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Understanding the effects of influenza hemagglutinin mutations on viral membrane fusion

Final Report Summary - INFLUENZA FUSION (Understanding the effects of influenza hemagglutinin mutations on viral membrane fusion)

Every enveloped virus fuses its membrane with a host cell membrane, thereby releasing its genome into the cytoplasm and initiating the viral replication cycle. The merger of the two membranes does not occur spontaneously, but require the action of specific virus fusion proteins or fusion-protein complexes. The purpose of this work is to explain how different conditions such as membrane composition and pH relate to observed but largely unexplained differences in fusogenic phenotypes. My methods range from bioinformatics and modeling to steered molecular dynamics.
During the project three aims were set up that have been addressed. The first aim was to define a structural model for how influenza hemagglutinin achieves viral fusion. Using a combination of literature studies, as well as building on earlier results from the University of Virginia, the hypothesis we worked out for this implicates lipid tail protrusion to be important. Lipid tail protrusion is an uncommon but not rare event when the tails of the lipid molecules point out away from the interior of the lipid bilayer. This hypothesis was investigated in aim 2, where we studied the ability of certain mutants to influence tail protrusion. It was found that influenza mutants that have been shown to be defective in their ability to cause membrane fusion in vivo, also exhibits a weaker ability to induce lipid tail protrusion.
In addition, as was stressed in Annex 1, the work specified allowed for ample opportunity for the researcher to become involved in other collaborations, and strengthen and broaden the horizons. To this end, we also used the theoretical methods described in Annex 1 to study the structure and dynamics of an outer membrane protein (Opa60) of the Neisseria bacterial species. This work highlighted a number of interesting features that would have been left unnoticed had we not used theory and experiment together.
Further, we also expanded our hypothesis to include other enveloped viruses, e.g. the Ebola virus. This was done in collaboration with experimental researchers at the University of Virginia, and was not included in Annex 1. The result of this was yet again a publication (Ebolavirus entry requires a compact hydrophobic fist at the tip of the fusion loop) submitted to PNAS.
We have also expanded our work to include studies of the insertion mechanisms of other enveloped viruses similar to influenza, here primarily dengue virus and tick-borne encephalitis. These two viruses are interesting because they have very distinct requirements on membrane composition to be able to achieve fusion, and thus allows for a very nice test-bed to investigate in detail the influence of membrane composition on membrane fusion. The simulations where evaluated for the ability of the peptides to insert into the bilayer, and subsequent events upon membrane association. As hypothesized, dengue fusion peptides show a greater amount of insertion into bilayers consisting of a mix of POPC and POPG or POPS lipids, as opposed to pure POPC bilayers. This work also resulted in a methods-oriented research paper concerning practical issues when doing simulations with different and varying lipid compositions.