Background:  Previous studies have described primary (N155H, Q148R(H)(K),Y143R(C)) and secondary (V72I, L74I, T97A, V151I, V201I, S230N) raltegravir (RAL) resistance mutations. Several secondary mutations exist as common polymorphisms, and in isolation, do not alter RAL susceptibility. In this study we compared the frequency of V72I, L74I(V)(M), T97A, V151I, G163E(R) and S230N(R) in RAL susceptible and RAL resistant viruses, and the effects of combinations of these mutations on RAL susceptibility.

Methods:  Phenotypic (PhenoSense® Integrase) and genotypic (Geneseq® Integrase) analyses were performed on 593 RAL susceptible and 175 RAL resistant patient viruses as well as a panel of isogenic viruses engineered to contain one or more secondary mutations. RAL resistance was defined as a change in IC₅₀ > 1.5-fold.

Results:  A higher frequency of integrase substitutions L74I(V)(M), T97A, V151I, G163E(R) was observed in RAL resistant viruses compared to RAL susceptible viruses, but not for substitutions at V72I, V201I or S230N(R). Irrespective of overall frequency, certain substitutions were enriched in RAL resistant viruses: S230R was only detected in RAL resistant virus, and L74M and G163R were more common in resistant viruses (6, 9 and 12 percent) versus sensitive viruses (0, 0.5 and 0.3 percent), respectively. Based on studies using a panel of site directed mutant viruses, substitutions V72I, L74M, T97A, V151I, G163R and S230R alone generally did not reduce susceptibility to RAL. However some combinations of these substitutions resulted in reductions in RAL susceptibility. This observation was consistent with a patient virus that exhibited a 9-fold reduction in RAL susceptibility and contained V72I, T97A and G163R in the absence of a primary mutation. An additional four patient viruses that exhibited reduced RAL susceptibility (range of IC₅₀ fold change = 2 to 80) also lacked primary RAL resistance mutations, but all contained at least one secondary mutation.

Conclusions:  As previous groups have shown, viruses containing individual IN polymorphisms remain susceptible to RAL, however certain substitutions increase in frequency under RAL selective pressure. Combinations of these secondary mutations can result in reduced susceptibility to RAL, however the selection of these combinations of secondary substitutions in the absence of primary RAL resistance mutations appears infrequent.