Dias de Castro Rodrigues, A.F.; (2007) Structural studies on the H3 influenza A virus haemagglutinin : Receptor binding and membrane fusion. Doctoral thesis , University of London.

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Abstract

In this project, the receptor binding and membrane fusion activities of the membrane-anchored glycoprotein haemagglutinin (HA), H3 subtype, of the influenza A virus were studied. Influenza viruses from different hosts can distinguish between a.2-3 and a2-6 linkages that sialic acid (Sia) forms with the penultimate saccharide residue of the receptor. Human viruses prefer a2-6 linkages and avian viruses ct2-3 linkages. The side chain of residue 226 of the HA receptor binding site (RBS) is involved in the specific recognition of those linkages. The HA of the 1968 Hong Kong (HK) pandemic virus (H3 subtype), contained in the recombinant X-31 virus, has a 226-leucine and prefers binding to a2-6 linkages. The L226Q HA of the variant virus X-31/horse serum (X-31/HS), corresponding to a single-site mutant of the X-31 HA having 226-glutamine, prefers a2-3 linkages. To define the molecular interactions of the L226Q HA with the virus receptor, the crystal structures of L226Q HA in complex with the receptor analogues lactoseries tetrasaccharide a (LSTa), a2-3-terminated, and lactoseries tetrasaccharide c (LSTc), a2-6-terminated, were determined. The structures show the saccharide residues Sia and galactose (Gal) of LSTa and only the Sia of LSTc bound on the L226Q HA RBS, which correlates with the higher affinity of L226Q HA for the ct2-3 linkage. However, the L226Q HA binds both the trans and cis configurations of the Sia-Gal glycosidic bond of LSTa, which has never been observed before. These results are discussed in comparison with data from binding assays and the available crystal structures of the X-31 HA, the H3 avian HA of the influenza virus A/duck/Ukraine/63, a potential precursor of the 1968 HK pandemic virus, and other HAs, of different subtypes and hosts of origin, in complex with LSTa and LSTc. Upon endocytosis, at the low endosomal pH, the HA undergoes an irreversible conformational change associated with the fusion of the viral and endosomal membranes, a process by which the virus enters the target cell. To gain further insights into the membrane fusion mechanism catalysed by the influenza HA, the purification and crystallisation trials of three different X-31 HA forms representative of the neutral pH and fusion pH-induced conformations, containing the membrane-interacting segments fusion peptide and transmembrane anchor, were carried out with the future aim of their crystal structure determination. The procedures for the purification and crystallisation trials of the X-31 HA proteins in different conformations are discussed in the context of the general purification and crystallisation of membrane proteins. No promising hint has yet been obtained in the crystallisation trials.

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