FIG 6.

Substitutions compensating for fitness impairment acted at different steps of the viral life cycle. The effects of combinations of substitutions at positions 18 and 155 (A), positions 54 and 98 (B), and positions 155 and 168 (C) on the HCV life cycle were studied by transfection of RNA transcripts from the indicated recombinants into S29 cells, followed by the determination of IC and EC core concentrations and infectivity titers as described in Materials and Methods. To account for possible differences in transfection efficiency, IC and EC core concentrations at 48 h were normalized to IC core concentrations at 4 h. To determine the effects of the indicated NS3P substitutions on viral fitness, normalized core values and infectivity titers of variant recombinants were related to the values of the respective original recombinants (original). 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, and EC core concentrations at 48 h ranged from 17.5 to 131.8 fmol/liter. The values shown for the control were normalized to the IC core concentrations at 4 h and were related to the values obtained for the respective original recombinants (original) as described for the NS3P variants. LOC indicates the lower cutoff of the infectivity titration assay. For automated counting of FFU, the LOCs for IC and EC infectivity titers were 1.5 log₁₀ FFU/well and 2.3 log₁₀ FFU/ml, respectively. In instances where low replication efficiency in S29 cells precluded automated counting, FFU were counted manually, and the resulting titers are indicated by asterisks. The LOCs for infectivity titers derived from manually counted FFU were 0.9 log₁₀ FFU/well for IC titers and 1.6 log₁₀ FFU/ml for EC titers. LOC values were related to the values obtained for the respective original recombinants (original) as described for NS3P variants. The NS3P substitutions in boldface were specifically selected in the indicated virus under PI treatment in the current study (Fig. 1; see also Tables S4, S5, S12, S14, and S16 in the supplemental material). For comparison, titer differences (Inf. titer diff.) observed following transfection of Huh7.5 cells are indicated above each variant. Titer differences were calculated as described in Materials and Methods. With some exceptions, titer differences were calculated using the titers determined in this study (Fig. 3) and are indicated in boldface. Titer differences for the genotype 2a (isolate JFH1) and genotype 6a (isolate HK6a) recombinants in panel C were calculated from infectivity titers determined in reference 28. For the genotype 3a (isolate 452) R155K recombinant, the titer difference was calculated on the basis of infectivity titers obtained from a transfection other than that for Fig. 3. The higher titer difference observed in this transfection might be due to slower acquisition of a compensatory NS3P substitution by the genotype 3a (isolate 452) R155K recombinant in this transfection or to reversion of the R155K substitution, as observed previously (28). The results obtained for genotype 1a (isolate TN) with V36M and R155K, previously suggested as a combination with compensatory effects, are shown in Fig. S5 in the supplemental material.