Identification of ENV determinants in V3 that influence the molecular anatomy of CCR5 utilization

The V3 loop of the ENV glycoprotein exerts a dominant influence on the inte raction of gp120 with coreceptors. Primary env genes cloned from sequential isolates from two seroconverters revealed Pro --> Ala conversion in the co nserved GPG motif of the V3 crown in seven of 17 R5 ENV. ENV containing the GPG motif in the V3 crown had fusogenic activity with chimeric receptors c ontaining either the N terminus or loops of CCR5, whereas those with the GA G variant utilized only the former. Site-directed mutagenesis of multiple p rimary and prototypic R5 env genes demonstrated that the GPG motif was nece ssary for dual utilization of the N terminus and body of CCR5 in both gain and loss-of-function experiments. All ENV containing the GPG V3 crown showe d CCR5 binding in the presence of soluble CD4, whereas it was not detected with the GAG variants. Molecular dynamic simulations of a V3 peptide predic ts that the Pro --> Ala substitution results in a conformational change wit h loss of the crown structure. These studies demonstrate that sequences in the third hypervariable region determine the specificity of coreceptor util ization for fusion, and that a conserved motif in the crown directly influe nces the molecular anatomy of the interaction between gp120 and CCR5. (C) 2 000 Academic Press.