Structure-function studies of HIV-1 envelope protein

Entry of HIV into target cells requires sequential interactions of the viral envelope protein (Env) with CD4 and appropriate chemokine receptor on the target cell. The expression and usage of various chemokine receptors on CD4-positive cells defines viral tropism at the level of entry, an important determinant of HIV pathogenesis. Two major projects are presented in this thesis examining the relationship between Env and coreceptors. The first project examines the coreceptor use by a number of Env isolates from pathogenic and non-pathogenic SHIV clones that evolved from a single, CXCR4-specific precursor. While some isolates continued to use CXCR4, one isolate was identified that used CCR2b but not CXCR4. A series of chimeras between these isolates mapped the determinants of coreceptor use to the V1/V2 and V3 regions of Env. In the second project, a CD4-independent strain of HIV was derived that could make direct interactions with CXCR4 and did not require CD4 for virus entry. A series of chimeras mapped the determinants of CD4 independence to the V3 and V4/C4 domains of Env. Optical biosensor studies showed that increased exposure of the chemokine receptor binding site in Env was responsible for CD4 independence. The increased neutralization sensitivity of this virus and exposure of the highly conserved chemokine receptor binding site, an important neutralization determinant, form the basis for the further evaluation of CD4 independent viruses in vaccine trials. The studies performed in this thesis provide important insight regarding the mechanism of HIV entry when coupled with recently derived structural information regarding HIV Env