Three main mutational pathways in HIV-2 lead to high-level raltegravir and elvitegravir resistance: implications for emerging HIV-2 treatment regimens

Abstract

Human immunodeficiency virus type 2 (HIV-2) is intrinsically resistant to non-nucleoside reverse transcriptase inhibitors and exhibits reduced susceptibility to several of the protease inhibitors used for antiretroviral therapy of HIV-1. Thus, there is a pressing need to identify new classes of antiretroviral agents that are active against HIV-2. Although recent data suggest that the integrase strand transfer inhibitors raltegravir and elvitegravir may be beneficial, mutations that are known to confer resistance to these drugs in HIV-1 have been reported in HIV-2 sequences from patients receiving raltegravir-containing regimens. To examine the phenotypic effects of mutations that emerge during raltegravir treatment, we constructed a panel of HIV-2 integrase variants using site-directed mutagenesis and measured the susceptibilities of the mutant strains to raltegravir and elvitegravir in culture. The effects of single and multiple amino acid changes on HIV-2 replication capacity were also evaluated. Our results demonstrate that secondary replacements in the integrase protein play key roles in the development of integrase inhibitor resistance in HIV-2. Collectively, our data define three major mutational pathways to high-level raltegravir and elvitegravir resistance: i) E92Q+Y143C or T97A+Y143C, ii) G140S+Q148R, and iii) E92Q+N155H. These findings preclude the sequential use of raltegravir and elvitegravir (or vice versa) for HIV-2 treatment and provide important information for clinical monitoring of integrase inhibitor resistance in HIV-2-infected individuals.

Figure 1. Single-cycle replication capacities of HIV-2 integrase variants.

Each datum point is the infectious titer [MAGIC-5A focus-forming units (FFU)/ml] produced by an independent transfection of full-length HIV-2 plasmid DNA into 293T-17 cells. Bars indicate the mean titers for each wild-type (WT) or mutant strain. Light-grey bars indicate variants that are significantly different from WT (P>0.05) and * indicates a significant difference between Q148R and G140S+Q148R HIV-2 (P>0.01) (ANOVA of log₁₀-transformed titers with Tukey’s post-test). Filled and open circles represent the titers produced by two independent plasmid DNA preparations for each genotype; titers from a third preparation of wild-type DNA are shown as inverted triangles. Error bars indicate standard deviations.

Figure 2. Susceptibility of HIV-2 integrase variants to raltegravir (RAL) and elvitegravir (EVG).

Panels A and C show the EC₅₀ values for wild-type (WT) HIV-2 ROD9 and each of 13 site-directed ROD9 integrase mutants tested against raltegravir and elvitegravir, respectively. Bars indicate the means of three or more independent dose-response experiments. Light, medium and dark-colored bars indicate low-level, moderate, and highlevel resistance (mean EC₅₀ values 2–5-fold, 6–15-fold and >15-fold relative to wild-type, respectively). With the exception of Q148H versus raltegravir and Y143C versus elvitegravir, EC₅₀ values for all strains shown in color were statistically greater than the corresponding values for wild-type ROD9 (P>0.05, ANOVA of log₁₀-transformed EC₅₀ values with Tukey's post-test). EC₅₀ values for T97A and G140S did not statistically differ from WT for either drug. Panels B and D show representative dose-response data for WT, Q148R, and G140S+Q148R versus raltegravir and WT, N155H, and E92Q+N155H versus elvitegravir, respectively. Titers are expressed as the percentage of those seen in the absence of drug (i.e., % of solvent-only controls) and are the means of three independent cultures at each drug concentration. Error bars in all panels indicate standard deviations.