Aminooxypentane-RANTES Induces CCR5 Internalization but Inhibits Recycling: A Novel Inhibitory Mechanism of HIV Infectivity

Abstract

CCR5, a chemokine receptor expressed on T cells and macrophages, is the principal coreceptor for M-tropic HIV-1 strains. Recently, we described an NH2-terminal modification of the CCR5 ligand regulated on activation, normal T cell expressed and secreted (RANTES), aminooxypentane-RANTES (AOP-RANTES), that showed potent inhibition of macrophage infection by HIV-1 under conditions where RANTES was barely effective. To investigate the mechanism of AOP-RANTES inhibition of HIV infectivity we examined the surface expression of CCR5 using a monoclonal anti-CCR5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% decrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. RANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycling of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged periods of time in the presence of RANTES, CCR5 expression is comparable to that seen on cells treated with control medium, whereas there is no CCR5 surface expression on cells cultured in the presence of AOP-RANTES. Immunofluorescence indicated that both AOP-RANTES and RANTES induced downmodulation of cell surface CCR5, and that the receptor was redistributed into endocytic organelles containing the transferrin receptor. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence of AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOST) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infection by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differences between AOP-RANTES and RANTES in their effect on receptor downregulation and recycling suggest a mechanism for the potent inhibition of HIV infection by AOP-RANTES. Moreover, these results support the notion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.

Distinct Mechanisms of Agonist-induced Endocytosis for Human Chemokine Receptors CCR5 and CXCR4

“…Agonist treatment induced reduction of high expressers, rather than quantitative loss of CCR5 on all these cells ( Figure 1A1). In agreement with earlier reports (Mack et al, 1998), the IC 50 and t 1/2 for AOP-RANTES was at least threefold better than for RANTES or MIP-1␤. …”

“…Likewise, agonist occupied CXCR4 underwent clathrindependent endocytosis in both primary cells and ectopic expression systems (Amara et al, 1997). Although these studies implied that agonist-driven endocytosis of CCR5 followed the clathrin pathway, a recent report suggested that CCR5 endocytosis followed both clathrin-and caveolae-dependent routes in CHO and HeLa cells (Mueller et al, 2002).Although previous studies (Amara et al, 1997;Mack et al, 1998) examined the rate and route of CCR5 and CXCR4 internalization in both T cells and stable cell lines, there has not been a quantitative head-to-head comparison of the two. Moreover, their localization to specific plasma membrane domains has not been clearly defined.…”

“…In both primary lymphocytes and expression systems, CCR5 has been reported to internalize upon binding agonists. In CHO cells, internalized CCR5 has been reported to colocalize in endosomes with transferrin receptor (Tfn-R), which was used to label the clathrin pathway (Mack et al, 1998). Likewise, agonist occupied CXCR4 underwent clathrindependent endocytosis in both primary cells and ectopic expression systems (Amara et al, 1997).…”

Rab11-Family Interacting Protein 2 and Myosin Vb Are Required for CXCR2 Recycling and Receptor-mediated Chemotaxis

“…Chemokine activation of these intracellular signals is often accompanied by chemokine receptor internalization and trafficking back to the cell membrane. The intracellular trafficking of chemokine receptors controls their activities, and the balance between the chemokine receptor recycling and degradation dictates the leukocyte responsiveness to chemokines (Sabroe et al, 1997;Asagoe et al, 1998;Khandaker et al, 1998;Mack et al, 1998).…”

“…Chemokine activation of these intracellular signals is often accompanied by chemokine receptor internalization and trafficking back to the cell membrane. The intracellular trafficking of chemokine receptors controls their activities, and the balance between the chemokine receptor recycling and degradation dictates the leukocyte responsiveness to chemokines (Sabroe et al, 1997;Asagoe et al, 1998;Khandaker et al, 1998;Mack et al, 1998).Ligand stimulated chemokine receptor internalization can be accomplished through the formation of clathrin-coated pits and/or through formation of lipid rafts (Signoret et al, 1997;Yang et al, 1999;Mueller et al, 2002;Venkatesan et al, 2003). The coated pits internalize as coated vesicles and the later fuse to early endosomes after uncoating (Wu et al, 2001).…”