Quantitative similarity of HIV gp160 V513E-dominant reduction of fusion and infection with fusion by the gp41 ectodomain hairpin supports an important fusion role for the final trimer-of-hairpins structure

The V513E mutation of the gp41 subunit of HIV gp160 has been known for thirty years to dominantly reduce HIV fusion and infection, which has been important to estimate the number of trimers needed for efficient infection. This mutation is at the N-terminus of the fusion peptide (Fp) region of gp41 that binds the target membrane. The structural basis for V513E-dominant reduction is not yet well-understood. In this study, mixtures of WT and V513E proteins of a large HIV gp41 ectodomain construct with final trimer-of-hairpins structure exhibit V513E-dominant reduction of vesicle fusion that is quantitatively-similar to that for gp160 fusion and infection. The gp41 ectodomain also exhibits V513E-dominant reduction of hairpin helicity. Global fitting of the gp41 ectodomain and gp160 data supports a requirement of six gp41/two trimers for most efficient fusion and infection. The data support a model in which the ∼25 kcal/mole free energy for close membrane apposition prior to membrane fusion is compensated by the thermostable hairpin in between Fp in target membrane and proximal and transmembrane domains in virus membrane. The Fp's are hypothesized to form an antiparallel β sheet with interleaved strands from two trimers. Relative to WT, sheets with V513E strands are longer because of salt bridges/hydrogen bonds between E2 and charged/polar residues near the Fp C-terminus. The longer V513E sheet results in shorter hairpin with consequent larger inter-membrane distance during initial apposition prior to membrane fusion. This increased distance is the hypothesized reason for reduced fusion and infection. Overall, these data support an important role for the final hairpin structure in membrane fusion.