PHORBOL ESTERS AND SDF-1 INDUCE RAPID ENDOCYTOSIS AND DOWN-MODULATIONOF THE CHEMOKINE RECEPTOR CXCR4

The chemokine receptor CXCR4 is required, together with CD4, for entry by some isolates of HIV-1, particularly those that emerge late in inf ection. The use of CXCR4 by these viruses likely has profound effects on viral host range and correlates with the evolution of immunodeficie ncy. Stromal cell-derived factor-1 (SDF-1), the ligand for CXCR4, can inhibit infection by CXCR4-dependent viruses. To understand the mechan ism of this inhibition, we used a monoclonal antibody that is specific for CXCR4 to analyze the effects of phorbol esters and SDF-1 on surfa ce expression of CXCR4. On human T cell lines SupT1 and BC7, CXCR4 und ergoes slow constitutive internalization (1.0% of the cell surface poo l/min). Addition of phorbol esters increased this endocytosis rate >6- fold and reduced cell surface CXCR4 expression by 60 to 90% over 120 m in. CXCR4 was internalized through coated pits and coated vesicles and subsequently localized in endosomal compartments from where it could recycle to the cell surface after removal of the phorbol ester. SDF-1 also induced the rapid down modulation (half time similar to 5 min) of CXCR4. Using mink lung epithelial cells expressing CXCR4 and a COOH-t erminal deletion mutant of CXCR4, we found that an intact cytoplasmic COOH-terminal domain was required for both PMA and ligand-induced CXCR 4 endocytosis. However, experiments using inhibitors of protein kinase C indicated that SDF-1 and phorbol esters trigger down modulation thr ough different cellular mechanisms. SDF-1 inhibited HIV-1 infection of mink cells expressing CD4 and CXCR4. The inhibition of infection was less efficient for CXCR4 lacking the COOH-terminal domain, suggesting at least in part that SDF-1 inhibition of virus infection was mediated through ligand-induced internalization of CXCR4. Significantly, ligan d induced internalization of CXCR4 but not CD4, suggesting that CXCR4 and CD4 do not normally physically interact on the cell surface. Toget her these studies indicate that endocytosis can regulate the cell-surf ace expression of CXCR4 and that SDF-1-mediated down regulation of cel l-surface coreceptor expression contributes to chemokine-mediated inhi bition of HIV infection.