Effects of partial deletions within the human immunodeficiency virus type 1 V3 loop on coreceptor tropism and sensitivity to entry inhibitors

Abstract

The human immunodeficiency virus type 1 (HIV-1) V3 loop is critical for coreceptor binding and principally determines tropism for the CCR5 and CXCR4 coreceptors. The recent crystallographic resolution of V3 shows that its base is closely associated with the conserved coreceptor binding site on the gp120 core, whereas more distal regions protrude toward the cell surface, likely mediating interactions with coreceptor extracellular loops. However, these V3-coreceptor interactions and the structural basis for CCR5 or CXCR4 specificity are poorly understood. Using the dual-tropic virus HIV-1(R3A), which uses both CCR5 and CXCR4, we sought to identify subdomains within V3 that selectively mediate R5 or X4 tropism. An extensive panel of V3 mutants was evaluated for effects on tropism and sensitivity to coreceptor antagonists. Mutations on either side of the V3 base (residues 3 to 8 and 26 to 33) ablated R5 tropism and made the resulting X4-tropic Envs more sensitive to the CXCR4 inhibitor AMD3100. When mutations were introduced within the V3 stem, only a deletion of residues 9 to 12 on the N-terminal side ablated X4 tropism. Remarkably, this R5-tropic Delta9-12 mutant was completely resistant to several small-molecule inhibitors of CCR5. Envs with mutations in the V3 crown (residues 13 to 20) remained dual tropic. Similar observations were made for a second dual-tropic isolate, HIV-1(89.6). These findings suggest that V3 subdomains can be identified that differentially affect R5 and X4 tropism and modulate sensitivity to CCR5 and CXCR4 inhibitors. These studies provide a novel approach for probing V3-coreceptor interactions and mechanisms by which these interactions can be inhibited.

FIG. 1.

Effects of mutations in the HIV-1_R3A V3 base on tropism. (A) Fusion activity in a cell-cell fusion assay is shown for Envs with small deletions and alanine substitutions (Table 1). Percent fusion was calculated by using luciferase activity normalized to R3A fusion on CD4⁺ CXCR4⁺ or CD4⁺ CCR5⁺ QT6 cells. The values are means plus standard errors of the mean (SEM). The data shown are the averages of three experiments. (B) Infectivities of the indicated Envs are shown using a single-cycle luciferase reporter virus on U87.CD4.CXCR4 and U87.CD4.CCR5 cells. Percent infection was calculated by using luciferase activity normalized to R3A infection on U87.CD4.CXCR4 cells or U87.CD4.CCR5 cells. The values are means plus SEM. The data shown are the averages of three experiments. (C) Growth curves for viruses containing the Δ5-6 and Δ28-29 Envs in comparison with parental R3A are shown on U87.CD4.CXCR4 cells (left) and U87.CD4.CCR5 cells (right). RT activity in culture supernatants was measured at the indicated time points. The results from one of two independent experiments are shown.

FIG. 2.

Sensitivities of V3 base mutants to AMD3100. (A) Sensitivities to AMD3100 of mutants containing two amino acid deletions or alanine substitutions in the V3 base are shown using a cell-cell fusion assay with CXCR4⁺ QT6 cells. Percent fusion was calculated by using luciferase activity normalized to fusion in the absence of inhibitor for each Env. The values are means ± standard errors of the mean (SEM). The data shown are the averages of three experiments. (B) Sensitivities of V3 base mutants to AMD3100 are shown using a pseudotype infection assay on U87.CD4.CXCR4 cells. Percent fusion was calculated by using luciferase activity normalized to infection in the absence of inhibitor for each virus. The values are means ± SEM. The data shown are the averages of three experiments.

FIG. 3.

Effects of mutations in the V3 crown and stem on tropism. (A) Effects of 2- and 4-amino-acid deletions in the V3 crown (residues 13 to 20) and stem (residues 9 to 12 and 21 to 25) on cell-cell fusion are shown. Percent fusion was calculated using luciferase activity normalized to R3A fusion on CD4⁺ CXCR4⁺ or CD4⁺ CCR5⁺ cells. The values are means plus standard errors of the mean (SEM). The data shown are the averages of three experiments. (B) Effects of deletions and alanine substitutions within the N-terminal side of the V3 stem (residues 9 to 12) on cell-cell fusion are shown. Percent fusion was calculated by using luciferase activity normalized to R3A fusion on CD4⁺ CXCR4⁺ or CD4⁺ CCR5⁺ cells. The values are means plus SEM. The data shown are the averages of three experiments. (C) Growth curves for infectious viruses containing the parental R3A and Δ9-12 Envs on SupT1 cells (CXCR4⁺ CCR5⁻) and SupCCR5 cells (CXCR4⁺ CCR5⁺) are shown. RT activity in culture supernatants was measured at the indicated time points. The results from one of two independent experiments are shown.

FIG. 4.

Sensitivities of the R5-tropic mutant Δ9-12 Env to CCR5 inhibitors. The sensitivities of R3A, ΔV3(9,9), and Δ9-12 to CMPD-167 (A), AD101 (B), Schering D (C), and Aplaviroc (D) are shown using a cell-cell fusion assay on CCR5⁺ cells. Percent fusion was calculated by using luciferase activity normalized to fusion in the absence of inhibitor for each Env. The values are means ± standard errors of the mean. The data shown are the averages of three experiments.

FIG. 5.

Sensitivities of HIV-1_R3A V3 crown and stem mutants to AD101. (A) Sensitivities to AD101 of mutants containing 2- or 4- amino-acid deletions in the V3 crown and stem are shown using a cell-cell fusion assay with CCR5⁺ cells. Percent fusion was calculated by using luciferase activity normalized to fusion in the absence of inhibitor for each Env. The values are means ± standard errors of the mean (SEM). The data shown are the averages of three experiments. (B) Sensitivities to AD101 of mutants containing small deletions or alanine substitutions within V3 stem residues 9 to 12 are shown using a cell-cell fusion assay with CCR5⁺ cells. Percent fusion was calculated by using luciferase activity normalized to fusion in the absence of inhibitor for each Env. The values are means ± SEM. The data shown are the averages of three experiments.

FIG. 6.

Effects of HIV-1_89.6 V3 deletion mutants on coreceptor tropism and sensitivity to coreceptor inhibitors. (A) Fusion activities of Envs containing deletions in the V3 base (Δ5-6 and Δ28-29) and within the N-terminal side of the V3 stem (Δ9-12) are shown in a cell-cell fusion assay. Percent fusion was calculated by using luciferase activity normalized to 89.6 fusion on CD4⁺ CXCR4⁺ or CD4⁺ CCR5⁺ cells. The values are means plus standard errors of the mean (SEM). The data shown are the averages of three experiments. (B) Sensitivities of V3 base deletion mutants (Δ5-6 and Δ28-29) to AMD3100 are shown using a cell-cell fusion assay with CXCR4⁺ cells. Percent fusion was calculated by using luciferase activity normalized to fusion in the absence of inhibitor for each Env. The values are means ± SEM. The data shown are the averages of three experiments. (C) Sensitivity of the V3 stem deletion mutant Δ9-12 to Schering D is shown using a cell-cell fusion assay on CCR5⁺ cells. Percent fusion was calculated by using luciferase activity normalized to fusion in the absence of inhibitor for each Env. The values are means ± SEM. The data shown are the averages of three experiments.

FIG. 7.

Model for coreceptor utilization and sensitivity to entry inhibitors for the dual-tropic Envs R3A and 89.6. (A) Although Env interactions with both the coreceptor N terminus and the ECLs occur, the interaction of the bridging sheet and V3 base with the coreceptor N terminus is critical for R5 tropism, while the interaction between more distal regions of V3 and the coreceptor ECLs is critical for X4 tropism. (B) Deletions at either side of the V3 base (residues 3 to 8 and 26 to 33, indicated by the white boxes) ablate R5 tropism, resulting in X4-tropic Envs with an increased reliance on the V3-ECL interaction. Because AMD3100 binds within the ECLs of CXCR4 and blocks this interaction, V3 base mutations increase sensitivity to this inhibitor. (C) A deletion of residues 9 to 12 on the N-terminal side of the V3 stem (white box) directly or indirectly disrupts a region that is critical for X4 tropism, resulting in a pure R5-tropic Env. However, this mutation also perturbs the V3-ECL interaction for CCR5, resulting in an increased reliance on the bridging-sheet-V3 base-N terminus interaction. Because CCR5 inhibitors also bind within the transmembrane helices of CCR5 to block the V3-ECL interaction, the Δ9-12 mutant is resistant to these compounds.