CCR5-mediated human immunodeficiency virus entry depends on an amino-terminal gp120-binding site and on the conformational integrity of all four extracellular domains

The human immunodeficiency virus type 1 coreceptor activity of CCR5 depends on certain polar and charged residues in its amino-terminal domain. Since studies of chimeric receptors have indicated that the extracellular loops o f CCR5 are also involved in viral fusion and entry, we have explored the ro le of bulky, polar and nonpolar residues in these regions. Selected amino a cids in the three extracellular loops were individually changed do alanines , and the coreceptor activities of the mutant CCR5 proteins were tested in a luciferase reporter virus-based entry assay. We found that the cysteines in the extracellular loops of CCR5 are essential for coreceptor activity. H owever, only minor (two- to threefold) effects on coreceptor function were noted for all of the other alanine substitutions. We also demonstrated that when the first 19 residues of the amino-terminal region were separated fro m the rest of CCR5; by insertion of glycine/serine spacers between proline 19 and cysteine 20, coreceptor function decreased. Together with our previo us studies, these data indicate that both an amino-terminal gp120-binding s ite and extracellular domain geometry play a role in viral entry.