Characterization of a human immunodeficiency virus type 1 V3 deletion mutation that confers resistance to CCR5 inhibitors and the ability to use aplaviroc-bound receptor

Abstract

The human immunodeficiency virus type 1 (HIV-1) V3 loop is essential for coreceptor binding and principally determines tropism for the CCR5 and CXCR4 coreceptors. Using the dual-tropic virus HIV-1(R3A), we previously made an extensive panel of V3 deletions and identified subdomains within V3 that could differentially mediate R5 and X4 tropism. A deletion of residues 9 to 12 on the N-terminal side of the V3 stem ablated X4 tropism while leaving R5 tropism intact. This mutation also resulted in complete resistance to several small-molecule CCR5 inhibitors. Here, we extend these studies to further characterize a variant of this mutant, Delta9-12a, adapted for growth in CCR5(+) SupT1 cells. Studies using coreceptor chimeras, monoclonal antibodies directed against the CCR5 amino terminus (NT) and extracellular loops, and CCR5 point mutants revealed that, relative to parental R3A, R5-tropic Delta9-12a was more dependent on the CCR5 NT, a region that contacts the gp120 bridging sheet and V3 base. Neutralization sensitivity assays showed that, compared to parental R3A, Delta9-12a was more sensitive to monoclonal antibodies b12, 4E10, and 2G12. Finally, cross-antagonism assays showed that Delta9-12a could use aplaviroc-bound CCR5 for entry. These studies indicate that increased dependence on the CCR5 NT represents a mechanism by which HIV envelopes acquire resistance to CCR5 antagonists and may have more general implications for mechanisms of drug resistance that arise in vivo. In addition, envelopes such as Delta9-12a may be useful for developing new entry inhibitors that target the interaction of gp120 and the CCR5 NT.

FIG. 1.

Effects of adaptation of the Δ9-12 mutant on coreceptor tropism. (A) Fusion activity in a cell-cell fusion assay is shown for parental R3A, unadapted Δ9-12, and the adapted Δ9-12a Env clone. Percent fusion was calculated by using luciferase activity normalized to R3A fusion on CD4⁺ CXCR4⁺ or CD4⁺ CCR5⁺ cells. The values are means plus the standard errors of the means. The data shown are the averages of three experiments. (B) The infectivities of the R3A, Δ9-12, and Δ9-12a Envs, obtained with a single-cycle luciferase reporter virus on Cf2Th cells transiently transfected with CD4 and the indicated coreceptors, are shown. Percent infection was calculated by using luciferase activity normalized to R3A infection on CD4⁺ CXCR4⁺ or CD4⁺ CCR5⁺ cells. The values are means plus the standard errors of the means. The data shown are the averages of three experiments. (C) Growth curves of viruses containing the R3A and Δ9-12a Envs on SupT1 and SupCCR5 cells are shown. RT activity in culture supernatants was measured at the indicated time points. The results of one of three independent experiments are shown. (D) A schematic representation of the R3A, Δ9-12, and Δ9-12a Envs is shown. Δ9-12 and Δ9-12a contain a four-residue deletion in V3, indicated by Δ. Δ9-12a contains four additional mutations: N406D in V4, E424K and A431T in the C4 domain of the β21 strand of the bridging sheet, and A509V at the amino terminus of gp41 (see the text for the corresponding positions in the HXB reference Env). GFP, green fluorescent protein.

FIG. 2.

Dependence of Δ9-12a on the CCR5 amino terminus. (A) The fusion activities of the R3A and Δ9-12a Envs with cells expressing CD4 and the indicated coreceptors are shown. Percent fusion was calculated by using luciferase activity normalized to R3A fusion on CD4⁺ CCR5⁺ cells. The values are means plus the standard errors of the means. The data shown are the averages of three experiments. (B) Infectivities of the R3A and Δ9-12a Envs in a pseudotype infection assay on QT6 cells expressing CD4 and the indicated coreceptors are shown. Percent infection was calculated by using luciferase activity normalized to R3A infection on CD4⁺ CCR5⁺ cells. The values are means plus the standard errors of the means. The data shown are the averages of three experiments. (C) NP2.CD4.CCR5 cells were preincubated with increasing concentrations of 3A9 and infected with R3A and Δ9-12a pseudovirions. Percent infection was calculated by using luciferase activity normalized to infection in the absence of antibody for each virus. The values are means ± the standard errors of the means. The data shown are the averages of three experiments. GFP, green fluorescent protein.

FIG. 3.

Effects of adaptive mutations in Δ9-12a on coreceptor tropism. Fusion activity on cells expressing CD4, CD4 plus 5222, or CD4 plus 2555 is shown for R3A, the panel of add-back mutations (N406D, E424K, A431T, and A509) in which Δ9-12a mutations were introduced individually into Δ9-12, and the panel of takeaway mutations (D406N, K424E, T431A, and V509A) in which Δ9-12a mutations were individually removed. Percent fusion was calculated by using luciferase activity normalized to Δ9-12a fusion on CD4⁺ 5222⁺ cells. The values are means plus the standard errors of the means. The data shown are the averages of four experiments.

FIG. 4.

Effects of CCR5 point mutations on Δ9-12a. (A) Cell-cell fusion events between the R3A or Δ9-12a Env and cells expressing the indicated CCR5 point mutants are shown. Percent fusion was calculated by using luciferase activity normalized to R3A fusion on CD4⁺ CCR5⁺ cells. The values are means plus the standard errors of the means. The data shown are the averages of three experiments. (B) The infectivities of the R3A and Δ9-12a Envs were determined by using a single-cycle luciferase reporter virus on Cf2Th cells expressing the indicated CCR5 point mutants. Percent infection was calculated by using luciferase activity normalized to R3A infection on CD4⁺ CCR5⁺ cells. The values are means plus the standard errors of the means. The data shown are the averages of three experiments. GFP, green fluorescent protein.

FIG. 5.

Sensitivity of the Δ9-12a mutant to a panel of broadly neutralizing monoclonal antibodies. The sensitivities of R3A, TA1, and Δ9-12a to IgG b12 (A), 17b (B), 4E10 (C), and 2G12 (D) determined in a pseudotype infection assay on NP2.CD4.CCR5 cells are shown. Percent infection was calculated by using luciferase activity normalized to infection in the absence of inhibitor for each virus. The values are means ± standard errors of the means. The data shown are the averages of three experiments.

FIG. 6.

Sensitivity of the Δ9-12a mutant to aplaviroc. (A) The sensitivities of the R3A and Δ9-12a Envs to aplaviroc in a cell-cell fusion assay on CCR5⁺ cells are shown. Percent fusion was calculated by using luciferase activity normalized to fusion in the absence of inhibitor for each Env. The values are means ± the standard errors of the means. The data shown are the averages of three experiments. (B) The sensitivities of R3A and Δ9-12a to aplaviroc in a pseudotype infection assay on NP2.CD4.CCR5 cells are shown. Percent infection was calculated by using luciferase activity normalized to infection in the absence of inhibitor for each virus. The values are means ± the standard errors of the means. The data shown are the averages of three experiments. (C) Growth curves of infectious viruses containing the Δ9-12a and YU2 Envs on SupCCR5 cells in the presence or absence of 10 μM aplaviroc (APL) are shown. RT activity in culture supernatants was measured at the indicated time points. The results of one of three independent experiments are shown.

FIG. 7.

Susceptibility of Δ9-12a to anti-CCR5 monoclonal antibodies 2D7 and 45531 in the presence of a saturating concentration of aplaviroc. (A) NP2.CD4.CCR5 cells were treated with aplaviroc, stained with the indicated antibodies, and analyzed by flow cytometry. The mean channel fluorescence (MCF) is plotted against increasing concentrations of aplaviroc. (B) NP2.CD4.CCR5 cells were preincubated in the presence or absence of 10 μM aplaviroc, preincubated with serial dilutions of 2D7, and then infected with R3A or Δ9-12a pseudovirions. Percent infection was calculated by using luciferase activity normalized to infection in the absence of inhibitor for each virus. The values are means ± the standard errors of the means. The data shown are the averages of three experiments. (C) NP2.CD4.CCR5 cells were preincubated in the presence or absence of 10 μM aplaviroc, preincubated with serial dilutions of 45531, and then infected with R3A or Δ9-12a pseudovirions. Percent infection was calculated by using luciferase activity normalized to infection in the absence of inhibitor for each virus. The values are means ± the standard errors of the means. The data shown are the averages of three experiments.