Relationships between CD4 independence, neutralization sensitivity, and exposure of a CD4-induced epitope in a human immunodeficiency virus type 1 envelope protein

A CD4-independent version of the X4 human immunodeficiency virus type 1 (HI V-1) HXBc2 envelope (Env) protein, termed 8x, mediates infection of CD4-neg ative, CXCR4-positive cells, binds directly to CXCR4 in the absence of CD I due to constitutive exposure of a conserved coreceptor binding site in the gp120 subunit, and is more sensitive to antibody-mediated neutralization, To study the relationships between CD4 independence, neutralization sensiti vity, and exposure of CD4-induced epitopes associated with the coreceptor b inding site, we generated a large panel of Env mutants and chimeras between 8x and its CD4-dependent parent, HXBc2, We found that a frameshift mutatio n just proximal to the gp41 cytoplasmic domain in 8x Env was necessary but not sufficient for CD4 independence and led to increased exposure of the co receptor binding site. In the presence of this altered cytoplasmic domain, single amino acid changes in either the 8x V3 (V320I) or V4/C4 (N386K) regi ons imparted CD4 independence, with other changes playing a modulatory role . The N386K mutation resulted in loss of an N-linked glycosylation site, bu t additional mutagenesis showed that it was the presence of a lysine rather than loss of the glycosylation site that contributed to CD4 independence. However, loss of the glycosylation site alone was sufficient to render Env neutralization sensitive, providing additional evidence that carbohydrate s tructures shield important neutralization determinants. Exposure of the CD4 -induced epitope recognized by monoclonal antibody 17b and which overlaps t he coreceptor binding site was highly sensitive to an R298K mutation at the base of the V3 loop and was often but not always associated with CD4 indep endence. Finally, while not all neutralization-sensitive Envs were CD4 inde pendent, all CD4-independent Envs exhibited enhanced sensitivity to neutral ization by HIV-l-positive human sera, indicating that the humoral immune re sponse can exert strong selective pressure against the CD4-independent phen otype in vivo. Whether this can be used to advantage in designing more effe ctive immunogens remains to be seen.