Dolutegravir Resistance Mutation R263K Cannot Coexist in Combination with Many Classical Integrase Inhibitor Resistance Substitutions

Abstract

The new integrase strand transfer inhibitor (INSTI) dolutegravir (DTG) displays limited cross-resistance with older drugs of this class and selects for the R263K substitution in treatment-experienced patients. We performed tissue culture selections with DTG, using viruses resistant to older INSTIs and infectivity and resistance assays, and showed that the presence of the E92Q or N155H substitution was compatible with the emergence of R263K, whereas the G140S Q148R, E92Q N155H, G140S, Y143R, and Q148R substitutions were not.A lthough considerable progress has been made in HIV therapy, drug resistance has emerged for every available drug class, threatening long-term care and the potential for a cure (1). The first two integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG), can be compromised by primary substitutions that generate resistance at the expense of fitness (2), the majority of which occur in the catalytic core domain of the integrase protein (IN). Although the newer INSTI dolutegravir (DTG) has not yielded resistance when used in initial therapy (3), our group selected an R263K substitution in HIV-1 IN in tissue culture with DTG (4). We showed that R263K, located in the C-terminal portion of the protein, confers moderate resistance to DTG and EVG, while also decreasing the biochemical activity of IN by ϳ30% (5). The R263K substitution was also found in two of four INSTInaïve treatment-experienced participants who failed DTG therapy in the SAILING clinical trial (of 354 patients) but otherwise continued to perform well (6). This suggests that R263K is a signature substitution for DTG.In contrast, 7/51 individuals who previously failed RALbased regimens and who possessed primary resistance mutations subsequently failed DTG-based therapy in the VIKING trial without R263K (7), suggesting that the substitutions that confer resistance to RAL or EVG might be incompatible with R263K.(This work was largely performed by K. Anstett in partial fulfillment of the requirements for a Ph.D. degree at McGill University.)To test this hypothesis, biochemical analyses of the strand transfer efficiencies of integrase proteins containing the E92Q, Y143R, Q148R, and N155H RAL or EVG resistance substitutions, both alone and in combination with R263K, were performed (Fig. 1). Site-directed mutagenesis was performed as described previously (4, 5) to introduce E92Q, Y143R, Q148R, and N155H into the wild-type (pET15b wt ) and mutant (pET15b R263K ) integrase subtype B expression vectors (primer sequences available upon request). Recombinant integrase proteins were expressed and purified and strand transfer assays were performed as published (4, 5). Table 1 summarizes the enzyme kinetics values K m and V max for each protein tested.Every single mutation had a significant negative impact on strand transfer activity, and this effect was exacerbated when these substitutions were combined with R263K. Among single mutant proteins, the Q148R-and N155H-containing integrases were the most impacted with regar...

Characterization of the Drug Resistance Profiles of Integrase Strand Transfer Inhibitors in Simian Immunodeficiency Virus SIVmac239

“…E92Q-mutated SIV possessed 40% diminished infectivity. This agrees with data that have been obtained for HIV-1 (27).…”

Dolutegravir-Selected HIV-1 Containing the N155H and R263K Resistance Substitutions Does Not Acquire Additional Compensatory Mutations under Drug Pressure That Lead to Higher-Level Resistance and Increased Replicative Capacity

“…The generation of the pNL4.3 IN(N155H/R263K) plasmid has been reported previously (10). Similar methods were used to generate other pNL4.3IN (mutant) plasmids through site-directed mutagenesis using the primers listed in Table 1.…”

“…Sense and antisense primer sequences are listed in Table 1. The generation of the construct bearing the N155H/R263K mutations has been described already (10). Recombinant integrase proteins were expressed in BL21(DE3) bacterial cells and purified as previously published (7).…”

“…There was, however, no selection of R263K in this trial, suggesting that RAL/EVG resistance substitutions are incompatible with the R263K DTG resistance pathway. Indeed, we have previously shown that the combination of R263K with the most common primary RAL/EVG resistance substitutions resulted in decreased enzymatic activity of IN as well as overall diminished viral infectivity (10). The only combination that appeared to increase viral infectivity and resistance to DTG compared to that of R263K was the combination of N155H with R263K (10).…”

Multifunctional facets of retrovirus integrase

“…The higher genetic barrier of DTG in patients may be due to its longer dissociative half-life from the HIV IN-DNA complex suggesting that DTG extended binding may be a significant factor for prevention of drug-resistance [86] ; also shown to be a factor in producing kinetically stabilized RSV SC ( Figure 9). HIV carrying the rare signature IN R263K mutation (others are H51Y and E138K) observed with DTG apparently cannot co-exist in combination with many of the above classical IN resistance mutations [87] . HIV is apparently unable to compensate for these mutations which induce a fitness cost that prevents the virus from evading inhibitor pressure.…”