Molecular anatomy of CCR5 engagement by physiologic and viral chemokines and HIV-1 envelope glycoproteins: Differences in primary structural requirements for RANTES, MIP-1 alpha, and vMIP-II binding

Molecular analysis of CCR5, the cardinal coreceptor for HIV-1 infection, ha s implicated the N-terminal extracellular domain (N-ter) and regions vicina l to the second extracellular loop (ECL2) in this activity. It was shown th at residues in the N-ter are necessary for binding of the physiologic logic ligands, RANTES (CCL5) and MIP-1 alpha (CCL3). vMIP-II, encoded by the Kap osi's sarcoma-associated herpesvirus, is a high affinity CCR5 antagonist, b ut lacks efficacy as a coreceptor inhibitor. Therefore, we compared the mec hanism for engagement by vMIP-II of CCR5 to its interaction with physiologi c ligands. RANTES, MIP-1 alpha, and vMIP-II bound CCR5 at high affinity, bu t demonstrated partial cross-competition. Characterization of 15 CCR5 alani ne scanning mutants of charged extracellular amino acids revealed that alte ration of acidic residues in the distal N-ter abrogated binding of RANTES, MIP-1 alpha, and vMIP-II. Whereas mutation of residues in ECL2 of CCR5 dram atically reduced the binding of RANTES and MIP-la and their ability to indu ce signaling, interaction with vMIP-II was not altered by any mutation in t he exoloops of the receptor. Paradoxically, monoclonal antibodies to N-ter epitopes, did not block chemokine binding, but those mapped to ECL2 were ef fective inhibitors. A CCR5 chimera with the distal N-ter residues of CXCR2 bound MIP-1a and vMIP-II with an affinity similar to that of the wildtype r eceptor. Engagement of CCR5 by vMIP-II, but not RANTES or MIP-1a blocked th e binding of monoclonal antibodies to the receptor, providing additional ev idence for a distinct mechanism for viral chemokine binding. Analysis of th e coreceptor activity of randomly generated mouse-human CCR5 chimeras impli cated residues in ECL2 between H173 and V197 in this function. RANTES, but not vMIP-II blocked CCR5 M-tropic coreceptor activity in the fusion assay. The insensitivity of vMIP-II binding to mutations in ECL2 provides a potent ial rationale to its inefficiency as an antagonist of CCR5 coreceptor activ ity. These findings suggest that the molecular anatomy of CCR5 binding play s a critical role in antagonism of coreceptor activity. (C) 2001 Academic P ress.