FIG 8.

Combinations of substitutions at positions 155 and 168 increased viral fitness but not PI resistance. RNA transcripts from the indicated genotype 2a (isolate JFH1) and genotype 3a (isolate 452) recombinants were transfected into S29 cells. Four hours later, cultures were treated with the indicated concentrations of paritaprevir (ABT-450) or boceprevir. IC core concentrations 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 IC and EC infectivity titers and EC core levels determined in these experiments are shown in Fig. S7 in the supplemental material. Transfections of recombinants of the same genotype (isolate) were carried out in the same experiment. “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 NS3P variants. The NS3P substitutions in boldface were specifically selected in the indicated virus under treatment with the indicated PI. For comparison, the fold resistance to paritaprevir or boceprevir, as determined in Huh7.5 cells, is indicated above each variant. The fold resistance values were calculated as described in Materials and Methods. Values for boceprevir determined in this study (Fig. 4) are indicated in boldface; values for paritaprevir were reported previously (28). NA (not applicable) indicates that for single R155T and R155K variants, fold resistance values in Huh7.5 cells could not be determined, because introduction of these single substitutions led to the acquisition of additional substitutions at position 168 in first-passage virus stocks used for treatment experiments (Fig. 3) (28).