AMINOOXYPENTANE-RANTES INDUCES CCR5 INTERNALIZATION BUT INHIBITS RECYCLING - A NOVEL INHIBITORY MECHANISM OF HIV INFECTIVITY

CCR5, a chemokine receptor expressed on T cells and macrophages, is th e principal coreceptor for M-tropic HIV-1 strains. Recently, we descri bed an NH2-terminal modification of the CCR5 ligand regulated on activ ation, normal T cell expressed and secreted (RANTES), aminooxypentane- RANTES (AOP-RANTES), that showed potent inhibition of macrophage infec tion by HIV-1 under conditions where RANTES was barely effective. To i nvestigate the mechanism of AOP-RANTES inhibition of HIV infectivity r ye examined the surface expression of CCR5 using a monoclonal anti-CCR 5 antibody, MC-1. We demonstrate that AOP-RANTES rapidly caused >90% d ecrease in cell surface expression of CCR5 on lymphocytes, monocytes/ macrophages, and CCR5 transfected Chinese hamster ovary (CHO) cells. R ANTES also caused a loss of cell surface CCR5, although its effect was less than with AOP-RANTES. Significantly, AOP-RANTES inhibited recycl ing of internalized CCR5 to the cell surface, whereas RANTES did not. When peripheral blood mononuclear cells are cultured for prolonged per iods 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-RANT ES. Immunofluorescence indicated that both AOP-RANTES and RANTES induc ed downmodulation of cell surface CCR5, and that the receptor was redi stributed into endocytic organelles containing the transferrin recepto r. When RANTES was removed, the internalized receptor was recycled to the cell surface; however, the receptor internalized in the presence o f AOP-RANTES was retained in endosomes. Using human osteosarcoma (GHOS T) 34/CCR5 cells, the potency of AOP-RANTES and RANTES to inhibit infe ction by the M-tropic HIV-1 strain, SF 162, correlated with the degree of downregulation of CCR5 induced by the two chemokines. These differ ences between AOP-RANTES and RANTES in their effect on receptor downre gulation and recycling suggest a mechanism for the potent inhibition o f HIV infection by AOP-RANTES. Moreover, these results support the not ion that receptor internalization and inhibition of receptor recycling present new targets for therapeutic agents to prevent HIV infection.