Mutations associated with failure of raltegravir treatment affect integrase sensitivity to the inhibitor in vitro

Abstract

Raltegravir (MK-0518) is a potent inhibitor of human immunodeficiency virus (HIV) integrase and is clinically effective against viruses resistant to other classes of antiretroviral agents. However, it can select mutations in the HIV integrase gene. Nine heavily pretreated patients who received salvage therapy including raltegravir and who subsequently developed virological failure under raltegravir therapy were studied. For each patient, the sequences of the integrase-coding region were determined and compared to that at the beginning of the treatment. Four different mutation profiles were identified in these nine patients: E92Q, G140S Q148H, N155H, and E157Q mutations. For four patients, each harboring a different profile, the wild-type and mutated integrases were produced, purified, and assayed in vitro. All the mutations identified altered the activities of integrase protein: both 3' processing and strand transfer activities were moderately affected in the E92Q mutant; strand transfer was markedly impaired in the N155H mutant; both activities were strongly impaired in the G140S Q148H mutant; and the E157Q mutant was almost completely inactive. The sensitivities of wild-type and mutant integrases to raltegravir were compared. The E92Q and G140S Q148H profiles were each associated with a 7- to 8-fold decrease in sensitivity, and the N155H mutant was more than 14-fold less sensitive to raltegravir. At least four genetic profiles (E92Q, G140S Q148H, N155H, and E157Q) can be associated with in vivo treatment failure and resistance to raltegravir. These mutations led to strong impairment of enzymes in vitro in the absence of raltegravir: strand transfer activity was affected, and in some cases 3' processing was also impaired.

FIG. 1.

Kinetics of HIV RNA copy numbers in plasma for nine patients failing raltegravir therapy. Dotted lines at 1.60 log₁₀ copies/ml (40 copies/ml) show the detection limit of the viral load assay. wt, wild type; NA, nonamplifiable.

FIG. 2.

3′ processing and strand transfer activities of recombinant INs. (A) Activities of recombinant INs. INs obtained from patient 3 on day zero (IN_WT) and at week 14 (IN_E92Q) were incubated for 1 h at 37°C with 10 nM substrate (21-mer) in the presence of various concentrations of raltegravir (given above the lanes). st., strand; 3′ pro, 3′ processing. (B) Comparison of inhibition curves obtained for isolates from patients 1, 2, and 3. RI, resistance index. Symbols: •, data obtained for wild-type virus at day zero; ▴, data obtained for resistant viruses.