Amino acid 324 in the simian immunodeficiency virus SIVmac V3 loop can confer CD4 independence and modulate the interaction with CCR5 and alternative coreceptors

Abstract

The V3 loop of the simian immunodeficiency virus (SIV) envelope protein (Env) largely determines interactions with viral coreceptors. To define amino acids in V3 that are critical for coreceptor engagement, we functionally characterized Env variants with amino acid substitutions at position 324 in V3, which has previously been shown to impact SIV cell tropism. These changes modulated CCR5 engagement and, in some cases, allowed the efficient usage of CCR5 in the absence of CD4. The tested amino acid substitutions had highly differential effects on viral infectivity. Eleven of sixteen substitutions disrupted entry via CCR5 or the alternative coreceptor GPR15. Nevertheless, most of these variants replicated in the macaque T-cell line 221-89 and some also replicated in rhesus macaque peripheral blood monocytes, suggesting that efficient usage of CCR5 and GPR15 on cell lines is not a prerequisite for SIV replication in primary cells. Four variants showed enhanced entry into the macaque sMagi reporter cell line. However, sMagi cells did not express appreciable amounts of CCR5 and GPR15 mRNA, and entry into these cells was not efficiently blocked by a small-molecule CCR5 antagonist, suggesting that sMagi cells express as-yet-unidentified entry cofactors. In summary, we found that a single amino acid at position 324 in the SIV Env V3 loop can modulate both the efficiency and the types of coreceptors engaged by Env and allow for CD4-independent fusion in some cases.

FIG. 1.

(A) Alignment of V3 sequences. An alignment of the V3 sequences of HIV-1 JR-FL (top) and SIVmac239 (bottom) is shown; the arrow indicates amino acid I324. (B) Env expression and virion incorporation. The following SIVmac239 proviral genomes were transiently transfected into 293T cells: mock (lane 1), ΔEnv (lane 2), wt (lane 3), 316Env (lane 4), I324S Env (lane 5), I324V Env (lane 6), I324L Env (lane 7), I324Y Env (lane 8), and I324H Env (lane 9). The transfected cells were lysed and Env expression in lysates was detected by Western blot with the DA6 monoclonal antibody (13) (top panel). Alternatively, viral supernatants normalized for equal amounts of p27 antigen were pelleted, lysed, and Env (middle panel) or p27 (lower panel) incorporation was assessed in parallel by Western blot analysis with DA6 (Env staining) or serum from SIVmac-infected rhesus macaques (p27 staining).

FIG. 2.

Amino acid substitutions at position 324 modulate CCR5 engagement. (A) Usage of wt CCR5 for cell-cell fusion. Effector cells were transiently transfected with the indicated Env expression plasmids and infected with vaccinia virus encoding T7 polymerase. Target cells were transfected with rhesus macaque CD4, human CCR5, and an expression plasmid encoding luciferase under control of the T7 promoter. Effector and target cells were mixed, and luciferase production, indicating cell-cell fusion, was quantified. The average ± the standard error of the mean (SEM) of three independent experiments carried out in triplicates is shown. (B) Usage of CCR5/CCR2 chimeric receptors for cell-cell fusion. The fusion assay was carried out as described above; however, the indicated CCR5/CCR2 chimeras were used as coreceptors. The average ± the SEM of four independent experiments carried out in triplicates is presented. (C) Impact of CCR5 inhibition on replication in T cells. The macaque T-cell line 221-89 was incubated with the indicated concentrations of the small-molecule inhibitor TAK-779 and infected with 5 ng of either SIVmac239wt or the I324Y and I324H variants. Inhibitor was replenished during cultivation, and reverse transcriptase activity in the culture supernatants was assessed. The average ± the SEM of three experiments performed in parallel is indicated.

FIG. 3.

Amino acid 324 can confer CD4-independent cell fusion. The ability of 239wt Env and the I324Y, I324H, and I324L Envs to mediate fusion via human and rhesus macaque CCR5 in the presence or absence of rhesus macaque CD4 was assessed in a cell-cell fusion assay as described in the legend to Fig. 2. The results from a representative experiment carried out in triplicate is shown, with error bars indicating the standard deviation. Comparable results were obtained in two different experiments.

FIG. 4.

Coreceptor determinants for SIV entry into sMagi cells. (A) Inhibition of infection by small-molecule inhibitors. P4R5 reporter cells expressing human CD4, CCR5, and CXCR4 and sMagi cells were infected with the indicated p24/p27-normalized virus stocks in the presence or absence (Mock) of the CCR5 inhibitor TAK-779 (5 μM) or the CXCR4 inhibitor AMD3100 (1 μM). The β-galactosidase activity as a readout for infection was determined 3 days later. The results of a representative experiment are shown, and similar results were obtained in an independent experiment. (B) Inhibition of entry into sMagi cells by different concentrations of TAK-779. rhCCR5 Magi cells, which express human CD4, together with pigtailed macaque CCR5, and sMagi cells were infected as described above. Similar results were obtained in an independent experiment.

FIG. 5.

Coreceptor expression in sMagi cells and coreceptor usage of the I324L variant. (A) Analysis of coreceptor expression in sMagi cells. Total RNA was prepared from sMagi cells, DNase treated, diluted as indicated, and subjected to RT-PCR analysis with primers specific for the indicated coreceptors. (B) Coreceptor usage of the I324L variant. 293T cells were transiently cotransfected with human CD4 and the indicated coreceptors of rhesus macaque origin. The transfected cells were infected with 50 ng of the indicated luciferase reporter viruses and luciferase activity quantified. The results are shown relative to SIVmac239 entry via CD4 and CCR5 and similar results were obtained in an independent experiment.