García Moro, Eva; (2023) Structural studies of the influenza haemagglutinin membrane fusion mechanism. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Infection by enveloped viruses requires fusion of viral and cellular membranes to enter cells. Influenza A virus (IAV) host cell entry is mediated by the viral glycoprotein hemagglutinin (HA), which has both receptor-binding and membrane-fusion activities. Activation of membrane fusion is triggered by low pH in late endosomes and results in extensive structural rearrangements in HA that eventually lead to merging of the viral and endosomal membranes and to the release of the virus contents into the host cell cytoplasm. Structures of HA at neutral pH and at the pH of membrane fusion have been characterised by X-ray crystallography and by cryo-EM, but details of how HA transitions between the different states are not completely understood yet. The purpose of this thesis is to further our understanding of the mechanism of HA-mediated membrane fusion from a structural perspective, in particular, by using single- particle cryo-EM and cryo-ET. HA is synthesised as a single-chain precursor (HA0) that has to be proteolytically cleaved into the disulfide-linked polypeptides HA1 and HA2 to become functionally active. The high-resolution structure of the X-31 HA0 at low pH, solved by single-particle cryo-EM, is presented here, which reveals extensive structural changes due to effects of low pH on the uncleaved precursor and a potential new intermediate state in the low-pH transition of HA. A novel helical structure adopted by the fusion peptide is also described, and complete reversion of the low pH-induced changes in the context of the covalently restricted HA0 is established. Additional experiments investigate whether the changes observed for the X-31 HA0 can be extended to other hemagglutinin subtypes. Finally, results on the orientation distribution of HA on the viral membrane studied by cryo-ET are presented, finding that the HA linkage to the envelope is flexible as previously suggested by in vitro studies of full-length HA, and implications of HA flexibility for membrane fusion are discussed.

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