Dynamics and Specificity of Influenza A Virus-Receptor Interactions

Abstract

Influenza A viruses (IAVs) are pathogens of birds and mammals, including humans. They cause a huge economic burden and major public health problems. All IAVs recognize terminal sialic acids (SIAs) as receptors. The hemagglutinin (HA) glycoprotein is responsible for binding to SIA receptors, whereas neuraminidase (NA) cleaves SIA receptors. HA binding, NA cleavage and the balance between the two (HA/NA balance) are of great importance in IAV entry, replication, release and transmission, and thus for host tropism. In this PhD project, we established a kinetic and label-free methodology to study virus-receptor interactions based on biolayer interferometry (BLI). Furthermore, we aimed to analyze the correlation between antigenic drift and virus-receptor binding and to study to what extent the emergence of novel highly pathogenic H5Nx viruses was accompanied with changes in virus receptor binding properties. By using BLI we were able to show that in the absence of NA activity viruses were dynamically, but irreversibly attached to the receptor-coated sensors. Low activity NA protein could contribute to the initial binding rate of virus particles. In the presence of NA activity, virus particles were rolling over the receptor-coated surface until the receptor density was sufficiently decreased to allow virus dissociation. Both antibodies and mucus decoy receptors were shown to interfere with the virus-receptor interactions. Strikingly, virus self-elution and elution driven by mucus or antibodies decreased upon increased virus-receptor binding times indicating virus binding to become much tighter with time. We refer to this process as the maturation of virus binding. Based on these observations, we propose that the HA/NA balance is a crucial determinant of viral fitness by enabling migration through mucus layers and over cell surfaces. Maturation of virus binding is proposed to result from multiple HA protomers in the HA trimer interacting with sialosides, which may be important for virus entry. BLI was also used to analyze to what extent changes in HA antigenicity are accompanied with changes in receptor binding. While antigenically distinct viruses display similar receptor-binding properties, some individual mutations affecting antigenicity increased or decreased binding. Potential compensatory effects on receptor binding were observed for some of the antigenically neutral mutations, suggesting they may function in limiting the changes in receptor binding properties resulting from antigenic changes. Finally, we also studied the evolutionary history of the HA proteins of novel highly pathogenic H5 subtypes that emerged in 2014 and that contained different NA subtypes (H5Nx viruses). We showed that these H5 genes form a monophyletic group that evolved from a clade 2.3.4 H5N1 variant. The H5 proteins were shown to be able to bind fucosylated receptors resulting from –for highly pathogenic H5 unique- substitutions at positions 222 and 227 in the receptor binding site. We hypothesize that the altered receptor-binding specificity of these novel H5 proteins has contributed to their ability to team up with different NA subtypes and their unprecedented emergence and spread.