Selection in culture of HIV resistance to dolutegravir by mutations at integrase positions R263K and H51Y that diminish viral replication fitness

Abstract

Purpose of the study We selected for resistance in tissue culture against dolutegravir (DTG), a second-generation HIV integrase strand transfer inhibitor, which is now in phase 2/3 clinical trials, in order to try to characterize the resistance profile of this compound. Methods HIV-1 of different subtypes was grown in both MT-2 cells and peripheral blood mononuclear cells over protracted periods, with the concentration of DTG being incrementally increased from an initial concentration of 0.05 nM, i.e. 4 times less than the EC 50 . After a total of 6 months of growth, a final drug concentration of 50Á100 nM was achieved, beyond which virus could no longer be grown. Viral DNA was then sequenced to reveal the presence of mutations that might confer resistance to DTG. The biological relevance of these mutations was confirmed through site-directed mutagenesis experiments in which individual mutations or combinations of mutations were studied in comparison with wild-type (wt) virus in tissue culture and with recombinant HIV integrase enzyme in biochemical assays. Summary of results The most common integrase resistance mutation to arise in subtype B and recombinant A/G viruses was R263K followed by H51Y. In the case of subtype C viruses, the most common mutation was G118R followed by H51Y. The presence of R263K alone conferred an approximate 3Á6-fold level of resistance to DTG in culture, a 30% drop in levels of recombinant integrase strand transfer activity, as well as an approximate 20Á30% loss in viral replicative capacity. Biochemical experiments indicated that the t ½ residency times of DTG for subtype B and C viruses for wt integrase were 26 h and 38 h, respectively, and for R263K, 16h and 22h, respectively. In contrast, H51Y by itself did not significantly affect either strand transfer activity or resistance to DTG. However, the combination of R263K together with H51Y led to an increase in levels of DTG resistance to about 15-fold accompanied by an Â50% loss in both viral replication capacity and integrase strand transfer activity.