HIV coreceptor downregulation as antiviral principle: SDF-1alpha-dependent internalization of the chemokine receptor CXCR4 contributes to inhibition of HIV replication

Abstract

Ligation of CCR5 by the CC chemokines RANTES, MIP-1alpha or MIP-1beta, and of CXCR4 by the CXC chemokine SDF-1alpha, profoundly inhibits the replication of HIV strains that use these coreceptors for entry into CD4(+) T lymphocytes. The mechanism of entry inhibition is not known. We found a rapid and extensive downregulation of CXCR4 by SDF-1alpha and of CCR5 by RANTES or the antagonist RANTES(9-68). Confocal laser scanning microscopy showed that CCR5 and CXCR4, after binding to their ligands, are internalized into vesicles that qualify as early endosomes as indicated by colocalization with transferrin receptors. Internalization was not affected by treatment with Bordetella pertussis toxin, showing that it is independent of signaling via Gi-proteins. Removal of SDF-1alpha led to rapid, but incomplete surface reexpression of CXCR4, a process that was not inhibited by cycloheximide, suggesting that the coreceptor is recycling from the internalization pool. Deletion of the COOH-terminal, cytoplasmic domain of CXCR4 did not affect HIV entry, but prevented SDF-1alpha-induced receptor downregulation and decreased the potency of SDF-1alpha as inhibitor of HIV replication. Our results indicate that the ability of the coreceptor to internalize is not required for HIV entry, but contributes to the HIV suppressive effect of CXC and CC chemokines.

Figure 1

Effect of SDF-1α stimulation on CXCR4 surface expression. CEM lymphoblastoid T cells (a), monocyte-depleted PBMC (b), or HeLa cells (c) were treated for 40 min at 37°C with 200 nM SDF-1α, 200 nM RANTES, or medium alone (UNTREATED). The cells were then washed with acidic glycine buffer, labeled at 4°C with anti-CXCR4 and a PE-conjugated secondary antibody, and analyzed by flow cytometry. In b, analysis was performed on gated CD4⁺ cells (FITC-conjugated anti-CD4). Control cells (CTRL) were labeled with the secondary antibody only. (d) Dependence of CXCR4 downregulation on SDF-1α concentration. CEM cells were incubated for 40 min at 37°C with increasing concentrations of SDF-1α, and surface expression of CXCR4 was determined. PTX: before incubation with SDF-1α, the cells were treated with 5 μg/ml pertussis toxin for 90 min. (e) Time course of CXCR4 downregulation. Cells were pre-incubated at 4°C for 60 min with 200 nM SDF-1α or RANTES. After washing, cells were cultured at 37°C for the indicated times in the absence of chemokines. (   f   ) Re-expression of CXCR4. The cells were incubated at 37°C for 40 min with 200 nM SDF-1α in the presence or absence of 100 μg/ml cycloheximide, washed in acidic glycine buffer and further cultured for up to 60 min at 37 °C with or without cycloheximide in the absence of SDF-1α. (d–f  ) Relative surface expression of CXCR4 was analyzed by flow cytometry as described for a.

Figure 2

Internalization of CXCR4 (a) and CCR5 (b and c) were analyzed by confocal laser scanning microscopy in CEM (a), CHO-CCR5-GFP (b), and HeLa-CCR5-GFP (c) cells after exposure to 200 nM SDF-1α, RANTES or RANTES(9-68) for 30 min at 37°C. –, untreated cells; CXCR4, cells labeled with anti-CXCR4 and a FITC-conjugated secondary antibody; CCR5, autofluorescence of CCR5-GFP; CXCR4+, Tf-RITC and CCR5+Tf-RITC, simultaneous detection of Tf-RITC (red ) and either CXCR4 or CCR5-GFP (  green). Yellow spots indicate colocalization of chemokine receptor and Tf-RITC.

Figure 2

Internalization of CXCR4 (a) and CCR5 (b and c) were analyzed by confocal laser scanning microscopy in CEM (a), CHO-CCR5-GFP (b), and HeLa-CCR5-GFP (c) cells after exposure to 200 nM SDF-1α, RANTES or RANTES(9-68) for 30 min at 37°C. –, untreated cells; CXCR4, cells labeled with anti-CXCR4 and a FITC-conjugated secondary antibody; CCR5, autofluorescence of CCR5-GFP; CXCR4+, Tf-RITC and CCR5+Tf-RITC, simultaneous detection of Tf-RITC (red ) and either CXCR4 or CCR5-GFP (  green). Yellow spots indicate colocalization of chemokine receptor and Tf-RITC.

Figure 2

Internalization of CXCR4 (a) and CCR5 (b and c) were analyzed by confocal laser scanning microscopy in CEM (a), CHO-CCR5-GFP (b), and HeLa-CCR5-GFP (c) cells after exposure to 200 nM SDF-1α, RANTES or RANTES(9-68) for 30 min at 37°C. –, untreated cells; CXCR4, cells labeled with anti-CXCR4 and a FITC-conjugated secondary antibody; CCR5, autofluorescence of CCR5-GFP; CXCR4+, Tf-RITC and CCR5+Tf-RITC, simultaneous detection of Tf-RITC (red ) and either CXCR4 or CCR5-GFP (  green). Yellow spots indicate colocalization of chemokine receptor and Tf-RITC.

Figure 3

Effect of deletion of the COOH-terminal cytoplasmic domain of CXCR4. (a) HeLa cells were transiently transfected with the CXCR4 WT or the CXCR4 ΔCyt expression vector, along with a plasmid encoding the reporter gene GFP (pEGFP). 24 h later, the cells were incubated for 45 min at 37°C in the presence or absence of 300 nM SDF-1α, labeled with anti-CXCR4, and analyzed by flow cytometry. Expression of GFP allows to distinguish transfected (GFP ⁺) and nontransfected (GFP ⁻) cells. After transfection with CXCR4 WT or CXCR4 Δcyt, SDF-1α–dependent downregulation of the endogenous and the transfected receptor were monitored in GFP⁻ and GFP⁺ cells, respectively. (–) HeLa cells were transiently transfected with the CXCR4 WT or CXCR4 ΔCyt expression vector, along with the pEGFP plasmid. 24 h later, the cells were incubated for 45 min at 37°C with 300 nM SDF-1α or with 20 ng/ml PMA, labeled with anti-CXCR4 and surface expression of CXCR4 was analyzed by flow cytometry in GFP⁺ cells. (c) CHO cells were transfected with either the CXCR4 WT or the CXCR4 ΔCyt expression vectors and were loaded 48 h later with Fura-2. CHO control cells were transfected with vector DNA alone (pcDNA3). Recordings of [Ca²⁺]_i changes after stimulation with 200 nM of SDF-1α are shown.

Figure 4

SDF-1α–dependent inhibition of HIV infection in cells expressing wild-type or COOH-terminally truncated CXCR4. U373-CD4 LTRlacZ were transfected with either CXCR4 WT or CXCR4 ΔCyt expression vector, plated in 96-well plates (10⁴ cells per well) and infected with the HIV-1_NL4-3 strain or the pseudotyped HIV-1(Δenv)G, in the presence of the indicated concentrations of SDF-1α or RANTES. HIV-1(Δenv)G–infected cells were treated with 500 nM SDF-1α. Cell cultures were carried out in triplicates. Surface expression of CXCR4 WT or CXCR4 ΔCyt was assessed 24 h after transfection by FACS^® analysis and amounted to 482 and 533 fluorescence units, respectively. HIV infection was revealed 24 h later by staining for β-galactosidase activity and scoring of positive cells. The numbers of HIV-infected cells per well in the absence of chemokines were 168 and 232 (mean of five experiments) for cultures transfected with CXCR4 WT and CXCR4 ΔCyt, respectively. In cultures infected with HIV-1(Δenv)G the average number of infected cells per well was 280. Shown are the percentages of infected cells in the presence of chemokines. Infection with HIV-1_NL4-3 or HIV-1(Δenv)G in the absence of chemokines was set to 100%.