Structure analysis of HIV-1 IN Y143C/R Raltegravir resistance mutation in association with the secondary mutation T97A

The HIV-1 integrase (IN) mutations Y143C/R are known as raltegravir (RAL) primary resistance mutations. In a previous study, we investigated the genetic pathway and the dynamics of emergence of the Y143C/R mutations in three patients failing on RAL-containing regimens. In these patients, Y143C/R was associated with the T97A mutation. The aim of the present biochemical and molecular studies in vitro were to evaluate whether the secondary mutation T97A associated with Y143C/R could increase the level of resistance to RAL and impact the IN activities. Site-directed mutagenesis experiments were performed on expression vectors harboring the region of pol gene coding for IN. With 3' end-processing assay, IC(50) were 1.2 μM, 1.2 μM, 2.4 μM (fold change (FC) =2) and 20 μM (FC=16.7) for IN WT, IN T97A, IN Y143C/T97A, and Y143R/T97A respectively. FC of 18 and 100 were observed with the strand transfer assay for IN Y143C/T97A and Y143R/T97A, with IC(50) of 0.625 μM and 2.5 μM respectively. In the strand transfer assay, IN Y143C or R combined with the secondary mutation T97A severely impaired susceptibility to RAL compared to IN Y143C or R alone. Assays without RAL suggested that T97A could rescue the catalytic activity which was impaired by the presence of Y143C/R. The combination of T97A with the primary RAL resistance mutations Y143C/R strongly reduces the susceptibility to RAL and rescues the catalytic defect due to the Y143C/R mutation. This indicates that the emergence of double mutation pattern Y143C/R/T97A in patients is a signature of high resistance level.