Fig 6.

Effect of nonspecific loss of protease activity on viral infectivity and inhibitor sensitivity. (A) Protease activity was reduced in a nonspecific way by transfecting the infectious molecular clone with an isogenic clone containing an inactivating mutation at the protease active site (D25A). Both the fraction of the D25A mutant and the calculated remaining protease activity in each preparation are indicated. The relative infectivity of each mixture was measured using the SpIn (filled circles) and RC (open circles) assays. (B) The effect of nonspecific loss of protease activity on sensitivity to inhibitors of HIV replication was studied by measuring the EC₅₀ for nucleoside reverse transcriptase inhibitors (tenofovir, AZT), nonnucleoside reverse transcriptase inhibitors (EFV), and protease inhibitors (LPV, darunavir [DRV]). Increasing the amount of mutant protease up to 50% (equivalent to 25% residual protease activity) had a systematic effect on the EC₅₀ value for the three classes of antiviral drugs tested. (C) The FC EC₅₀ of 50% D25A relative to the FC EC₅₀ of 0% D25A is shown for all the inhibitors tested. The values of three classes of inhibitors were compared by statistical analysis using a nonparametric Kruskal-Wallis test. The open arrowhead indicates the value from AZT. (D) Western blot analysis showing the extent of processing of p24 and RT in virus particle preparations containing either 100% WT or 50% inactive mutant protease.