Background: Interaction of HIV-1 and SIV gp120 with CD4 induces conformational change in gp41 leading to virus-cell membrane fusion. This “fusion-active” gp41 conformation constitutes a six-helix bundle in which three N-terminal helices form coiled-coil internal structure that is surrounded by three C-terminal helices in an antiparallel orientation. Sequences in the N- and C-terminal a-helices significantly influence the stability of the helical bundle and fusion activity of gp41. Hear we report that a single mutation that naturally occurred in the N-terminal a-helix of SIV gp41 expands coreceptor specificity of viral envelope.

Methods: An M-tropic SIV variant, termed SIVmac155/BR, was isolated from a brain of rhesus macaque that developed encephalitis following infection with T-tropic SIVmac239. NP-2 cells expressing CD4 and chemokine receptors were used to define viral coreceptor usage. Viral sequences encoding gp120 and gp41 of SIVmac155/BR were obtained by PCR and used to construct env recombinant viruses. Soluble CD4 (sCD4) and SIV-C34 peptide were used for virus neutralization assays.

Results: In a panel of NP-2 cells expressing CD4 and chemokine receptors, SIVmac239 replicated in the cells co-expressing CD4/CCR5; however SIVmac155/BR replicated in the cells expressing CD4/CCR5, CD4/CXCR4 and CD4/RDC-1. We found that gp120 of SIVmac155/BR recognized only CD4/CCR5, whereas gp41 of SIVmac155/BR conferred the ability on viral envelope to use CD4/CXCR4 and CD4/RDC-1 for virus entry. Genetic mapping using finer gp41 recombinants and site-specific mutants revealed that a single mutation of 586Thr/Val in the N-terminal a-helix of the gp41 was solely responsible for the recognition of CD4/CXCR4 and CD4/RDC-1. The 586Val/Thr mutation did not influence virus neutralization by sCD4 but significantly reduced sensitivity to neutralization by SIV-C34 peptide.

Conclusions: These data demonstrate that natural mutation of 586Thr/Val in the N-terminal a-helix of SIV gp41 induces a strong impact on viral envelope to expand its coreceptor repertoires. Thr586 is well conserved in SIV and located at the N-terminal haptad-repeat of hydrophobic residues, whereas HIV-1 contains hydrophobic Ile at this position. Of note, a substitution of Ile for Thr586 has been shown to increase the stability of SIV gp41 structure. Further studies will define the impact of 586Thr/Val mutation on the structure and fusion activity of SIV gp41, coreceptor usage and viral pathogenicity.