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

Amara A, Gall SL, Schwartz O, Salamero J, Montes M, Loetscher P, Baggiolini M, Virelizier JL, Arenzana-Seisdedos F - J. Exp. Med. (1997)

Bottom Line: We found a rapid and extensive downregulation of CXCR4 by SDF-1alpha and of CCR5 by RANTES or the antagonist RANTES(9-68).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.

View Article: PubMed Central - PubMed

Affiliation: Unité d'Immunologie Virale, Institut Pasteur, 75724 Paris, Cedex 15, France.

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.

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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.
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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.

Mentions: It was previously shown that endocytosis of IL-8 receptors requires an intact COOH-terminal cytoplasmic domain (26). The role of the corresponding domain of CXCR4 was, therefore, investigated using HeLa cells that were transiently transfected with a vector expressing a CXCR4 cDNA deleted of the last 41 amino acids (CXCR4 ΔCyt). HeLa cells were chosen because they constitutively express CXCR4, and allow to perform simultaneous analysis of SDF-1α effects on endogenous and transfected CXCR4. Cotransfection of either CXCR4 WT or CXCR4 ΔCyt with a GFP reporter vector permitted to distinguish transfected from nontransfected cells. Incubation with SDF-1α induced a profound downregulation of CXCR4 WT, but did not affect the surface expression of the COOH-terminally truncated receptor, CXCR4 ΔCyt (Fig. 3, a and b). As expected the endogenous CXCR4, in nontransfected cells which are identified by the lack of GFP fluorescence, was also markedly down-regulated by SDF-1α (Fig. 3 a). PMA had a similar effect. It downregulated CXCR4 WT (Fig. 3 b) as well as the endogenous CXCR4 (not shown), but not CXCR4 ΔCyt (Fig. 3 b). These results are in agreement with a previous report showing downregulation of CXCR4 by PMA in human T lymphocytes (27), and suggest that phosphorylation of serines and threonines in the COOH-terminal region are involved in internalization. It has been shown that phosphorylation of the COOH-terminal domain is essential for arrestin-mediated uptake of seven-transmembrane domain receptors via clathrin-coated pits (28, 29).


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

Amara A, Gall SL, Schwartz O, Salamero J, Montes M, Loetscher P, Baggiolini M, Virelizier JL, Arenzana-Seisdedos F - J. Exp. Med. (1997)

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.
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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.
Mentions: It was previously shown that endocytosis of IL-8 receptors requires an intact COOH-terminal cytoplasmic domain (26). The role of the corresponding domain of CXCR4 was, therefore, investigated using HeLa cells that were transiently transfected with a vector expressing a CXCR4 cDNA deleted of the last 41 amino acids (CXCR4 ΔCyt). HeLa cells were chosen because they constitutively express CXCR4, and allow to perform simultaneous analysis of SDF-1α effects on endogenous and transfected CXCR4. Cotransfection of either CXCR4 WT or CXCR4 ΔCyt with a GFP reporter vector permitted to distinguish transfected from nontransfected cells. Incubation with SDF-1α induced a profound downregulation of CXCR4 WT, but did not affect the surface expression of the COOH-terminally truncated receptor, CXCR4 ΔCyt (Fig. 3, a and b). As expected the endogenous CXCR4, in nontransfected cells which are identified by the lack of GFP fluorescence, was also markedly down-regulated by SDF-1α (Fig. 3 a). PMA had a similar effect. It downregulated CXCR4 WT (Fig. 3 b) as well as the endogenous CXCR4 (not shown), but not CXCR4 ΔCyt (Fig. 3 b). These results are in agreement with a previous report showing downregulation of CXCR4 by PMA in human T lymphocytes (27), and suggest that phosphorylation of serines and threonines in the COOH-terminal region are involved in internalization. It has been shown that phosphorylation of the COOH-terminal domain is essential for arrestin-mediated uptake of seven-transmembrane domain receptors via clathrin-coated pits (28, 29).

Bottom Line: We found a rapid and extensive downregulation of CXCR4 by SDF-1alpha and of CCR5 by RANTES or the antagonist RANTES(9-68).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.

View Article: PubMed Central - PubMed

Affiliation: Unité d'Immunologie Virale, Institut Pasteur, 75724 Paris, Cedex 15, France.

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.

Show MeSH
Related in: MedlinePlus