Volume 141, Issue 3, Pages 1067-1079 (September 2011) Differential Efficacy of Protease Inhibitors Against HCV Genotypes 2a, 3a, 5a, and 6a NS3/4A Protease Recombinant Viruses  Judith M. Gottwein, Troels K.H. Scheel, Tanja B. Jensen, Lubna Ghanem, Jens Bukh  Gastroenterology  Volume 141, Issue 3, Pages 1067-1079 (September 2011) DOI: 10.1053/j.gastro.2011.06.004 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Viability of J6/JFH1-based recombinants with genotype-specific NS3P/NS4A following transfection of Huh7.5 cells. Cells were split every 2–3 days. Percentage of HCV NS5A antigen positive cells in the transfection cultures was determined by immunostaining. T1, T2, T3; First, second, third transfection experiment, respectively. 3a(S52), 5a(SA13), and 6a(HK6a) spread only after long-term culture suggesting adaptation. Supernatant taken after virus spread was used to passage virus to naïve Huh7.5 cells; the open reading frame sequences of virus derived from such subsequent viral passage experiments are shown in Supplementary Tables 4–6. Several replicate transfections, including 2 for 5a(SA13), not leading to viral spread are not shown; for details, see Supplementary Results, “Viability of genotype 1a, 1b, 4a, and 7a NS3P/NS4A Recombinants in Huh7.5 Cells.” Gastroenterology 2011 141, 1067-1079DOI: (10.1053/j.gastro.2011.06.004) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Identification of adaptive mutations leading to efficient growth of genotype 3a (A), 5a (B), and 6a (C) NS3P/NS4A recombinants. Huh7.5 cells were transfected with RNA transcripts of genotype(isolate) as indicated with or without engineered putative adaptive mutations as indicated by dots; mutants are specified by numbers. T1, T2; First and second transfection experiments, respectively. Infectivity titers in culture supernatant are means of 3 replicates with standard error of mean. The lower limit of detection in the experiments shown was up to 2.4-log10 focus forming units (FFU)/mL, indicated by y-axis break; lower titers are shown as < 2.4-log10 FFU/mL. ♦, culture stopped because of cell death due to viral infection.18 #Supernatants used for viral passage; open reading frame sequences and peak infectivity titers of passaged viruses are shown in Supplementary Tables 4–6. Numbers above graphs indicate days, at which viral spread to the complete cell culture occurred; nd, no spread during an observation period of 15–30 days. Gastroenterology 2011 141, 1067-1079DOI: (10.1053/j.gastro.2011.06.004) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 NS3P/NS4A recombinants of genotypes 2a, 3a, 5a, and 6a prototype isolates or with resistance mutations showed differential sensitivity to protease inhibitors but not to interferon-alfa2. Cultures were infected with culture adapted NS3P/NS4A recombinants of the indicated genotype(isolate) or 2a(JFH1) with putative resistance mutations, 2a(JFH1)RES; for details on virus stocks see Table 2 footnotes. Cultures were treated with protease inhibitor (A) VX-950, (B) Sch503034, (C) TMC435350, (D) ITMN-191, (E) MK-7009, or (F) interferon-alfa2b and evaluated as described in Materials and Methods section. Values are means of 3 replicates with standard error of mean. Median EC50 values from these and replicate experiments are shown in Tables 2 and 3. Gastroenterology 2011 141, 1067-1079DOI: (10.1053/j.gastro.2011.06.004) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Compared with genotype 2a, genotype 3a isolates were less sensitive to protease inhibitors. Cultures were infected with culture adapted NS3P/NS4A recombinants of the indicated 2a and 3a isolates; for details on virus stocks, see Table 2 footnotes. Cultures were treated with (A) VX-950, (B) Sch503034, (C) TMC435350, (D) ITMN-191, (E) MK-7009, or (F) interferon-alfa2 and evaluated as described in Materials and Methods section. Values are means of 3 replicates with standard error of mean. Median EC50 values from these and replicate experiments are shown in Tables 2 and 3. *Data also shown in Figure 3 are included for comparison. #For VX-950, these experiments were carried out with a delay of ∼12 months compared with other experiments. Apparently, VX-950 was only partially stable in dimethyl sulfoxide during this period because efficacy against the 2a(JFH1) control was decreased ∼4.4-fold. For these experiments, values were normalized with a factor (=4.4) compared with direct calculations. Gastroenterology 2011 141, 1067-1079DOI: (10.1053/j.gastro.2011.06.004) Copyright © 2011 AGA Institute Terms and Conditions