Influenza A surface glycosylation and vaccine design

Abstract

We have shown that glycosylation of influenza A virus (IAV) hemagglutinin (HA), especially at position N-27, is crucial for HA folding and virus survival. However, it is not known whether the glycosylation of HA and the other two major IAV surface glycoproteins, neuraminidase (NA) and M2 ion channel, is essential for the replication of IAV. Here, we show that glycosylation of HA at N-142 modulates virus infectivity and host immune response. Glycosylation of NA in the stalk region affects its structure, activity, and specificity, thereby modulating virus release and virulence, and glycosylation at the catalytic domain affects its thermostability; however, glycosylation of M2 had no effect on its function. In addition, using IAV without the stalk and catalytic domains of NA as a live attenuated vaccine was shown to confer a strong IAVspecific CD8+ T-cell response and a strong cross-strain as well as cross-subtype protection against various virus strains.influenza A virus | glycosylation | vaccine design I nfluenza A viruses (IAV) belong to the Orthomyxoviridae family and can circulate widely and cross interspecies barriers through the highly antigenic drift and shift of the 18 subtypes of HA and 11 subtypes of neuraminidase (NA) (1, 2). In addition, the posttranslational modification of the IAV surface proteins is important to circumvent host defense and support the virus life cycle (3).HA is a major surface glycoprotein of IAV and is involved in viral infection via binding to sialic acid (SA)-containing glycans on the surface of host cell (3). The other major glycoprotein of IAV, NA, is involved in the cleavage of SA on the host cell receptor to facilitate the release of viral particles to infect other cells (4). M2, the third surface protein of IAV, has ion channel activity to regulate virus penetration and uncoating (1). All surface proteins interact with M1 protein for virus assembly and release (5).The modification of HA and NA by N-glycosylation is important in the IAV life cycle (1, 6-8). Previously, we have shown that the glycosylation of HA affects its receptor binding, immune response, and structural stability, and glycosite 27 is essential for retaining the structural integrity of HA and its receptor binding (6, 9). In addition, using the monoglycosylated HA as immunogen, it showed a broader protection against various IAV subtypes compared with the fully glycosylated version (10). However, it is not clear whether the other specific glycosites and their glycan structures on HA regulate its functions. With regard to NA, the functional roles of its glycosylation are not well understood, though N-glycosylation was reported to stabilize the protein from protease digestion and may affect the enzyme activity (11,12). The aim of this study was to understand the functional effects of glycosylation on HA, NA, and M2, and how glycosylation of the surface proteins affects the life cycle of IAV. ResultsGlycosylation of HA at N-142. Because several glycosylation sites (glycosites 27, 40, 176, 303, and 497) on HA are ...

Global aspects of viral glycosylation

“…Prominent examples include the Ebola virus glycoprotein modified by a very high content of O-linked glycans, and the HIV-1 glycoprotein gp160 that is glycosylated by the addition of multiple N-linked glycans (Jeffers et al 2002;Pabst et al 2012;Yang W et al 2014). Although specific functional roles have been assigned to distinct glycosites of multiple viruses (Goffard et al 2005;Falkowska et al 2007;Helle et al 2010;Wang et al 2013Wang et al , 2017Lennemann et al 2014;Bradel-Tretheway et al 2015;Luo et al 2015;Orlova et al 2015;Suenaga et al 2015;Stone et al 2016;Wu et al 2017), it is generally believed that the high levels of glycosylation serve as a protective shield from the host's immune system (Francica et al 2010;Helle et al 2011;Sommerstein et al 2015;Behrens et al 2016;Gram et al 2016;Walls et al 2016). The other major role of glycosylation occurs during virus entry where glycans on the host cell represent viral receptors interacting with carbohydrate binding proteins on the viral surface.…”

Glycomic analysis of host-response reveals high mannose as a key mediator of influenza severity

“…N -glycosylation of HA is important in receptor binding, immune response, and viral stability (33). Glycosylation of NA is essential for the enzyme activity (33). M2, a small transmembrane protein of about 11 KD, is at 10-100 fold lower abundance than HA on the virus surface and has a putative N -glycosylation site at which glycosylation is not commonly observed (33–36).…”

“…Glycosylation of NA is essential for the enzyme activity (33). M2, a small transmembrane protein of about 11 KD, is at 10-100 fold lower abundance than HA on the virus surface and has a putative N -glycosylation site at which glycosylation is not commonly observed (33–36). To determine whether the increase in high mannose was due to influenza virus glycoproteins or to changes in glycosylation of host proteins, we performed lectin blot analysis in tandem with Western blot analysis of HA and NA, the major influenza virus glycoproteins ( Fig.…”

Differential Disease Severity and Whole Genome Sequence Analysis for Human Influenza A/H1N1pdm Virus in 2015-2016 Influenza Season

“…Glycosylation on HA proteins has been proposed to prevent antibody recognition and viruses often accumulate secondary mutations at antigenic or receptor binding sites to adapt to the presence of new glycans [20]. Glycosylation on the NA stalk has been shown to be important for NA activity and virulence [21] and glycosylation near the NA enzyme sites (NA-245) alters NA enzymatic activity while also preventing the binding of antibodies (Powell et al, BioRxiv). There are 8 potential N-linked glycosylation sites on the HA of A/California/7/2009.…”