The S230R Integrase Substitution Associated With Virus Load Rebound During Dolutegravir Monotherapy Confers Low-Level Resistance to Integrase Strand-Transfer Inhibitors

Abstract

Background: Dolutegravir (DTG) is an integrase strand-transfer inhibitor (INSTI) used for treatment of human immunodeficiency virus (HIV)-infected individuals. Owing to its high genetic barrier to resistance, DTG has been clinically investigated as maintenance monotherapy to maintain viral suppression and to reduce complication and healthcare costs. Our study aims to explain the underlying mechanism related to the emergence of a S230R substitution in patients who experienced virologic failure while using DTG monotherapy.

Methods: We evaluated the effect of the S230R substitution in regard to integrase enzyme activity, viral infectivity, replicative capacity, and susceptibility to different INSTIs by biochemical and cell-based assays.

Results: The S230R substitution conferred a 63% reduction in enzyme efficiency. S230R virus was 1.29-fold less infectious than wild-type virus but could replicate in PM1 cells without significant delay. Resistance levels against DTG, cabotegravir, raltegravir, and elvitegravir in tissue culture were 3.85-, 3.72-, 1.52-, and 1.21-fold, respectively, in virus with the S230R substitution.

Conclusions: Our data indicate that the S230R substitution is comparable to the previously reported R263K substitution in some respects. Virologic failure during DTG monotherapy can occur through the development of the S230R or R263K mutation, without the need for high-level DTG resistance.

Figure 1.

Mean strand-transfer activities (±standard errors of the mean) of purified recombinant wild-type integrase (WT) and integrase bearing the S230R substitution (S230R). *P < .05 compared to WT, by the Student t test (OpenEpi Toolkit). A, Strand-transfer activity in the presence of 20 nM target DNA and increasing concentrations of purified integrases. For each protein concentration, assays were performed twice independently, in triplicate, yielding 6 independent values. B, Strand-transfer activity in the presence of 400 nM purified WT or S230R integrase and increasing concentrations of target DNA. For each protein, assays were performed 3 times independently, in triplicates, yielding 9 independent values for each target DNA concentration and protein. C, Calculated maximum strand-transfer activities (V_max) for WT and S23OR integrases, using protein concentrations specified in panel B. D, Half of the concentration of DNA necessary to reach V_max (K_m) for purified WT and S23OR integrases, using values from panel B.

Figure 2.

A, Mean relative viral infectivity (±standard error of the mean [SEM]) in TZM-bl cells of wild-type (WT) and S230R viruses, measured by luciferase fluorescence, 48 hours after infection. Values were calculated from 3 independent experiments, each performed in triplicate, yielding 9 values per point. B, Viral replication kinetics over a 21-day period in PM1 cells. PM1 cells were infected with the same amount of reverse transcriptase (RT) for all viral stocks. Levels of RT in cell-free culture fluids were monitored by quantitative RT assays. Means (±SEMs) of 3 different experiments, each performed in triplicate, for each virus are presented. EC₅₀, 50% effective concentration; RLU, relative luminescence units; RTU, reverse transcriptase units. ^aCompared to wild-type genotype, by the Student t test (OpenEpi Toolkit). A value < .05 is considered statistically significant.

Figure 3.

Dose-response infectivity curves in the presence of dolutegravir (DTG), cabotegravir (CAB), raltegravir (RAL), and elvitegravir (EVG) for wild-type (WT) and S230R viruses. Data reflect viral resistance to RAL, EVG, DTG, and CAB in TZM-bl cells. TZM-bl cells were infected with NL4.3_IN(WT) or NL4.3_IN(S230R) virus over 48 hours, and luciferase levels were measured. Infectivity of NL4.3_IN(WT) virus is shown for comparison. Dose-response curves were produced from the compilation of 3 independent experiments, each performed in triplicate. Error bars indicate means (±standard errors of the mean). RLU, relative luminescence units.