The molecular basis of differences in the receptor-binding properties of antigenically distinct influenza haemagglutinins.

Schafer, Kathrin Andrea (2006). The molecular basis of differences in the receptor-binding properties of antigenically distinct influenza haemagglutinins. PhD thesis The Open University.



The uptake of influenza viruses into host cells is initiated by binding of their haemagglutinins (HA) to terminal sialic acids on receptors. After infection, the immune system produces anti-HA antibodies that block receptor-binding activity and thereby neutralise virus infectivity. As a consequence, antigenic variation is necessary for a new influenza epidemic to occur. Since many epitopes are close to the receptor-binding site (RBS) of HA, it was the aim of this thesis to investigate whether changes in antigenicity correlated with differences in receptor-binding properties.
A microscale-assay using surface plasmon resonance (SPR) was developed to study the interaction of virus particles with receptor analogues. This technique provides information on kinetic rate constants and affinities for biomolecular interactions. The conditions of the assay were established using bovine fetuin, which contains sialic acid in the a(2,3)- and a(2,6)-linkage to terminal galactose of oligosaccharides. Viruses isolated from different hosts vary in their ability to recognise sialic acid in these linkages. Therefore, the assay was performed using fetuins derivatised to contain sialic acid either in the a(2,3)- or the a(2,6)-linkage, in order to determine the linkage-specificity of influenza viruses.
Various antigenically distinct HI and H3 subtype viruses, chosen on the basis of reactivity with post-infection ferret sera, were tested for their receptor-binding properties. It was shown that antigenic evolution of HA for both subtypes was associated with changes in affinities for receptor analogues. Sequencing of the gene coding for HA revealed that the changes correlated with amino acid substitutions close to or in the RBS. The importance of the variant residues for the observed changes in receptor-binding properties is discussed using the previously solved X-ray structures of HAs complexed with receptor analogues.
Finally, the molecular basis for the evolution of avian H3 HAs to recognise sialic acid in the a(2,6)-linkage characteristic of human HAs was studied by in vitro selection of receptor-binding variant viruses. Although selection of an avian HA with a substitution at residue 226 previously reported by Rogers et al. (1985) was not achieved, a variant with a substitution at residue 201 was identified.

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