Abstract
Cellular entry of influenza virus is mediated by the viral protein hemagglutinin (HA), which forms an initial complex of three HA1 and three HA2 subunits. Each HA2 includes a fusion peptide (FP), a soluble ectodomain (SE), and a transmembrane domain. HA1 binds to cellular sialic acids, followed by virus endocytosis, pH reduction, dissociation of HA1, and structural rearrangement of HA2 into a final trimer-of-SE hairpins. A decrease in pH also triggers HA2-mediated virus/endosome membrane fusion. SE hairpins have an interior parallel helical bundle and C-terminal strands in the grooves of the exterior of the bundle. FPs are separate helical hairpins. This study compares wild-type HA2 (WT-HA2) with G1E(FP) and I173E(SE strand) mutants. WT-HA2 induces vesicle fusion at pH 5.0, whereas the extent of fusion is greatly reduced for both mutants. Circular dichroism for HA2 and FHA2≡FP+SE constructs shows dramatic losses of stability for the mutants, including a T(m) reduced by 40 °C for I173E-FHA2. This is evidence of destabilization of SE hairpins via dissociation of strands from the helical bundle, which is also supported by larger monomer fractions for mutant versus WT proteins. The G1E mutant may have disrupted FP hairpins, with consequent non-native FP binding to dissociated SE strands. It is commonly proposed that free energy released by the HA2 structural rearrangement catalyzes HA-mediated fusion. This study supports an alternate mechanistic model in which fusion is preceded by FP insertion in the target membrane and formation of the final SE hairpin. Less fusion by the mutants is due to the loss of hairpin stability and consequent reduced level of membrane apposition of the virus and target membranes.