Molecular analysis of CCR5, the cardinal coreceptor for HIV-1 infection, has 
implicated the N-terminal extracellular domain (N-ter) and regions vicinal to 
the second extracellular loop (ECL2) in this activity. It was shown that 
residues in the N-ter are necessary for binding of the physiologic ligands, 
RANTES (CCL5) and MIP-1 alpha (CCL3). vMIP-II, encoded by the Kaposi's 
sarcoma-associated herpesvirus, is a high affinity CCR5 antagonist, but lacks 
efficacy as a coreceptor inhibitor. Therefore, we compared the mechanism for 
engagement by vMIP-II of CCR5 to its interaction with physiologic ligands. 
RANTES, MIP-1 alpha, and vMIP-II bound CCR5 at high affinity, but demonstrated 
partial cross-competition. Characterization of 15 CCR5 alanine scanning mutants 
of charged extracellular amino acids revealed that alteration 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 dramatically reduced the binding of 
RANTES and MIP-1 alpha and their ability to induce signaling, interaction with 
vMIP-II was not altered by any mutation in the exoloops of the receptor. 
Paradoxically, monoclonal antibodies to N-ter epitopes did not block chemokine 
binding, but those mapped to ECL2 were effective inhibitors. A CCR5 chimera with 
the distal N-ter residues of CXCR2 bound MIP-1 alpha and vMIP-II with an 
affinity similar to that of the wild-type receptor. Engagement of CCR5 by 
vMIP-II, but not RANTES or MIP-1 alpha blocked the binding of monoclonal 
antibodies to the receptor, providing additional evidence for a distinct 
mechanism for viral chemokine binding. Analysis of the coreceptor activity of 
randomly generated mouse-human CCR5 chimeras implicated 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 potential rationale to its inefficiency as an 
antagonist of CCR5 coreceptor activity. These findings suggest that the 
molecular anatomy of CCR5 binding plays a critical role in antagonism of 
coreceptor activity.