E resistance pathway against DTG, our results provide an explanation for
E resistance pathway against DTG, our results provide an explanation for the absence of drug resistance mutations in drug-naive patients who have been treated with DTG.Table 2 Effects of the H51Y and R263K mutations on HIV replication capacity and susceptibility to dolutegravir (DTG), raltegravir (RAL), and elvitegravir (EVG) as measured by the PhenoSenseW Integrase assay (Monogram Biosciences)DTG Backbone pNL4-3 Genotype WT H51Y R263K H51Y/R263K Fold BQ-123 cost change 0.92 1.25 1.95 6.95 RAL Fold change 0.91 1.11 1.21 2.94 EVG Fold change 1.03 2.06 3.28 41.5 100 89 70 11 Replication capacityResultsThe addition of H51Y to R263K increases resistance against dolutegravirWe have previously shown and confirm here that the unique R263K mutation confers low-level resistance (10-fold) to DTG (Table 1) [17]. Now, by introducing the H51Y mutation alone or in combination with R263K into pNL4.3 proviral PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28192408 DNA, we show that the combination of H51Y and R263K increased resistance to DTG (FC=16.5-fold), whereas H51Y alone did not confer resistance to this drug (Table 1). Similar experiments with RAL showed that the combination of both mutations conferred low-level resistance to this drug (2.1-fold, Table 1) while the individual H51Y and R263K mutations were innocuous. Notably, the fold change in RAL susceptibility observed with H51Y (1.2-fold) was not significant in our experiments but was identical to results from another study [20]. As expected, HIV susceptibility to the non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) was unaltered by these mutations alone or in combination. Since levels of drug resistance can vary depending on differences in assays or target cells, HIV susceptibility to RAL, EVG, and DTG PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 was also tested in viruses containing the H51Y and R263K mutations using the PhenoSenseW Integrase phenotypic assay (Monogram Biosciences) (Table 2). In this assay, R263K conferred low-level resistance to DTG ( 2-fold) and the addition of H51Y to R263K further increased HIV resistance to 7-fold. These mutations tested individually had no impact on HIV susceptibility to RAL whereas the H51Y/R263K combination conferred an approximate 3-fold resistance to this compound. Both H51Y and R263K individuallyconferred low-level resistance to EVG (2.06 and 3.28-fold, respectively) while the combination dramatically increased the IC50 for this drug by 41.5-fold.The addition of H51Y to R263K decreases integrase strand-transfer activityOur previous work showed that the primary resistance mutation R263K decreased integrase activity in cell-free assays [17]. Now, experiments with purified recombinant wild-type (wt) and mutated integrase proteins, i.e. INWT, INH51Y, INR263K, and INH51Y/R263K at varying concentrations (Figure 1A and B) showed that INWT and INH51Y had comparable maximal strand transfer activity (100?.7 and 107.7?0.7 , respectively) whereas INH51Y/R263K maximal activity was severely diminished (20.01?.9 ). Similar observations were made in the presence of variable concentrations of DNA substrate (Figure 1C). The sequential addition of R263K and the H51Y/R263K mutations resulted in an incremental loss in enzyme activity, while the effect of H51Y alone was innocuous.The addition of H51Y to R263K decreases HIV replication capacityTo determine whether this biochemical defect also applied to replication capacity in cell culture, we assessed viruses that were either wt or contained these various mutations, i.e. pNL4.3IN(WT), pNL4.3IN(H51Y), pNL4.3IN.
