Background:  The V3 loop of envelope glycoprotein (Env) is the major determinant for co-receptor utilization, but the structural basis for this specificity remains to be defined. Multiple individual domains of CCR5 were previously suggested to influence co-receptor activity. These observations led to our hypothesis that R5 Env variants may differ in the mechanism for utilization of CCR5 and the molecular anatomy of CCR5 usage may have important implications for viral pathogenesis.

Methods:  To test our hypothesis, cell–cell fusion, pseudotyped infection and gp120 binding assays, combining mutageneses, molecular modeling, and comprehensive sequence analyses, were employed to characterize a set of naturally occurring R5 Env variants cloned from semen and blood samples as well as multiple prototypic R5 Env from different subtypes.

Results:  We demonstrate that Asp-324 in the conserved IIGDIR motif of the V3 loop C-terminal segment regulates the molecular anatomy of CCR5 utilization. Whereas gp120 subunits with Asp or Asn at position 324 were fusogenic with co-receptor chimeras containing either the N-ter domain or the body of CCR5, substitution of charged or small hydrophobic residues resulted in complete loss of fusogenic activity with the N-ter and markedly reduced utilization of the body of CCR5. This phenotypic conversion was confirmed in both gain and loss of function experiments using Env from multiple subtypes. Alignment of sequences of R5 V3 loops (n = 599) from the HIV database revealed that the mutation of Asp-324 in the conserved IIGDIR motif is restricted to Asn-324, with proportions of 71.5% and 28%, respectively. The CD4-dependent gp120 binding to CCR5 was decreased when Asp-324 was replaced with a charged or hydrophobic residue, but unchanged when replaced with Asn. Computational modeling predicts that Asp/Asn-324 form H-bond with Asn-300 that presumably are critical for maintaining the overall conformation of V3 loop. 

Conclusions:  These findings indicate that Asp or Asn at position 324 of the V3 stem stabilizes the conformation of V3 loop and hence influences the intensities of interaction between CD4-activated gp120 and CCR5, which results in viral entry. Our study illustrates the power of characterizing co-receptor function with chimeras and provides insight into the role of the V3 loop in the association of gp120 with the CCR5 co-receptor.

Keywords: HIV-1; Envelope glycoprotein; CCR5