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13 Determinants in the HIV-1 Cytoplasmic Tail That Induce Exposure of CD4-Induced Epitopes in gp120
S. Wyss*1, T. Edwards1, F. Baribaud1, J. Romano1, P. J. Vance1, S. Zolla-Pazner2, R. W. Doms1, and J. A. Hoxie1
1Univ. of Pennsylvania, Philadelphia, USA and 2VA Med. Ctr., New York, NY, USA
Background: We described an X4-tropic, CD4-independent variant of HIV-1/IIIB, termed 8x. Mutations in the 8x Env were shown to expose CD4-induced epitopes partially overlapping the chemokine receptor binding site on gp120 as determined by mAbs 17b and 48d. Determinants for CD4-independent fusion were shown to map to mutations in V3 and V4/C4 on gp120 and remarkably to a frameshift mutation in the gp41 membrane-spanning domain that generated a divergent and prematurely truncated cytoplasmic tail of only 27 amino acids. Interestingly, the 8x frame shift mutation alone was sufficient to induce 17b and 48d reactivity and also conferred increased fusogenicity and neutralization sensitivity using cell to cell fusion assays.
Methods: To understand how the 8x frameshift mutation apparently altered the conformation of the Env ectodomain, site-directed mutations were introduced in the HXBc2 cytoplasmic tail, and reactivity to 17b and 48d was determined by radioimmunoassays of Envs expressed on 293T cells.
Results: Premature termination codons introduced at positions 733, 753, and 764 were found to be sufficient to expose 17b and 48d epitopes, whereas reactivity with mAbs to the C5 and V3 domains of gp120 were unaffected. Stop codons at position 771 and 808 did not produce this effect. Although Cys residues at positions 764 and 837 have been shown to be palmitoylated and implicated in membrane associations, mutations that eliminated these sites failed to confer 17b or 48d reactivity. In general, Envs with 17b and 48d reactivity were also more fusogenic in fusion assays.
Conclusions: Truncations in the gp41cytoplasmic domain can alter the conformation of the Env ectodomain and increase exposure of CD4-induced epitopes, including those overlapping with the chemokine receptor binding site. Because similar changes were not seen for mAbs to C5 or V3, these effects could not be accounted for simply by differences in Env surface expression. Ongoing studies with additional mutants will help to establish the mechanism for this novel effect and determine the extent to which these conformational changes in gp120 can be exploited in the development of novel Env-based immunogens.
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