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, 89 (1), 833-43

Mutation V111I in HIV-2 Reverse Transcriptase Increases the Fitness of the Nucleoside Analogue-Resistant K65R and Q151M Viruses

Collaborators, Affiliations

Mutation V111I in HIV-2 Reverse Transcriptase Increases the Fitness of the Nucleoside Analogue-Resistant K65R and Q151M Viruses

Ilona P Deuzing et al. J Virol.

Abstract

Infection with HIV-2 can ultimately lead to AIDS, although disease progression is much slower than with HIV-1. HIV-2 patients are mostly treated with a combination of nucleoside reverse transcriptase (RT) inhibitors (NRTIs) and protease inhibitors designed for HIV-1. Many studies have described the development of HIV-1 resistance to NRTIs and identified mutations in the polymerase domain of RT. Recent studies have shown that mutations in the connection and RNase H domains of HIV-1 RT may also contribute to resistance. However, only limited information exists regarding the resistance of HIV-2 to NRTIs. In this study, therefore, we analyzed the polymerase, connection, and RNase H domains of RT in HIV-2 patients failing NRTI-containing therapies. Besides the key resistance mutations K65R, Q151M, and M184V, we identified a novel mutation, V111I, in the polymerase domain. This mutation was significantly associated with mutations K65R and Q151M. Sequencing of the connection and RNase H domains of the HIV-2 patients did not reveal any of the mutations that were reported to contribute to NRTI resistance in HIV-1. We show that V111I does not strongly affect drug susceptibility but increases the replication capacity of the K65R and Q151M viruses. Biochemical assays demonstrate that V111I restores the polymerization defects of the K65R and Q151M viruses but negatively affects the fidelity of the HIV-2 RT enzyme. Molecular dynamics simulations were performed to analyze the structural changes mediated by V111I. This showed that V111I changed the flexibility of the 110-to-115 loop region, which may affect deoxynucleoside triphosphate (dNTP) binding and polymerase activity.

Importance: Mutation V111I in the HIV-2 reverse transcriptase enzyme was identified in patients failing therapies containing nucleoside analogues. We show that the V111I change does not strongly affect the sensitivity of HIV-2 to nucleoside analogues but increases the fitness of viruses with drug resistance mutations K65R and Q151M.

Figures

FIG 1
FIG 1
Relative fitness of wild-type (WT) and mutant viruses. The SupT1 T-cell line was infected with equal amounts of two viruses, and the frequencies of the viral genotypes were assessed at each passage by population sequencing of the RT gene. The percentage of each virus in the population was calculated from the Sanger sequencing electropherograms. Shown are pairwise competition experiments between the wild-type and V111I (A), M184V and M184V V111I (B), K65R and K65R V111 (C), and Q151M and Q151M V111I (D) viruses. All experiments were repeated at least three times, and representative experiments are shown.
FIG 2
FIG 2
Molecular dynamics simulations of the wild-type and V111I HIV-2 RT enzymes. (A) Crystal structure of the polymerase domain of HIV-2 RT, with the palm (yellow), thumb (red), and fingers (blue) subdomains. The position of the 110-to-115 loop is shown in pink. The second loop conformation, observed only during simulations of the wild-type RT, is shown in cyan. The location of residue 111 within the loop is indicated by a sphere (placed on the carbon alpha atom). The location of residue D185 in the polymerase active site is marked by a black sphere. (B) Structure of HIV-1 RT bound to DNA/dATP (PDB code 3KK2) indicating the location of the dNTP binding pocket relative to the polymerase active site (which is highly conserved between HIV-1 and HIV-2). The subdomain coloring and orientation are the same as for HIV-2 RT; DNA is shown in light blue, and dATP in green. (C) The RMSD of the 110-to-115 loop plotted against the RMSD of residue D185 in the polymerase active site over the simulations (relative to the position in the HIV-2 crystal structure). Two conformations of the 110-to-115 loop are observed for wild-type RT (red), whereas only a single cluster of values is observed for V111I RT (black). The flexibility of the loop is associated with displacement of catalytic residue D185 in the wild-type simulations.
FIG 3
FIG 3
Polymerase activities of wild-type and mutant virus-derived HIV-2 RT enzymes. (A) The polymerase activities of the wild-type, Q151M, K65R, V111I, Q151M V111I, and K65R V111I HIV-2 RT enzymes were determined using an HIV-1 RNA template and an end-labeled DNA primer complementary to the PBS. The primer was heat annealed onto the template and extended by the addition of virion-derived RT and dNTPs. Formation of the 202-nt cDNA product (cDNA) was monitored after 30, 60, and 120 min of incubation time (t). Increasing incubation time is marked by a triangle. A DNA size marker (M) was loaded as a reference (nt). Similar results were obtained in at least three independent experiments. (B) The amount of cDNA production was quantified, and the polymerase activity of the wild-type virus-derived RT was set at 100%. (C) Polymerase activities of wild-type M184V, V111I, and M184V V111I virus-derived RT enzymes. (D) Quantification of the cDNA products, with the activity of wild-type RT set at 100%. Representative gels are shown, and similar results were obtained in at least two independent experiments and were repeated using HIV-2 RNA templates.
FIG 4
FIG 4
Fidelity of the wild-type and V111I HIV-2 RT enzymes. (A) Single-nucleotide incorporation assays were performed using an RNA template encompassing the HIV-2 PBS region and a labeled DNA primer complementary to the PBS. Virion-derived wild-type or V111I RT was added and incubated with increasing concentrations of dATP as the correct nucleotide or dTTP as the incorrect nucleotide (marked by triangles). Single-nucleotide incorporation was quantified in three independent experiments (Table 5).

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