Abstract
Fusion of the human immunodeficiency virus (HIV) with target cells is mediated by the gp41 subunit of the envelope protein. Mutation and deletion studies within the transmembrane domain (TMD) of intact gp41 influenced its fusion activity. In addition, current models suggest that the TMD is in proximity with the fusion peptide (FP) at the late fusion stages, but there are no direct experimental data to support this hypothesis. Here, we investigated the TMD focusing on two regions: the N-terminal containing the GxxxG motif and the C-terminal containing the GLRI motif, which is conserved among the TMDs of HIV and the T-cell receptor. Studies utilizing the ToxR expression system combined with synthetic peptides and their fluorescent analogues derived from TMD revealed that the GxxxG motif is important for TMD self-association, whereas the C-terminal region is for its heteroassociation with FP. Functionally, all three TMD peptides induced lipid mixing that was enhanced significantly upon mixing with FP. Furthermore, the TMD peptides inhibited virus-cell fusion apparently through their interaction with their endogenous counterparts. Notably, the R2E mutant (in the GLRI) was significantly less potent than the two others. Overall, our findings provide experimental evidence that HIV-1 TMD contributes to membrane assembly and function of the HIV-1 envelope. Owing to similarities between functional domains within viruses, these findings suggest that the TMDs and FPs may contribute similarly in other viruses as well.