATGTCTCTCTCAGTGAC-3'

2nd round PCR

Forward Reverse

JF6177 JR7881

5'-AGCGTGTGACCCAACTACTTG-3' 5'-TTGATGTCCTTTAAGACTGAGTC-3'

AC C

EP

TE D

Reverse Transcription

1

ACCEPTED MANUSCRIPT

a

Mean EC50 for NS5A inhibitors (nM) (95% CI) Genotype Isolate

Daclatasvir

Ledipasvir

Ruzasvir

Velpatasvir

)

0.004 ( 0.003 - 0.005 ) 0.01 ( 0.01 - 0.01 )

0.02 ( 0.01 - 0.02 0.03 ( 0.02 - 0.03

)

0.02 - 0.02

Pibrentasvir

0.07 ( 0.05 - 0.08 ) 0.04 ( 0.03 - 0.05 ) ) 13 ( 10 - 17

0.04 ( 0.03 - 0.05 ) 0.008 ( 0.006 - 0.011 ) ) 1.0 - 1.6 1.3 (

0.01 ( 0.008 - 0.012 ) 0.008 ( 0.006 - 0.009 ) 0.07 ( 0.06 - 0.09 )

0.006 ( 0.004 - 0.009 ) 0.003 ( 0.002 - 0.004 ) 0.04 ( 0.03 - 0.05 )

0.03 ( 0.02 - 0.04 ) 0.02 ( 0.01 - 0.04 ) 0.01 ( 0.01 - 0.02 )

1b

J4 DH1

0.03 ( 0.027 - 0.031 ) 0.02 ( 0.016 - 0.018 )

0.001 ( 0.001 -0.002 ) 0.003 ( 0.003 -0.004 )

0.001 ( 0.001 -0.002 ) 0.001 ( 0.001 - 0.001 )

0.006 ( 0.005 - 0.007 ) 0.002 ( 0.002 - 0.003 )

0.01 ( 0.01 - 0.01 ) 0.003 ( 0.002 - 0.004 )

0.02 ( 0.02 - 0.02 ) 0.007 ( 0.005 - 0.011 )

0.008 ( 0.006 - 0.009 ) 0.003 ( 0.002 - 0.003 )

2a

JFH1 J6

0.1 ( 0.11 - 0.13 ) ) 21 ( 18 - 24

22 ( 20 - 24 726 ( 560 - 943

0.08 ( 0.07 - 0.10 ) ) 9 - 13 11 (

0.05 ( 0.04 - 0.05 ) 6.0 ( 5.3 - 6.9 )

0.02 ( 0.02 - 0.03 ) 0.2 ( 0.1 - 0.2 )

0.01 ( 0.01 - 0.02 ) 0.02 ( 0.01 - 0.02 )

2b

J8

109 (

98 - 122

)

3a

S52

2.6 (

2.2 - 3.0

)

4a

ED43

5a

RI PT

0.01 - 0.02

SC

0.01 ( 0.02 (

0.007 ( 0.006 - 0.008 ) 0.008 ( 0.007 - 0.008 )

0.07 ( 0.06 - 0.07 b 0.1 ( 0.11 - 0.14 ) b ) ( 0.8 - 1.6 1.1

) )

0.006 0.007 0.004 0.005 0.004

( 0.005 - 0.007 ) ( 0.006 - 0.009 ) ( 0.003 - 0.006 ) ( 0.003 - 0.008 ) ( 0.003 - 0.005 ) -

0.02 ( 0.09 (

0.02 - 0.03

)

)

0.07 - 0.12

)

1656 ( 1195 - 2293 )

0.03 (

0.02 - 0.05

)

6.0 (

4.9 - 7.5

)

6.7 (

6.1 - 7.3

)

0.5 (

0.4 - 0.6

)

0.02 ( 0.02 - 0.02 )

0.1 - 0.2

)

0.3 (

0.2 - 0.3

)

0.4 (

0.3 - 0.4

)

0.1 (

0.1 - 0.1

)

0.01 ( 0.01 - 0.01 )

0.01 ( 0.01 - 0.01 )

0.01 ( 0.01 - 0.01 )

0.004 ( 0.004 - 0.005 )

0.01 ( 0.01 - 0.01

0.02 ( 0.02 - 0.03

)

0.007 ( 0.006 - 0.008 )

0.06 ( 0.05 - 0.06 )

0.01 ( 0.01 - 0.01 )

)

263 ( 229 - 301

)

0.03 ( 0.02 - 0.03 )

0.6 (

0.5 - 0.7

)

0.001 ( 0.001 - 0.001 )

SA13

0.02 ( 0.02 - 0.03

)

0.6 (

0.4 - 0.8

)

0.009 ( 0.008 - 0.011

6a

HK6a

0.04 ( 0.03 - 0.04 )

2.7 (

2.2 - 3.3

)

9.8 (

7a

QC69

0.08 ( 0.06 - 0.10 )

29 (

25 - 33

)

0.06 (

0.006 ( 0.006 - 0.007 )

)

0.02 ( 0.02 - 0.02

8.4 - 11.4

)

0.02 ( 0.02 - 0.02 )

0.02 ( 0.01 - 0.02 )

0.05 - 0.07

)

0.04 ( 0.04 - 0.05 )

0.02 ( 0.02 - 0.02 )

TE D

0.1 (

M AN U

) )b

0.01 ( 0.009 - 0.012 ) 0.009 ( 0.005 - 0.015 )

Elbasvir

H77 TN DH6 HCV1 J1

1a

)

Ombitasvir

0.06 ( 0.06 - 0.07 0.03 ( 0.02 - 0.04

)

)

0.3 (

0.2 - 0.3

)

0.02 ( 0.02 - 0.02 )

Supplemental Table 2: Efficacy of NS5A-inhibitors against HCV genotypes 1-7. , Original HCV recombinants expressing NS5A of the indicated genotypes and isolates were subjected to treatment assays with the

EP

a

AC C

indicated NS5A-inhibitors and concentration-response curves were fitted as described in Materials and Methods. Log10 median effective concentration (EC50) and standard error (SE) of log10 EC50 from replicate experiments were used to calculate inverse variance weighted means of log10 EC50 with SE and 95% confidence interval (CI). Inverse logarithmic transformation rendered median EC50 with 95% CI. Reported data are based on one to five experiments with three replicates per concentration. For data of representative experiments see

2

ACCEPTED MANUSCRIPT

Figure 1. Direct sequence analysis of virus stocks revealed that the following variants had acquired additional substitutions in NS5A domain I, estimated to be present in at least 50% of viral genomes: DH6: Y182C/Y; DH1: Y85C/y.

EP

TE D

M AN U

SC

RI PT

, Previously reported data.1

AC C

b

3

AC C

)Y 93 H

EP

)Y 93 JF H H1 )Y 93 2a H (J 6) Y 3a 93 (S H 52 )Y 4a 93 (E H D4 3) 5a Y9 (S 3H A1 3) 6a T9 (H 3H K6 a) T9 3H

2a (

(H 77

1b (J 4

1a

c

c

c

c

TE D

c

c

c

c

RI PT

First timepoint of peak of infection (day post treatment)

SC

(H 77 )a 1a (T N a 1a (H ) C V1 )b 1a (J 1) b 1a (D H 6) b 1b (J 4) a 1a (D H 1) c ,d 2a (J FH 1) a 2a (J 6 2b ) a (J 8) a ,d 3a (S 52 4a )a (E D 43 5a )a (S A 13 6a )a (H K 6a 7a )a (Q C 69 )a

1a

M AN U

First timepoint of peak of infection (day post treatment)

ACCEPTED MANUSCRIPT

4

ACCEPTED MANUSCRIPT Supplemental Figure 1. Induction of viral escape of HCV genotype 1-7 NS5A recombinants from daclatasvir. Huh7.5 cells were infected with HCV recombinants expressing NS5A of the indicated genotype(isolate) without (A) or with (B) T/Y93H.1 Cultures were treated with the

RI PT

indicated concentrations of daclatasvir, expressed as fold-EC50 (for EC50 values see 1). Shown is the first time-point following initiation of treatment, at which at least 80% of culture cells had become HCV positive by immunostaining.

SC

♦, viral suppression, no HCV antigen positive cells were observed for at least 2 weeks. *, concentration resulted in cytotoxicity. a

b

c

M AN U

, infection with 0.05 MOI, treatment initiated day 1 postinfection (10-30% cells infected). , infection with <0.05 MOI; treatment initiated day 1 postinfection (2-5% cells infected).

, infection with ≤0.05 MOI; treatment initiated day 1-6 postinfection, (20-40% cells infected; for

3a(S52)Y93H, 70% cells infected).

EP

TE D

, for EC50 values see Supplemental Table 2.

AC C

d

5

100000

D L

10000 O

1000 100

E

10 1

RI PT

Fold resistance

ACCEPTED MANUSCRIPT

R

V

P

M AN U

100000 10000 1000 100 10 1

O E R V P

AC C

EP

*

D L

TE D

Fold resistance

SC

*

Fold resistance

100000

10000

D

1000

L O

100 E

10 R

1 V

* P

*

6

ACCEPTED MANUSCRIPT Supplemental Figure 2. Efficacy of NS5A-inhibitors against engineered HCV genotype 1-7 escape variants. Fold resistance values (y-axis, logarithmic scale) were calculated by relating EC50 of variants of the indicated genotype(isolate) with indicated substitutions (x-axis) to that of

RI PT

the original viruses treated in the same experiment for the NS5A-inhibitors daclatasvir (D), ledipasvir (L), ombitasvir (O), elbasvir (E), ruzasvir (R), velpatasvir (V), and pibrentasvir (P) (zaxis) based on one representative experiment. Striped pyramids indicate that the respective variant

SC

was not inhibited by the highest non-cytotoxic concentration of the respective inhibitor applied; a minimum fold resistance value was calculated using this concentration. For variants with <80-fold

M AN U

resistance to daclatasvir, not included here, for additional aa changes acquired by the treated virus stocks in NS5A domain I and for an overview according to NS5A amino acid position, see Figure 4. *, double mutants with at least 4-fold increased resistance to daclatasvir compared to the most resistant of the respective single mutants. Fold resistance values for daclatasvir against

AC C

EP

TE D

1a(H77)Y93H, 1b(J4)Y93H and 2a(J6)Y93H were previously reported.1

7

ACCEPTED MANUSCRIPT

RI PT

10000000 100000 10000 1000 100

SC

Fold resistance

1000000

10

M AN U

1

D L O

E R V P

TE D

Supplemental Figure 3. Efficacy of NS5A-inhibitors against HCV variants with clinically relevant RAS not selected in escape variants. Fold resistance values (y-axis, logarithmic scale) were calculated by relating EC50 of variants with the indicated substitutions and of the indicated

EP

genotype(isolate) (x-axis) to that of the original viruses treated in the same experiment for the NS5A-inhibitors daclatasvir (D), ledipasvir (L), ombitasvir (O), elbasvir (E), ruzasvir (R),

AC C

velpatasvir (V), and pibrentasvir (P) (z-axis) based on one representative experiment; treatment assays were done with two replicates per concentration. Original 4a(ED43) had M at position 31, while original viruses of the other genotypes(isolates) shown here had L. Striped pyramids indicate that the respective variant was not inhibited by the highest non-cytotoxic concentration of the respective inhibitor applied; a minimum fold resistance value was calculated using this concentration. For additional aa changes acquired by the treated virus stocks in NS5A domain I see Figure 5. Data for 5a(SA13)L31V are reproduced from Supplemental Figure 2.

8

ACCEPTED MANUSCRIPT genetic

fold resistance

(log10 FFU/ml)

stabilityc

to daclatasvird

-0.1

stable

30

Y93C

0

stable

553

Y93D

-1

D93V

na

Y93E

-1.1

E93V

na

Y93F

-0.2

stable

29

Y93G

-0.4

stable

16048

Y93H

-0.1

additional N69N/t

4000

Y93I

-0.2

stable

2059

Y93K

-0.2

stable

2184

Y93A

b

0.4

stable

1879

0.5

stable

637

Y93N

0

stable

Y93P

non viable

na

Y93Q

0

stable

Y93R

0

stable

16888

Y93S

0.5

stable

6122

SC

Y93L Y93M

17725 na

M AN U

1798

Y93T

0

stable

931

Y93V

-0.2

stable

1379

Y93W

-0.6

stable

5197

Titer difference (log10 FFU/ml)

strong fitness impairment moderate fitness impairment comparable fitness increased fitness

TE D

<-0.9 ≥-0.9 to ≤-0.5 >-0.5 to <+0.5 ≥+0.5

RI PT

titer difference substitutiona

AC C

EP

Fold resistance compared to original recombinant ≤3 no resistance low resistance 4-79 80-999 intermediate resistance high resistance ≥1000

Supplemental Figure 4. Effect of various substitutions at NS5A position 93 on fitness and resistance of 1a(H77). a

, Huh7.5 cells were transfected with HCV RNA transcripts of 1a(H77) specified by the substitution

at NS5A position 93. b

, infectivity titer differences (log10 FFU/ml) were calculated as described in Materials and

Methods.

9

ACCEPTED MANUSCRIPT c

, Results of direct sequence analysis of NS5A domain I of first viral passage virus stocks.

d

, Viruses were subjected to treatment assays with daclatasvir and fold resistance values were

calculated by relating EC50 of variants to that of original viruses treated in the same experiment as

RI PT

described in Materials and Methods.

AC C

EP

TE D

M AN U

SC

na, non applicable.

10

ACCEPTED MANUSCRIPT

Supplemental Table 3. Substitutions identified in NS5A domain I of 1a(H77) recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 1a(H77) plasmid

6357 6358 6417 6474 6545 6546 6581 6589 6749

Nucleotide Identityb

Direct Sequence Analysis

1a(H77) plasmid

A

A

A

A

T

A

A

Subclonal Analysis

G

T

Daclatasvir concentration

Day PT

None None

14 16

●

●

●

●

●

●

A/g G/A

●

●

●

●

●

●

4xEC50 4xEC50

26 28

G/A

●

A/g

A/c

T/a

●

●

●

●

A/g/t G

●

●

16xEC50 16xEC50

28 30

●

C/A C/a

G/A G/a

●

● ●

G/A G/A

●

●

64xEC50 64xEC50

28 30

●

C C

●

●

T/c

A/g

●

●

256xEC50 256xEC50

35 37

●

●

●

●

●

●

●

T C

●

c

Q-R/He

2026 2045 2022 2041 50 69 D-G

N-T

G/t ●

T/c C

●

●

●

●

●

●

A/g

●

●

G/t G/t

C/t C/T

●

●

●

C

●

●

●

●

2069 2065 93

AC C

Amino Acid Change d

2006 2002 30

C/t

TE D

●

●

G/t T/G

Q30H+D50G (8/10); Y93C (2/10)

EP

●

●

M AN U

SC

1a(H77) escape cultures

Amino Acid Position 1a(H77) polyprotein H77 abs ref # H77 rel ref #

RI PT

daclatasvir.

Y-C/Hf

2081 2083 2137 2077 2079 2133 105 107 161 N-D

K-N

F-L

The NS5A domain I sequence of viral genomes derived from supernatant collected at the peak of infection from viral escape experiments (Supplemental Figure 1) was determined by direct sequence analysis; in selected cases subclonal analysis was carried out.

11

ACCEPTED MANUSCRIPT

a

, Nucleotide positions are numbered according to the HCV sequence of the specified plasmid. Positions, at which coding nucleotide

changes were identified, are shown. , Nucleotide identity at the respective position of the plasmid or of viral genomes recovered from supernatants of escape cultures. Each

RI PT

b

culture is specified by the applied concentration of daclatasvir. Further, the day posttreatment (Day PT) at which viral genomes were

SC

derived, is indicated. For direct sequencing results, positions with quasispecies are written with two capital letters in case of a 50/50 quasispecies and with capital letter/lowercase letter in case of a dominant and a minor sequence. In selected cases, subclonal analysis was

M AN U

carried out. Results of this analysis are indicated in the far right column. For subclonal analysis, amino acid substitutions at positions, at which substitutions were also detected in direct sequence analysis, and combinations thereof are shown; the number of subclones with the respective substitutions of the total number of subclones analysed is indicated in parenthesis. In case direct sequence analysis revealed

c

TE D

quasispecies nucleotide changes at several positions of one codon, only amino acid changes confirmed by subclonal analysis are indicated. , Amino acid positions are numbered according to the polyprotein sequence encoded by the plasmid and with absolute as well as relative

, Amino acid changes indicate first the amino acid found at the respective position of the polyprotein encoded by the plasmid and second

AC C

d

EP

H77 (GenBank ID AF009606) reference numbers.2

the amino acid found at the respective position of the polyprotein encoded by viral genomes recovered from escape cultures. e

, A6357G results in: Q30R; A6358C or A6358T result in Q30H

f

, T6545C results in Y93H; A6546G results in Y93C

12

ACCEPTED MANUSCRIPT

Supplemental Table 4. Substitutions identified in NS5A domain I of 1a(TN) recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 1a(TN) plasmid

6356 6357 6358 6380 6473 6474 6545 6546 6890

Nucleotide Identityb

Direct Sequence Analysis

1a(TN) plasmid

C

A

A

T

A

A

Subclonal Analysis

T

A

C

Day PT

None None

5 7

●

●

●

●

●

●

●

●

●

●

A

●

●

●

4xEC50 4xEC50

7 10

●

●

●

●

●

●

●

●

A/g

●

●

●

●

●

16xEC50 16xEC50

12 14

●

G/A

●

●

A/c

●

●

●

●

●

●

●

●

●

64xEC50 64xEC50

12 14

●

G/A A/g

A/c/t A/c/t

●

●

●

●

●

256xEC50 256xEC50

14 17

A/C C/a

A/g A/g

●

●

●

●

●

●

Q-R/H/Ke

2014 2010 38 S-T

●

●

●

●

A/g

●

G/A G/a

C/t

●

●

●

A/c A/c

T/c/a ●

●

A/g G/A

●

G/A G/A

●

●

●

2045 2041 69

2069 2065 93

2184 2180 208

N-H/Tf

Y-C/Hg

H-Y

AC C

Amino Acid Change d

2006 2002 30

●

EP

c

●

●

TE D

Daclatasvir concentration

M AN U

SC

1a(TN) escape cultures

Amino Acid Position 1a(TN) polyprotein H77 abs ref # H77 rel ref #

RI PT

daclatasvir.

For Table footnotes compare Supplemental Table 3. e

, C6356A results in Q30K; A6357G results in Q30R; A6358T results in Q30H

f

, A6473C results in N69H; A6474C results in N69T 13

Q30R(4/10);Q30H(1/10);Q30R+N69T(1/10);Y93C(4/10) Q30K(2/10);Q30R(4/10);Q30R+N69T(1/10);Y93H(2/10);Y93C(1/10)

ACCEPTED MANUSCRIPT

g

AC C

EP

TE D

M AN U

SC

RI PT

, T6545C results in Y93H ; A6546G results in Y93C

14

ACCEPTED MANUSCRIPT

Supplemental Table 5. Substitutions identified in NS5A domain I of 1a(HCV1) recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 1a(HCV1) plasmid

6357 6404 6509 6545 6546

Nucleotide Identityb

Direct Sequence Analysis

1a(HCV1) plasmid

A

G

A

T

A

●

●

●

●

●

●

●

●

●

●

RI PT

daclatasvir.

Daclatasvir concentration

Day PT

None None

7 9

4xEC50 4xEC50

16 19

G G/A

●

●

●

●

●

A/t

C/T

●

16xEC50 16xEC50

16 19

G/a G/a

●

● ●

T/c T/c

●

●

64xEC50 64xEC50

16 19

G/a G/a

●

●

●

●

G/a

●

T/a/c

●

256xEC50 256xEC50

23 26

●

●

●

●

●

●

●

●

C C

Amino Acid Change d

Q-R

V-I

EP

TE D

●

2069 2065 93

AC C

Amino Acid Positionc 1a(HCV1) polyprotein 2006 2022 2057 H77 abs ref # 2002 2018 2053 30 46 81 H77 rel ref #

M AN U

SC

1a(HCV1) escape cultures

R-W

Y-H/N/Se

For Table footnotes compare Supplemental Table 3. e

, T6545C results in Y93H; T6545A results in Y93N; A6546C results in Y93S

15

ACCEPTED MANUSCRIPT

Supplemental Table 6. Substitutions identified in NS5A domain I of 1a(J1) recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 1a(J1) plasmid

6341 6351 6428 6521 6545

Nucleotide Identityb

Direct Sequence Analysis

1a(J1) plasmid

A

T

C

A

T

RI PT

daclatasvir.

Day PT

None None

9 12

●

●

●

●

●

●

●

●

●

●

4xEC50 4xEC50

19 21

G G

●

●

●

●

●

C/g

●

●

16xEC50 16xEC50

19 21

●

●

●

●

●

●

●

●

C C

64xEC50 64xEC50

63 65

G G

C C

●

A/g G/a

●

●

256xEC50 256xEC50

23 26

●

●

●

●

●

●

●

●

C C

EP

●

TE D

Daclatasvir concentration

M AN U

SC

1a(J1) escape cultures

Amino Acid Change d

T-A

M-T

AC C

Amino Acid Positionc 1a(J1) polyprotein 2001 2004 2030 2061 2069 H77 abs ref # 1997 2000 2026 2057 2065 25 28 54 85 93 H77 rel ref # H-D

S-G

Y-H

For Table footnotes compare Supplemental Table 3.

16

ACCEPTED MANUSCRIPT

Supplemental Table 7. Substitutions identified in NS5A domain I of 1a(DH6) recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 1a(DH6) plasmid Nucleotide Identityb 1a(DH6) plasmid

6527

RI PT

daclatasvir.

6546

Direct Sequence Analysis A

A

Daclatasvir concentration

Day PT

None None

9 12

●

●

●

●

4xEC50 4xEC50

9 12

●

G G

16xEC50 16xEC50

9 12

●

64xEC50 64xEC50

12 14

●

256xEC50 256xEC50

16 19

A/g

G G G G G G

Amino Acid Positionc 1a(DH6) polyprotein H77 abs ref # H77 rel ref #

2063 2059 87

2069 2065 93

Amino Acid Change d

T-A

AC C

●

●

TE D

●

EP

●

M AN U

SC

1a(DH6) escape cultures

Y-C

For Table footnotes compare Supplemental Table 3.

17

ACCEPTED MANUSCRIPT

Supplemental Table 8. Substitutions identified in NS5A domain I of 1b(J4) recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 1b(J4) plasmid

6350

Nucleotide Identityb

6545

6878

Direct Sequence Analysis

1b(J4) plasmid

C

T

A

Subclonal Analysis

SC

1b(J4) escape cultures Day PT

None None

5 7

●

●

●

●

●

●

4xEC50 4xEC50

12 17

●

C C

●

16xEC50 16xEC50

14 17

●

A/C C

●

64xEC50 64xEC50

17 17

●

C/A C

●

●

256xEC50 256xEC50

26 28

A/C A

A A

G/A

2004 2000 28

2069 2065 93

2180 2176 204

L-M

Y-N/He

T-A

●

●

L28M+Y93N(6/6)

●

AC C

c

Amino Acid Change d

TE D

●

●

EP

●

M AN U

Daclatasvir concentration

Amino Acid Position 1b(J4) polyprotein H77 abs ref # H77 rel ref #

RI PT

daclatasvir.

For Table footnotes compare Supplemental Table 3. e

, T6545C results in Y93H; T6545A results in Y93N

18

ACCEPTED MANUSCRIPT

Supplemental Table 9. Substitutions identified in NS5A domain I of 1b(DH1) recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 1b(DH1) plasmid

6284 6358 6359 6521 6522 6545 6677

Nucleotide Identityb

Direct Sequence Analysis

1b(DH1) plasmid

A

G

T

T

A

T

A

G G

●

●

●

●

RI PT

daclatasvir.

Day PT

None None

4 6

●

●

●

●

●

●

●

●

4xEC50 4xEC50

9 11

●

T T

●

C C

●

●

●

●

●

●

16xEC50 16xEC50

9 11

●

T T

●

C C

●

●

●

●

●

●

64xEC50 64xEC50

14 16

A/g

T/G G/t

●

●

●

C/t C/t

G/A G/A

256xEC50 256xEC50

16 18

G/a G/a

T T

A A

C C

●

●

c

Amino Acid Change d

1982 2006 2007 1978 2002 2003 6 30 31 R-G

Q-H

●

L-I

C/T C/t

G/A G/A

●

●

●

●

EP

●

2061 2057 85

2069 2113 2065 2109 93 137

AC C

Amino Acid Position 1b(DH1) polyprotein H77 abs ref # H77 rel ref #

G/A

●

TE D

Daclatasvir concentration

M AN U

SC

1b(DH1) escape cultures

Y-C/He

Y-H

N-D

For Table footnotes compare Supplemental Table 3. e

, T6521C results in Y85H; A6522G results in Y85C.

19

ACCEPTED MANUSCRIPT

Supplemental Table 10. Substitutions identified in NS5A domain I of 2a(JFH1) recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 2a(JFH1) plasmid

6351

Nucleotide Identityb

Direct Sequence Analysis

2a(JFH1) plasmid

6359

6545

RI PT

daclatasvir.

6825

T

C

T

C

Subclonal Analysis

SC

2a(JFH1) escape cultures Day PT

None None

7 10

●

●

●

●

●

●

●

●

4xEC50 4xEC50

12 14

C/t

C/a

●

●

●

●

C

●

16xEC50 16xEC50

12 14

T/c C/T

C/a C/a

T/c C/T

● ●

F28S(4/6);L31M(1/6);Y93N(1/6)

64xEC50 64xEC50

12 14

C/T C/t

●

C/T T/c

●

original(1/10);F28S(7/10);Y93H(2/10)

●

256xEC50 256xEC50

12 14

C C

●

●

C/t

●

●

●

2004 2000 28

2007 2003 31

2069 2065 93

2162 2158 186

F-S

L-M

Y-H

S-F

c

Amino Acid Change d

TE D

EP

●

AC C

Amino Acid Position 2a(JFH1) polyprotein H77 abs ref # H77 rel ref #

M AN U

Daclatasvir concentration

For Table footnotes compare Supplemental Table 3.

20

ACCEPTED MANUSCRIPT

Supplemental Table 11. Substitutions identified in NS5A domain I of 2a(J6) recovered from Huh7.5 cell cultures treated with daclatasvir.

6338 6350 6351 6452 6489 6542 6545 6549 6578 6636 6654 6678 6698 6866 6872

Nucleotide Identityb

Direct Sequence Analysis

2a(J6) plasmid

A

T

T

A

T

T

T

C

C

A

A

C

A

A

Subclonal Analysis

Daclatasvir concentration

Day PT

None None

5 7

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

4xEC50 4xEC50

7 10

●

●

●

●

●

●

●

●

A/g

●

●

●

●

●

●

●

16xEC50 16xEC50

12 14

T/A A/t

T/a

T/c T/c

A/g

T/c

T/a

●

●

T/c C/T/a

●

●

64xEC50 64xEC50

14 17

●

●

A/g G/A

●

●

T/c T/c

●

●

256xEC50 256xEC50

19 21

●

T/c

●

●

C/t C

2004 2000 28

Amino Acid Change d T-A/Se F-I/L/Sf

●

●

●

A/g

●

●

●

●

●

●

●

A/g

●

●

●

●

●

●

●

●

●

●

●

A/g

●

●

●

●

●

●

●

●

●

●

●

G/A

●

●

●

●

C/g

●

●

TE D C/t C/t

EP

●

●

AC C

Amino Acid Positionc 2a(J6) polyprotein 2000 H77 abs ref # 1996 24 H77 rel ref #

●

M AN U

SC

2a(J6) escape cultures

A

RI PT

Nucleotide Positiona 2a(J6) plasmid

C/a

C/t

●

●

●

●

A/g

●

●

●

●

●

●

●

●

●

G/A A/g

●

●

●

●

●

●

●

●

●

●

●

●

●

T/c

●

●

●

●

●

●

●

●

2038 2050 2068 2069 2070 2080 2099 2105 2113 2120 2176 2178 2034 2046 2064 2065 2066 2076 2095 2101 2109 2116 2172 2174 62 74 92 93 94 104 123 129 137 144 200 202 N-D

I-T

C-S Y-H/Ng T-K

P-S Q-R Y-C N-S

21

L-V M-V M-V

original(2/15);T24S(2/15) F28S(4/15);F28L+Y129C(1/15) F28I+N62D+Y93N(1/15) Y93H(2/15);Y93N(2/15) Y93H+L144V(1/15) F28S+Y93H(1/10);Y93H(3/10) N62D+Y93H(6/10) F28L(1/10);F28L+Y93H+Q123R(2/10) F28S(4/10);F28S+Q123R(3/10)

ACCEPTED MANUSCRIPT

For Table footnotes compare Supplemental Table 3. e

, A6338G results in T24A; A6338T results in T24S

, T6350C results in F28L; T6350A results in F28I; T6351C results in F28S

g

AC C

EP

TE D

M AN U

SC

, T6545C results in Y93H; T6545A results in Y93N

RI PT

f

22

ACCEPTED MANUSCRIPT

Supplemental Table 12. Substitutions identified in NS5A domain I of 2b(J8) recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 2b(J8) plasmid

6338 6339 6368 6542 6545

Nucleotide Identityb

Direct Sequence Analysis

2b(J8) plasmid

T

C

A

T

T

Subclonal Analysis

Daclatasvir concentration

Day PT

None None

7 9

●

●

●

●

A/g A/g

●

●

●

●

4xEC50 4xEC50

16 19

●

●

●

●

●

C C

●

●

16xEC50 16xEC50

23 26

●

T/C T/C

A/g

●

●

●

C C

64xEC50 64xEC50

23 26

A/t A/t

●

●

●

●

●

●

Amino Acid Change d

S-F/Te

2010 2068 2069 2006 2064 2065 34 92 93 I-V

C-R

TE D

2000 1996 24

C C

S24F(1/9);S24F+Y93H(6/9);I34V+Y93H(2/9) S24T+Y93H(6/7);Y93H(1/7)

EP

Amino Acid Positionc 2b(J8) polyprotein H77 abs ref # H77 rel ref #

●

Y-H

AC C

●

M AN U

SC

2b(J8) escape cultures

For Table footnotes compare Supplemental Table 3. e

RI PT

daclatasvir.

, T6338A results in S24T; C6339T results in S24F

23

ACCEPTED MANUSCRIPT

Supplemental Table 13. Substitutions identified in NS5A domain I of 3a(S52) recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 3a(S52) plasmid

6276

Nucleotide Identityb

Direct Sequence Analysis

3a(S52) plasmid

6359

6545

6563

A

C

T

A

Subclonal Analysis

SC

3a(S52) escape cultures Day PT

None None

2 5

G G

●

●

●

●

●

●

4xEC50 4xEC50

9 12

G G

●

C C/t

G

16xEC50 16xEC50

9 12

G G

●

C C

●

64xEC50 64xEC50

9 12

G G

●

C C/T

●

256xEC50 256xEC50

9 12

G G

●

C C

●

●

1979 1975 3

2007 2003 31

2069 2065 93

2075 2071 99

D-G

L-F

Y-H

T-A

T/C

●

TE D

●

Y93H+T99A(6/6) L31F(2/10);Y93H(8/10)

●

L31F(2/10);Y93H(7/10);L31F+Y93H(1/10)

●

●

EP

C/t

M AN U

Daclatasvir concentration

AC C

c

Amino Acid Position 3a(S52) polyprotein H77 abs ref # H77 rel ref # Amino Acid Change d

RI PT

daclatasvir.

For Table footnotes compare Supplemental Table 3. Green shading indicates cell culture adaptive substitution D3G1.

24

ACCEPTED MANUSCRIPT

Supplemental Table 14. Substitutions identified in NS5A domain I of 4a(ED43) recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 4a(ED43) plasmid

6291 6303 6335 6356 6357 6441 6443 6545 6726

Nucleotide Identityb

Direct Sequence Analysis

4a(ED43) plasmid

T

T

C

C

T

C

● ●

T/c

●

●

●

●

●

●

A

●

●

●

T/c

Subclonal Analysis

T

T

T

SC

4a(ED43) escape cultures Day PT

None None

5 7

4xEC50 4xEC50

14 17

T/c

●

●

●

●

●

●

●

●

●

●

T/c

T/c T/c

16xEC50

14

T/c

●

●

●

A

●

T/c

16xEC50

17

C/T

T/c

●

●

●

T

T/c

●

●

64xEC50 64xEC50

14 20

●

●

●

●

●

●

●

●

C C/T

●

C/T

T T/C

T/C

C/T

●

●

256xEC50 256xEC50

17 20

●

C C

●

T T

C C

●

C/T

●

T/g

●

●

●

●

c

V-A

V-A

2006 2002 30

L-I

L-H/Se

●

●

●

T/g

C/t T/c

T/g

●

T/g

TE D ●

●

2034 2035 2069 2129 2030 2031 2065 2125 58 59 93 153

AC C

Amino Acid Change d

1984 1988 1999 1980 1984 1995 8 12 23

EP

●

M AN U

Daclatasvir concentration

Amino Acid Position 4a(ED43) polyprotein H77 abs ref # H77 rel ref #

RI PT

daclatasvir.

P-L

C-R

Y-H

For Table footnotes compare Supplemental Table 3. e

, T6357A results in L30H; C6356T+T6357C results in L30S 25

V-G

V8A+L30H(6/10) L30H(3/10);Y93H(1/10)

V8A+P58L(2/9);L30S(7/9)

ACCEPTED MANUSCRIPT

Supplemental Table 15. Substitutions identified in NS5A domain I of 5a(SA13) recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 5a(SA13) plasmid

6284

Nucleotide Identityb

Direct Sequence Analysis

5a(SA13) plasmid

6359

6581

6653

A

C

A

T

Subclonal Analysis

Day PT

None None

10 12

G G

●

●

●

●

●

●

4xEC50 4xEC50

19 21

G G

T T

●

●

●

●

16xEC50 16xEC50

19 21

G G

T T

●

T/c C/T

64xEC50 64xEC50

21 24

G G

T T/g

A/c

●

●

●

256xEC50 256xEC50

21 24

G G

T T

●

●

●

●

1982 1978 6

2007 2003 31

2081 2077 105

2105 2101 129

R-G

L-F/Ve

N-H

Y-H

Amino Acid Change d

L31F(9/9)

AC C

c

L31F(5/10);L31F+Y129H(5/10)

EP

●

TE D

Daclatasvir concentration

M AN U

SC

5a(SA13) escape cultures

Amino Acid Position 5a(SA13) polyprotein H77 abs ref # H77 rel ref #

RI PT

daclatasvir.

For Table footnotes compare Supplemental Table 3. Green shading indicates cell culture adaptive substitution R6G1. e

, C6359T results in L31F ; C6359G results in L31V

26

ACCEPTED MANUSCRIPT

Supplemental Table 16. Substitutions identified in NS5A domain I of 6a(HK6a) recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 6a(HK6a) plasmid

6350 6357 6359 6440 6441 6902

Nucleotide Identityb

Direct Sequence Analysis

6a(HK6a) plasmid

RI PT

daclatasvir.

Subclonal Analysis

C

G

C

A

C

G

Day PT

None None

2 5

●

●

●

●

●

G/a

●

●

●

●

●

●

4xEC50 4xEC50

14 16

●

●

●

●

●

●

A/g G/A

●

●

●

●

16xEC50 16xEC50

16 19

T/C T/C

●

●

●

●

●

●

●

●

A/C

●

64xEC50 64xEC50

19 21

●

G/a

●

●

C/a A/c

A/g

●

●

●

256xEC50 256xEC50

21 23

●

●

●

●

A A/c

●

●

●

●

Amino Acid Change d

L-F

R-H

● ●

EP

2004 2006 2007 2000 2002 2003 28 30 31

●

●

2034 2030 58

2188 2184 212

T-A/Ne

E-K

AC C

Amino Acid Position 6a(HK6a) polyprotein H77 abs ref # H77 rel ref #

c

L-M

L28F(5/9);T58N(3/9);T58A(1/9)

TE D

Daclatasvir concentration

M AN U

SC

6a(HK6a) escape cultures

For Table footnotes compare Supplemental Table 3. e

, A6440G results in T58A; C6441A results in T58N

27

ACCEPTED MANUSCRIPT

Supplemental Table 17. Substitutions identified in NS5A domain I of 7a(QC69) recovered from Huh7.5 cell cultures treated with daclatasvir.

6339 6356 6359 6395 6434 6459 6615 6735 6740 6858 6870 6887

Nucleotide Identityb

Direct Sequence Analysis

7a(QC69) plasmid

A

A

C

T

A

C

C

T

A

A

Subclonal Analysis

T

SC

7a(QC69) escape cultures

A

RI PT

Nucleotide Positiona 7a(QC69) plasmid

Day PT

None None

5 7

●

●

●

●

●

●

●

A/g

●

●

●

●

●

●

4xEC50

19

G/A

●

T

T/C

●

●

T/C

4xEC50

21

G

G/a

T/C

●

●

16xEC50 16xEC50

19 21

G G

G G

●

●

●

●

●

64xEC50 64xEC50

19 21

G G

●

T T

●

256xEC50 256xEC50

40 42

C/A G/C/A

●

T T

●

●

●

●

●

●

●

●

A/t

●

T/c

●

●

●

C/T

K24R+L31F+T116M+S207P(4/13) K24R+L31F+Y43H+T116M+S207P(2/13) L31F(2/13);L31F+V156A(4/13) L31F+V156A+S207P(1/13) original(3/7);K24R+S30G(2/7) K24R+L31F+T116M(1/7) K24R+L31F+Y43H+T116M+S207P(1/7)

TE D

●

●

T/C

●

●

●

●

●

●

●

A/g

●

●

●

●

T T

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

C/t

●

●

●

●

●

●

●

A/g

●

●

●

●

●

●

●

●

EP

●

●

AC C

●

M AN U

Daclatasvir concentration

Amino Acid Positionc 7a(QC69) polyprotein 2000 2006 2007 2019 2032 2040 2092 2132 2134 2173 2177 2183 H77 abs ref # 1996 2002 2003 2015 2028 2036 2088 2128 2130 2169 2173 2179 30 31 43 56 64 116 156 158 197 201 207 H77 rel ref # 24 Amino Acid Change d K-R/Te S-G

L-F

Y-H

K-E

A-V

T-M

V-A

28

I-V

Q-L

Q-L

S-P

K24R+L31F(6/6)

ACCEPTED MANUSCRIPT

For Table footnotes compare Supplemental Table 3. e

AC C

EP

TE D

M AN U

SC

RI PT

, A6339G results in K24R; A6339C results in K24T.

29

ACCEPTED MANUSCRIPT

Nucleotide Positiona 1a(H77) plasmid

6381 6440 6474 6511 6545 6546 6665 6713

Nucleotide Identityb

Direct Sequence Analysis

1a(H77) plasmid

C

C

A

G

T

A

A

C C

●

●

G/A

●

C C

G G

●

T

Day PT 7 9

●

●

A/c

●

●

●

●

●

4xEC50 4xEC50

21 24

●

●

●

●

●

●

●

G/c

16xEC50 16xEC50

21 24

T T/c

●

●

●

C/g

●

●

Amino Acid Positionc 1a(H77) polyprotein H77 abs ref # H77 rel ref #

2014 2034 2045 2057 2010 2030 2041 2053 38 58 69 81

Amino Acid Change d

S-F

N-T

R-S

● ●

●

●

●

●

●

●

C/t

2069 2065 93

2109 2125 2105 2121 133 149

Y-H/Re

M-V

F-L

AC C

H-D

C C

A/g

●

TE D

None None

EP

Daclatasvir concentration

M AN U

1a(H77)Y93H escape cultures

SC

with daclatasvir.

RI PT

Supplemental Table 18. Substitutions identified in NS5A domain I of 1a(H77)Y93H recovered from Huh7.5 cell cultures treated

For Table footnotes compare Supplemental Table 3. Blue shading indicates engineered substitution Y93H1. e

, T6545C: Y93H; T6545C+A6546G: Y93R

30

ACCEPTED MANUSCRIPT

Supplemental Table 19. Substitutions identified in NS5A domain I of 1b(J4)Y93H recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 1b(J4) plasmid

6359

Nucleotide Identityb

6361

6545

RI PT

daclatasvir.

6666

Direct Sequence Analysis

1b(J4) plasmid

T

A

T

T

C C

●

Day PT

None None

10 12

●

●

●

●

4xEC50 4xEC50

35 38

G/A G/A

●

C C

●

●

16xEC50 16xEC50

35 38

G/T G/T

T/A T/a

C C

●

64xEC50 64xEC50

38 40

G G

●

C C

256xEC50 256xEC50

42 45

G G

●

Amino Acid Change d

L-I/Ve

TE D T/c C

C C

c

2007 2003 31

●

● ●

EP

●

●

2069 2065 93

2109 2105 133

Y-H

M-T

AC C

Amino Acid Position 1b(J4) polyprotein H77 abs ref # H77 rel ref #

●

●

M AN U

Daclatasvir concentration

SC

1b(J4)Y93H escape cultures

For Table footnotes compare Supplemental Table 3. Blue shading indicates engineered substitution Y93H1. e

, T6359G results in L31V; T6359A results in L31I.

31

ACCEPTED MANUSCRIPT

Supplemental Table 20. Substitutions identified in NS5A domain I of 2a(JFH1)Y93H recovered from Huh7.5 cell cultures treated

Nucleotide Positiona 2a(JFH1) plasmid

6351 6353 6501 6545 6677

Nucleotide Identityb

Direct Sequence Analysis

2a(JFH1) plasmid

T

C

A

T

A

C C

A/g

Subclonal Analysis

Day PT

None None

10 12

●

●

●

●

●

●

4xEC50 4xEC50

10 12

G/t G

●

● ●

C C

●

●

16xEC50 16xEC50

14 17

G/T G/T

T/c T/c

A/g A/g

C C

●

64xEC50 64xEC50

17 19

●

T T

●

C C

●

●

F28C+Y93H(5/8);P29S+Y93H(1/8);P29S+K78R+Y93H(2/8)

●

●

c

Amino Acid Change d

2004 2005 2054 2069 2113 2000 2001 2050 2065 2109 28 29 78 93 137 F-C

P-S

K-R

Y-H

N-D

TE D

●

●

EP

Amino Acid Position 2a(JFH1) polyprotein H77 abs ref # H77 rel ref #

●

M AN U

Daclatasvir concentration

SC

2a(JFH1)Y93H escape cultures

RI PT

with daclatasvir.

AC C

For Table footnotes compare Supplemental Table 3. Blue shading indicates engineered substitution Y93H1.

32

ACCEPTED MANUSCRIPT

Supplemental Table 21. Substitutions identified in NS5A domain I of 2a(J6)Y93H recovered from Huh7.5 cell cultures treated with

Nucleotide Positiona 2a(J6) plasmid

RI PT

daclatasvir.

6350 6353 6358 6381 6452 6544 6545 6857

b

Direct Sequence Analysis

Nucleotide Identity 2a(J6) plasmid

T

C

G

Subclonal Analysis

C

A

T

T

G

Day PT

None None

7 10

●

●

●

●

●

●

●

●

T/c

●

●

C C

●

●

4xEC50

14

C/T

C/t

G/c

C/t

G/a

●

C

G/a

4xEC50

17

C/t

C/t

G/c

C/t

G/A

T/g

C

●

c

Amino Acid Change d

2004 2005 2006 2014 2038 2068 2069 2173 2000 2001 2002 2010 2034 2064 2065 2169 28 29 30 38 62 92 93 197 F-L

P-S

K-N

S-F

N-D C-W

F28L+Y93H(1/7);F28L+N62D+Y93H(5/7);F28L+C92W+Y93H(1/7) F28L+Y93H(1/7);F28L+N62D+Y93H(5/7);F28L+C92W+Y93H(1/7)

TE D

Amino Acid Position 2a(J6) polyprotein H77 abs ref # H77 rel ref #

●

M AN U

Daclatasvir concentration

SC

2a(J6)Y93H escape cultures

Y-H

D-N

AC C

EP

For Table footnotes compare Supplemental Table 3. Blue shading indicates engineered substitution Y93H1.

33

ACCEPTED MANUSCRIPT

Supplemental Table 22. Substitutions identified in NS5A domain I of 3a(S52)Y93H recovered from Huh7.5 cell cultures treated

Nucleotide Positiona 3a(S52) plasmid

6276 6359 6492 6545 6563 6713 6714

Nucleotide Identityb

Direct Sequence Analysis

3a(S52) plasmid

A

C

C

T

A

T

C C

●

C/T

●

G/a

●

●

●

●

●

●

●

T/a

RI PT

with daclatasvir.

T

Day PT

None None

5 7

G G

●

●

●

●

4xEC50 4xEC50

14 12

G G

G G

C/t

C C

●

Amino Acid Positionc 3a(S52) polyprotein 1979 2007 2051 2069 2075 H77 abs ref # 1975 2003 2047 2065 2071 3 31 75 93 99 H77 rel ref # Amino Acid Change d

D-G

L-V

A-V Y-H T-A

2125 2121 149

TE D

Daclatasvir concentration

M AN U

SC

3a(S52)Y93H escape cultures

F-L/Ye

e

AC C

indicates engineered substitution Y93H.1

EP

For Table footnotes compare Supplemental Table 3. Green shading indicates cell culture adaptive substitution D3G1. Blue shading

, T6713C results in F149L; T6714A results in F149Y.

34

ACCEPTED MANUSCRIPT

Supplemental Table 23. Substitutions identified in NS5A domain I of 4a(ED43)Y93H recovered from Huh7.5 cell cultures treated

Nucleotide Positiona 4a(ED43) plasmid

6308 6312 6356 6357 6440 6443 6545 6636 6726

b

Direct Sequence Analysis

Nucleotide Identity 4a(ED43) plasmid

RI PT

with daclatasvir.

A

T

C

T

C

Subclonal Analysis

T

T

G

T

T/g

Day PT

None None

12 14

●

●

●

●

●

●

●

●

●

●

T/c

C C

●

●

4xEC50 4xEC50

16 19

G/A A/g

T/c T/c

●

C/T C/T

T/C T/C

T/c T/c

C C

●

16xEC50 16xEC50

19 23

●

●

● ●

T/c T/c

C C

●

●

A A

●

●

64xEC50 64xEC50

26 28

●

●

C C

C C

G/a

●

●

T T

●

●

●

●

256xEC50 256xEC50

28 30

●

●

●

●

T/C

C C

●

●

A A/c

●

●

●

●

Amino Acid Change d

T-A

V-A

●

T/c T/c T/c T/c

L-H/P/Se

●

●

T14A+L30H+Y93H(1/7);T14A+P58S+Y93H(1/7) L30P+Y93H(1/7);V15A+L30P+Y93H(3/7) P58S+Y93H(1/7)

●

● ●

TE D

●

●

●

EP

2006 2002 30

●

2034 2035 2069 2099 2129 2030 2031 2065 2095 2125 58 59 93 123 153

AC C

Amino Acid Positionc 4a(ED43) polyprotein 1990 1991 H77 abs ref # 1986 1987 14 15 H77 rel ref #

●

●

M AN U

Daclatasvir concentration

SC

4a(ED43)Y93H escape cultures

P-S

C-R

Y-H

R-K

V-G

For Table footnotes compare Supplemental Table 3. Blue shading indicates engineered substitution Y93H1. e

, T6357C results in L30P; T6357A results in L30H; C6356T+T6357C results in L30S.

35

ACCEPTED MANUSCRIPT

Supplemental Table 24. Substitutions identified in NS5A domain I of 5a(SA13)T93H recovered from Huh7.5 cell cultures treated

Nucleotide Positiona 5a(SA13) plasmid

6284 6353 6358

6359

6363 6545 6546 6547 6677 6678 6740 6764 6812 6893

b

Direct Sequence Analysis

Nucleotide Identity 5a(SA13) plasmid

A

C

A

C

C

A

C

A

A

A

Day PT

None None

7 9

G G

●

●

●

●

●

●

●

●

C C

A A

4xEC50

14

G

C/t

A/c/t

C/a/g

T/C

C

A

4xEC50

16

G

●

A/c

C/a/g/t

●

C

A

16xEC50 16xEC50

14 16

G G

●

A/c A/c

A/c/t A/T/c

●

●

C C

64xEC50 64xEC50

19 21

G G

●

● ●

T/g T

●

●

C C

256xEC50 256xEC50

19 21

G G

●

●

●

●

P-S

A

T

A

2007 2003 31

●

2008 2004 32

Q-H L-F/I/Ve P-L

C C

●

●

●

●

●

G/A A/g

●

●

●

●

●

C

●

●

A/g

●

●

●

C

●

●

G/A

●

●

●

A A

C C

●

●

●

●

●

●

●

●

G/A

●

●

●

A A

C C

●

●

●

●

●

G/A G/A

●

●

●

A/T

●

A A

C C

A/g A/g

A/g

G/A G/A

A/g

●

●

●

●

A/g

TE D

●

AC C

Amino Acid Positionc 5a(SA13) polyprotein 1982 2005 2006 H77 abs ref # 1978 2001 2002 6 29 30 H77 rel ref #

●

EP

T T

●

M AN U

Daclatasvir concentration

R-G

A

Subclonal Analysis

SC

5a(SA13)Y93H escape cultures

Amino Acid Change d

RI PT

with daclatasvir.

C C

2069 2065 93

2113 2109 137

T-H

N-D

●

original(3/9);Q30H+T93H(3/9) L31V+T93H(2/9);P32L+T93H(1/9) Q30H+T93H(2/7);L31I+T93H(2/7) L31F+T93H(2/7);P32L+T93H(1/7) Q30H+T93H(1/8) L31I+T93H(4/8);L31F+T93H(3/8)

2134 2142 2158 2185 2130 2138 2154 2181 158 166 182 209 I-V

N-D

F-I

I-V

For Table footnotes compare Supplemental Table 3. Green shading indicates cell culture adaptive substitution R6G1. Blue shading indicates engineered substitution T93H1. 36

ACCEPTED MANUSCRIPT

e

AC C

EP

TE D

M AN U

SC

RI PT

, C6359A results in L31I; C6359G results in L31V; C6359T results in L31F.

37

ACCEPTED MANUSCRIPT

Supplemental Table 25. Substitutions identified in NS5A domain I of 6a(HK6a)T93H recovered from Huh7.5 cell cultures treated

Nucleotide Positiona 6a(HK6a) plasmid

6308 6350 6357 6440 6441 6545 6546 6647 6902

Nucleotide Identityb

Direct Sequence Analysis

6a(HK6a) plasmid

A

Subclonal Analysis

C

G

A

C

A

C

T

G

A/G A

●

●

A A

●

●

C C

●

●

C/t

●

A A/G

●

●

A A

●

●

C C

●

●

●

●

A A

●

G/a

●

●

Daclatasvir concentration

Day PT

None None

7 10

●

●

●

●

4xEC50 4xEC50

7 10

●

●

●

●

16xEC50 16xEC50

10 12

64xEC50 64xEC50 256xEC50 256xEC50

M AN U

SC

6a(HK6a)Y93H escape cultures

●

A A

●

●

●

●

●

C C

14 17

●

C/a A/C

A A

A/g A/g

C/g C/g

C C

A/g A

●

●

●

●

G/a

14 17

●

●

G/A G/a

C/g C/g

C C

A/g A

●

●

A A

●

●

●

G/a

T-A

L-I

EP

2034 2030 58

AC C

Amino Acid Positionc 6a(HK6a) polyprotein 1990 2004 2006 H77 abs ref # 1986 2000 2002 14 28 30 H77 rel ref #

TE D

●

G/A

Amino Acid Change d

RI PT

with daclatasvir.

R-H

T-A

2069 2065 93

T-H/Re

L28I+R30H*+T93H(5/8) R30H*+Y93R(1/8);R30H*+T58A+T93H(2/8) R30H*+T58A+T93H(8/8)

2103 2188 2099 2184 127 212 C-R

E-K

For Table footnotes compare Supplemental Table 3. Blue shading indicates engineered substitution T93H1. *R30H was listed despite occurrence in non-treated cultures. 38

ACCEPTED MANUSCRIPT

e

AC C

EP

TE D

M AN U

SC

RI PT

, A6545C+C6546A results in T93H; A6545C+C6546G results in T93R

39

ACCEPTED MANUSCRIPT

RI PT

Reference List

1. Scheel TK, Gottwein JM, Mikkelsen LS, et al. Recombinant HCV variants with NS5A from genotypes 1-7 have different sensitivities

SC

to an NS5A inhibitor but not interferon-alpha. Gastroenterology 2011;140:1032-1042.

2. Simmonds P, Bukh J, Combet C, et al. Consensus proposals for a unified system of nomenclature of hepatitis C virus genotypes.

AC C

EP

TE D

M AN U

Hepatology 2005;42:962-973.

40