FIG 7.

Key resistance substitutions at NS3P positions 155, 156 and 168 rescued the replication of genotype 2a (isolate JFH1) under treatment with newer PIs. RNA transcripts from the indicated genotype 2a (isolate JFH1) recombinants were transfected into S29 cells, and 4 h later, cultures were treated with the indicated concentrations of grazoprevir (MK-5172) (A) or paritaprevir (ABT-450) (B). IC core concentrations and infectivity titers were determined as described in Materials and Methods. To account for possible differences in transfection efficiency, IC core concentrations at 48 h were normalized to IC core concentrations at 4 h. The EC core concentrations and infectivity titers determined in these experiments are shown in Fig. S6 in the supplemental material. Transfections of recombinants treated with paritaprevir were carried out in the same experiment. Transfections of recombinants treated with grazoprevir were carried out in two different experiments. For each experiment, the original genotype 2a (isolate JFH1) recombinant was included. “Control” indicates the replication-deficient genotype 2a (isolate JFH1) GND negative-control virus. For this control, IC core concentrations at 48 h ranged from 9.4 × 10³ to 25.0 × 10³ fmol/liter. The values shown were normalized to 4-h IC core values as described for the NS3P variants. The break in the y axis indicates the lower cutoff (LOC) of the infectivity titration assay. For automated counting of FFU, the LOC for IC infectivity titers was 1.5 log₁₀ FFU/well. For genotype 2a (isolate JFH1) A156V treated with grazoprevir, low replication efficiency in S29 cells precluded automated counting. FFU were counted manually, and the resulting titers are indicated by an asterisk. The LOC for IC infectivity titers derived from manually counted FFU was 0.9 log₁₀ FFU/well. NS3P substitutions in boldface were specifically selected in the indicated virus under PI treatment in the current study. ■, the substitution was identified in escape variants emerging under treatment with the newer PIs (Fig. 2). For comparison, the fold resistance to grazoprevir (A) or paritaprevir (B) as determined in Huh7.5 cells (28) is indicated above each variant. Instances in which the fold resistance values could not be determined because the specified recombinant could not be grown in Huh7.5 cells (Fig. 3) (28) are indicated by NA (not applicable).