Zidovudine (AZT) monotherapy selects for the A360V mutation in the connection domain of HIV-1 reverse transcriptase

Abstract

Background: We previously demonstrated in vitro that zidovudine (AZT) selects for A371V in the connection domain and Q509L in ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase (RT) which, together with the thymidine analog mutations D67N, K70R and T215F, confer greater than 100-fold AZT resistance. The goal of the current study was to determine whether AZT monotherapy in HIV-1 infected patients also selects the A371V, Q509L or other mutations in the C-terminal domains of HIV-1 RT.

Methodology/principal findings: Full-length RT sequences in plasma obtained pre- and post-therapy were compared in 23 participants who received AZT monotherapy from the AIDS Clinical Trials Group study 175. Five of the 23 participants reached a primary study endpoint. Mutations significantly associated with AZT monotherapy included K70R (p = 0.003) and T215Y (p = 0.013) in the polymerase domain of HIV-1 RT, and A360V (p = 0.041) in the connection domain of HIV-1 RT. HIV-1 drug susceptibility assays demonstrated that A360V, either alone or in combination with thymidine analog mutations, decreased AZT susceptibility in recombinant viruses containing participant-derived full-length RT sequences or site-directed mutant RT. Biochemical studies revealed that A360V enhances the AZT-monophosphate excision activity of purified RT by significantly decreasing the frequency of secondary RNase H cleavage events that reduce the RNA/DNA duplex length and promote template/primer dissociation.

Conclusions: The A360V mutation in the connection domain of RT was selected in HIV-infected individuals that received AZT monotherapy and contributed to AZT resistance.

Figure 1. ATP-mediated AZT-MP excision activity and RNase H activity of wildtype, A360V, TAM-1 and TAM-1/A360V HIV-1 RT.

A) Isotherms of ATP-mediated AZT-MP excision reactions carried out by wildtype and mutant HIV-1 RT on a DNA/DNA T/P. Data are the mean ± standard deviation from at least three independent experiments. Reaction times were: wildtype and A360V = 10, 20, 30, 45, 60, 75, 90, 105 min; TAM-1 and TAM-1/A360V = 3, 7.5, 15, 25, 35, 45, 60, 75 min. B) Isotherms of ATP-mediated AZT-MP excision reactions carried out by wildtype and mutant HIV-1 RT on an RNA/DNA T/P. Data are the mean ± standard deviation from at least three independent experiments. Reaction times were: wildtype and A360V = 15, 30, 45, 60, 75, 90, 105, 120 min; TAM-1 and TAM-1/A360V = 3, 7.5, 15, 25, 35, 45, 60, 75 min. C) Representative autoradiogram of the RNase H cleavage activity of the wildtype and mutant HIV-1 RTs. Experiments were carried out as described in the Materials and Methods. The reaction times were wildtype and A360V = 15, 30, 45, 60, 75, 90, 105, 120 min; TAM-1 and TAM-1/A360V = 3, 7.5, 15, 25, 35, 45, 60, 75 min. D) Isotherms for the accumulation of the −10 product formed by wildtype and mutant HIV-1 RT during AZT-MP excision.