Structure and function of influenza virus membrane fusion protein

The entry of influenza virus into a host cell is mediated by the viral protein hemagglutinin (HA), which forms an initial complex that contains three HA1 subunits in association with a bundle containing the ectodomains of three HA2 subunits. Each ectodomain has a N-terminal 250303-residue fusion peptide (FP) and 1603030-residue soluble ectodomain (SE). HA2 also has a 250303-residue transmembrane domain (TM) and a 100303-residue endodomain. Entry begins with HA1 binding to target cell sialic acids, followed by endocytosis of the virus and endosome maturation with pH reduction to < 6. The HA1 subunits move away from the HA2 bundle, which then undergoes a large structural rearrangement to a final hyperthermostable state with a trimer-of-SE hairpins. The pH reduction is also correlated with HA2-mediated joining/fusion of the virus and endosome membranes, and consequent deposition of the viral nucleocapsid in the cytoplasm. The FP and TM are the only HA2 segments that are deeply inserted in the fused membrane. Residues 38-105 from the SE hairpins form an interior parallel helical bundle, while 154-176 are strands within the grooves of the bundle exterior. The FP forms a separate hairpin structure with antiparallel and close packing of two FP helices. My research focuses on better understanding the HA2 structure and fusion mechanism, which may aid the development of antiviral drugs and vaccine. One main project in this dissertation compares WT-HA2 with G1E(FP) and I173E(SE strand) mutants. WT-HA2 induces vesicle fusion at pH 5.0, whereas fusion is greatly reduced for both mutants. Circular dichroism for HA2 and FHA2 2261 FP + SE constructs show dramatic losses in stability for the mutants, including a melting temperature reduced by 40 °C for I173E-FHA2. This evidences destabilization of SE hairpins via dissociation of strands from the helical bundle, which is also supported by larger monomer fractions for mutant vs. 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. The present 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. Lower fusion by the mutants is due to loss of hairpin stability and consequent reduced membrane apposition of the virus and target membranes. Another project describes the hydrogen-deuterium exchange-mass spectrometry (HDX-MS) on HA2. Fusion models often depict a FP/TM complex in the final HA2 state with membrane traversal by both domains, and a role for this complex in membrane pore expansion. The HDX-MS data are inconsistent with this complex, and show respective high FP and low TM aqueous exposures. The data support independent FP and TM with respective membrane interfacial and traversal locations. The data also support low aqueous exposure of the 22-38 segment, consistent with a structured and protected region and with retention of the 23-35 antiparallel sheet observed in the HA2/HA1 complex. The sheet is a semi-rigid connector between FP and SE that helps maintain close membrane apposition during fusion. The 22-69 and 150-191 regions exhibit much greater HDX for the I173E mutant vs. WT, which correlates with dissociation of C-terminal strands from interior N-helices of the trimer-of-hairpins. Similar trends are observed for G1E mutant vs. WT, as well as smaller HDX for G1E FP, which correlates with unfolding of the G1E FP helical hairpin and FP/SE binding. Both mutants exhibit highly-impaired fusion that may be due to increased distance between the initial apposed membranes and for G1E, FP binding to SE rather than target membrane.