Mutation T74S in HIV-1 subtype B and C proteases resensitizes them to ritonavir and indinavir and confers fitness advantage.

Objectives: Several drug resistance and secondary mutations have been described in HIV-1 viruses from patients undergoing antiretroviral therapy. In this study, we assessed the impact of the protease substitution T74S on the phenotype and on the replicative fitness in HIV-1 subtypes B and C. Methods: HIV-1 molecular clones carrying subtype B or C proteases had these coding regions subjected to site-directed mutagenesis to include T74S alone or in combination with four known protease inhibitor (PI) primary drug resistance mutations. All clones were used in a phenotypic assay to evaluate their susceptibility to most commercially available Pis. The impact of T74S on virus fitness was also assessed for all viruses through head-to-head competitions and oligonucleotide ligation assays to measure the proportion of each virus in culture. Results: Viruses of both subtypes carrying T74S did not have their susceptibility altered to any tested PI. Viruses with the four resistance mutations showed strong resistance to most Pis with fold changes ranging from 5 to 300 times compared with their wild-type counterparts. Surprisingly, the addition of T74S to the multiresistant clones restored their susceptibilities to indinavir and ritonavir and partially to lopinavir, close to those of wild-type viruses. Most 74S-containing viruses were more fit than their 74T counterparts. Conclusions: Our results suggest that T74S is not a major drug resistance mutation, but it resensitizes multiresistant viruses to certain Pis. T74S is a bona fide accessory mutation, restoring fitness of multidrug-resistant viruses in both subtypes B and C. T74S should be further studied in clinical settings and considered in drug resistance interpretation algorithms.