Tuning of influenza A virus neuraminidase activity

Abstract

Influenza A viruses (IAVs) are zoonotic pathogens that constantly circulate in a wide variety of species, including birds, pigs and humans. In humans, IAVs cause seasonal epidemics and occasional influenza pandemics. Annual epidemics caused by seasonal IAVs usually lead to millions of human infections, posing great threats to public health and cause large economic burdens. Influenza pandemic occurs when animal viruses managed to cross the host species barrier and became transmissible among humans. IAV pandemics have occurred four times in the 100 years, causing millions of deaths and global devastating effects. IAVs are enveloped, segmented negative-strand RNA viruses belonging to the Orthomyxoviridae family. There are two main important glycoproteins in the IAV virus membrane, hemagglutinin (HA) and neuraminidase (NA), both of which recognize sialic acids (SIAs). The HA protein is responsible for virus-cell attachment via binding to sialylated receptors at the cell surface. The NA protein is the receptor-destroying enzyme and responsible for removing SIA from host glycoproteins as well as glycolipids, thereby allowing release of progeny virions from cells and decoy receptors and preventing virus self-aggregation. A functional balance between the HA and NA proteins is of importance for maintaining optimal virus replication as well as transmission across different host species<!--[if supportFields]>ADDIN RW.CITE{{36 Xu,R. 2012; 37 Yen,H.L. 2011; 38 de Wit,E. 2010}}. While HA receptor-binding avidity and specificity have been studied in detail, much less is known about the molecular determinants that mediate (changes in) the specificity and activity of IAV NA proteins. The overall aim of this thesis was to unravel to what extent and how IAVs modulate the activity of their NA proteins during virus evolution. To this end, NA functionality, mainly enzymatic activity has been extensively investigated by using an optimized recombinant soluble protein approach. By doing so, we not only identified an optimal recombinant soluble NA expression approach, but also identified residues that affected NA folding and/or enzymatic activity. With the established recombinant soluble approach, we further found an important role of the 2nd SIA-binding site in NA enzymatic activity. Mutation of the 2nd SIA binding site provides viruses with an additional mechanism to manipulate the enzymatic activity of their NA proteins without having to mutate their active site residues directly. The studies presented in this thesis also highlight some complexities of HA/NA balance during virus evolution. Different NA phenotypic properties of H1N1pdm09 virus were found to be intertwined, with several NA substitutions affecting more than one phenotypic characteristic. The phenotypic changes of NA are probably also linked to the properties of the HA protein and corresponding HA/NA balance, which makes evolution of HA and NA more difficult to understand.