Surface Glycoproteins

There are currently 18 subtypes of HA and 11 subtypes of NA have been identified, all of which have been found in the natural host of these viruses, water fowl, apart from the recently discovered H17N10 and H18N11 which were isolated from bats (Fouchier et al. 2005; Tong et al. 2012; Tong et al. 2013). The zoonotic potential of influenza viruses is dependent on the ability of the surface glycoproteins to allow for cell entry and egress following successful replication.

HA

The first step during viral infection is viral entry, which requires attachment of the virus to the host cell, a process mediated by the viral HA protein. HA is the most

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abundant surface glycoprotein and is a type I integral membrane protein which undergoes a number of post-translational modifications such as glycosylation and acetylation of the cytoplasmic tail (de Graaf and Fouchier 2014). HA is initially translated and incorporated into virus particles as a single polypeptide, HA0, which is then cleaved extracellularly after virus release by host proteases to form two subunits HA1, which contains the receptor-binding domain of HA, and HA2, which contains the fusion subdomain. For attachment to the host cell HA targets sialylated glycan receptors on the cell surface and in 1983 it was shown that human and animal influenza viruses exhibited different preferences in the receptor specificity of HA (Rogers and Paulson 1983). Avian influenza viruses have a binding preference for sialic acid bound to galactose via an alpha 2,3 linkage, whereas human influenza viruses have a preference for alpha 2,6 linked sialic acid. However, viruses can easily evolve to change their receptor-binding specificity as it was recently shown that as few as 4 amino acid changes in an avian H5N1 HA was capable of switch specificity from alpha 2,3 to alpha 2,6, thereby allowing for successful airborne transmission of an avian virus among ferrets (Herfst et al. 2012).

The second major function of HA is low pH triggered fusion, which is required for the release of the viral genome. The low pH found in the endosome leads to a significant structural change in HA with the most prominent change in the position of the fusion peptide. Due to the hydrophobic characteristics of the fusion peptide the conformational change allows this peptide to bury itself in the endosomal membrane, thereby bringing the viral and endosomal membranes into close enough proximity for membrane fusion to occur. The presence of more than one HA trimer stimulates the production of a fusion pore, thereby connecting the viral interior with the host cell

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cytoplasm and allowing the vRNP to exit the virus particle and enter the cytoplasm (Palese and Shaw 2007).

HA is also recognized by the adaptive immune system, which leads to the production of neutralizing antibodies raised against the numerous antigenic sites found on the globular head region of the HA1 domain. This helps to drive the evolution of the virus as due to the infidelity of the polymerase, a viral quasi-species is produced, with many of the mutant viruses containing mutations within the antigenic sites of HA1. Changes that allow the virus to go unnoticed by the immune system are termed ‘escape mutants’ which over time become fixed changes defining the antigenic drift of the virus (Smith et al. 2004).

NA

The NA protein is the second most abundant surface glycoprotein on influenza A virions and is a type II integral membrane protein which forms tetramers on the surface of the virion and infected cells (Eichelberger and Wan 2015). The native tetramer is required for enzyme activity and each monomer has a distinctive 6-bladed beta propeller (Russell et al. 2006). There is less NA than HA on the virion surface, however this is virus dependent. For example, in most viruses the ratio is ~ 5:1 HA to NA but the 2009 H1N1 pandemic virus had a ratio nearer to 2:1 (Getie-Kebtie et al. 2013).

NA has a number of essential roles throughout the virus life cycle. )E!"*!-!*"-."5-*4! ZN"<N! <.4-#4*! +.J<=*"5"<! .",P-+4! Q41Z44,! 1N4! *"-."<! X)M-<41J.,43A-2","<Y! -<"5! -,5! -,! -5[-<4,1! +-.-<1=*4! A4*"534! X0N1JAJ-\! K=<N-.=#-\! -,5! S=#",! &IIRY% NA

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enables incoming virions to access respiratory tract epithelial cells by destroying decoy receptors present on mucins (Matrosovich et al. 2004). However, it is unlikely that this receptor-destroying activity prevents HA from binding and infecting epithelial cells due to the increased ratio of HA:NA and because also NA appears to be segregated from HA on the virus surface (Calder et al. 2010). NA is essential for the release of nascent virus particles as shown by experiments in which reduced enzymatic activity of NA led to a reduction in viral plaque size due to inefficient cell- to-cell spread (Kilbourne et al. 1968). NA also removes sialic acids from its own glycoproteins, which is thought to prevent aggregation of nascent virions at the cell surface and may explain the relationship between NA activity and transmissibility as free, non-aggregated virions are more likely to have improved transmission in aerosol droplets that are inhaled into the lower respiratory tract (Lakdawala et al. 2011; Yen et al. 2011).

1.3.3.4 Matrix Proteins