Two mechanisms for human immunodeficiency virus type 1 inhibition by N-terminal modifications of RANTES - PubMed (original) (raw)

Two mechanisms for human immunodeficiency virus type 1 inhibition by N-terminal modifications of RANTES

Cristina Pastore et al. Antimicrob Agents Chemother. 2003 Feb.

Abstract

C-C chemokine receptor 5 (CCR5) is the primary coreceptor for human immunodeficiency virus type 1 (HIV-1) infection. Native chemokines that bind to CCR5 inhibit HIV-1 infection, albeit weakly, but chemically modified chemokines inhibit infection more efficiently. We have investigated the inhibitory mechanism of three N-terminally modified RANTES variants (AOP-, NNY-, and PSC-RANTES) with the MT-2 human T-cell line stably expressing either native or mutated CCR5. The RANTES analogues showed the same rank order (PSC > NNY > AOP) in their capacity to induce prolonged CCR5 internalization, inhibit surface reexpression, and prevent HIV-1 infection on MT-2 cells expressing wild-type CCR5 or CCR5 with four C-terminal serine phosphorylation sites mutated to alanine. None of the RANTES analogues caused internalization of a C-terminal cytoplasmic domain deletion mutant of CCR5, and each derivative had equal potency in inhibiting HIV-1 infection of MT-2 cells expressing this mutant. We conclude that the C-terminal cytoplasmic residues of CCR5 are necessary for receptor sequestration by RANTES analogues but that the process and the relative activity of each derivative are not dependent upon phosphorylation of the C-terminal serine residues. Two mechanisms of antiviral activity are demonstrated: receptor blockade and receptor sequestration. Potency correlates with the ability to induce CCR5 sequestration but not with receptor binding, suggesting that sequestration may make the greater contribution to antiviral activity.

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Figures

FIG. 1.

FIG. 1.

Flow cytometry of MT-2 cells stably transduced with CCR5 (MT-2-R5lo and MT-2-R5hi), CCR5Δcyt (MT-2-R5Δcyt), or serine mutant CCR5 (MT-2-R5S4A). Staining with a CCR5 antibody is shown by a bold line; the negative control (MT-2 cells) is shown by a dashed line.

FIG. 2.

FIG. 2.

Fusion inhibition by Met-, AOP-, NNY-, and PSC-RANTES. HeLa-P5L and HeLa-Env-ADA cells were exposed to the indicated concentration of modified RANTES, and fusion efficiency was determined by expression of β-galactosidase activity as described before (20).

FIG. 3.

FIG. 3.

Internalization rate of CCR5 in MT-2-R5lo (A), MT-2-R5hi (B), MT-2-R5S4A (C), and MT-2-R5Δcyt cells (D) during treatment with AOP-, NNY-, or PSC-RANTES. The four MT-2 clones were treated with the different analogues (100 nM), and CCR5 surface expression was analyzed by flow cytometry at different time intervals. The internalization rate is shown as a percentage of the CCR5 expression level in untreated cells.

FIG. 4.

FIG. 4.

Extended-incubation experiment with RANTES analogues. MT-2-R5lo (A), MT-2-R5hi (B), MT-2-R5S4A (C), or MT-2-R5Δcyt (D) cells were incubated with 10 nM AOP-, NNY-, or PSC-RANTES for 8 days, and the expression of CCR5 was analyzed by flow cytometry at 1, 3, 4, 6, and 8 days. The reexpression rate is shown as a percentage of the CCR5 expression level in untreated cells.

FIG. 5.

FIG. 5.

CCR5 reexpression rate after ligand-mediated internalization. MT-2-R5lo (A), MT-2-R5hi (B), and MT-2-R5S4A (C) clones were treated with the three analogues (10 nM) and washed three times with and resuspended in fresh medium. CCR5 reexpression was monitored by flow cytometry at 1, 3, and 16 h.

FIG. 6.

FIG. 6.

Infection of MT-2 clones with HIV-1. MT-2-R5lo (A), MT-2-R5hi (B), MT-2-R5S4A (C), or MT-2-R5Δcyt (D) cells were incubated with different concentrations of AOP-, NNY-, or PSC-RANTES for 2 h before infection with the R5 virus BaL. The cells were washed after 24 h and incubated again with the RANTES analogues. The level of replication was assessed 5 days after infection by a p24 enzyme-linked immunosorbent assay of the cell culture medium. The results are shown as percent inhibition compared to the relevant untreated control.

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