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14 Modifications in the Cytoplasmic Domain of HIV-1 Env Allow Exposure of the Conserved Coreceptor-Binding Site and Enhance Neutralization Sensitivity
F. Baribaud*1, T. G. Edwards1, S. Wyss1, J. D. Reeves1, S. Zolla-Pazner2, J. A. Hoxie1, and R. W. Doms1
1Univ. of Pennsylvania, Philadelphia, USA and 2VA Med. Ctr., New York, NY, USA
Background: We recently described a CD4-independent variant of HXBc2 termed IIIBx. IIIBx was generated by serial in vivo passage of HXBc2 on CD4-negative, CXCR4-positive BC7 cells. A molecular clone from this swarm, designated 8x, was PCR amplified and was shown to bind directly to CXCR4 and to mediate CD4-independent virus infection. Determinants for CD4 independence map to residues in the V3 and V4/C4 domains together with the transmembrane (TM) domain of 8x. Sequence analysis revealed that 8x has a single nucleotide deletion in the TM domain, which introduces a frameshift (FS) at position 706. This FS results in a truncated cytoplasmic domain of only 27 amino acids. Using the CD4-induced MAbs 17b or 48d as surrogates for measuring the exposure of the coreceptor binding site, we showed that 8x binds both MAbs.
Methods: We used a fusion assay and a radioimmunoassay to understand how the 8x frameshift mutation alters the conformation of the Env ectodomain.
Results: We demonstrate here that introduction of the 8x FS mutation into heterologous R5, X4, or R5/X4 Envs directly impacts the conformation of the gp120 surface subunit, exposing the conserved coreceptor binding site. Interestingly, truncation of gp41 also impacted neutralization sensitivity of all of these Envs. Indeed, constructs bearing the FS mutation were severalfold more sensitive to neutralization by HIV+ sera than were the parental Envs. Also, we found that the FS constructs were more sensitive to neutralization by 48d, indicating that the coreceptor binding site is a major target for neutralizing antibodies. Finally, we find that all HIV+ human sera tested neutralize these modified Envs even when they were obtained from patients in advanced stages of AIDS (CD4 counts < 100).
Conclusions: These results demonstrate that a humoral response targeting the conserved coreceptor binding site is present but not sufficient to neutralize parental viruses at all stages of the disease. These results give new insights in the continuous efforts to design modified Envs as immunogens to elicit a potent neutralizing response.
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