Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin

Abstract

The role of three N-linked glycans which are conserved among various hemagglutinin (HA) subtypes of influenza A viruses was investigated by eliminating the conserved glycosylation (cg) sites at asparagine residues 12 (cgl), 28 (cg2), and 478 (cg3) by site-directed mutagenesis. An additional mutant was constructed by eliminating the cg3 site and introducing a novel site 4 amino acids away, at position 482. Expression of the altered HA proteins in eukaryotic cells by a panel of recombinant vaccinia viruses revealed that rates and efficiency of intracellular transport of HA are dependent upon both the number of conserved N-linked oligosaccharides and their respective positions on the polypeptide backbone. Glycosylation at two of the three sites was sufficient for maintenance of transport of the HA protein. Conserved glycosylation at either the cgl or cg2 site alone also promoted efficient transport of HA. However, the rates of transport of these mutants were significantly reduced compared with the wild-type protein or single-site mutants of HA. The transport of HA proteins lacking all three conserved sites or both amino-terminally located sites was temperature sensitive, implying that a polypeptide folding step had been affected. Analysis of trimer assembly by these mutants indicated that the presence of a single oligosaccharide in the stem domain of the HA molecule plays an important role in preventing aggregation of molecules in the endoplasmic reticulum, possibly by maintaining the hydrophilic properties of this domain. The conformational change observed after loss of all three conserved

Influenza A virus in Taiwan, 1980–2006: Phylogenetic and antigenic characteristics of the hemagglutinin gene

“…Compared to the results from phylogenetic analyses and antigenic subtypes, the data described in this article suggest a strong association between HA genetic variation and antigenic changes in influenza isolates used to link epidemic and vaccine strains. Due to the limited availability of HI testing equipment in clinical virology laboratories in Taiwan, there may be a greater reliance at present on HA sequencing for evaluating and Surface protein glycosylation plays an important role in the biology of many viruses, including the severe acute respiratory syndrome coronavirus (SARS-CoV) ], hepatitis C virus [Goffard et al, 2005], West Nile virus [Hanna et al, 2005], HIV-1 [Bashirova et al, 2001], and influenza viruses [Roberts et al, 1993]. Surface glycosylation may help these viruses enter target cells and evade host immune responses [Fournillier et al, 2001;Abe et al, 2004].…”

“…HIV-1 and influenza A viruses have been described as using glycosylated gp120 and HA proteins to interact with and mediate entry via DC-SIGN and mannose molecules on dendritic cells [Reading et al, 2000;Bashirova et al, 2001;Wang et al, 2008]. However, N-linked glycosylation on the HA proteins of influenza viruses has been reported as serving four important biological functions: (a) adding carbohydrates to antigenic sites, which interferes with neutralizing antibody access [Skehel et al, 1984]; (b) influencing virus pathogenicity when it occurs near cleavage sites [Ohuchi et al, 1989]; (c) sialylating oligosaccharide chains near receptor-binding sites, which interferes with viral binding [Ohuchi et al, 1995]; and (d) promoting the proper folding and maintenance of protein conformation or stability [Roberts et al, 1993]. According to the present results, N-linked glycosylation sites on partial HA1 proteins in both H1N1 and H3N2 have gradually increased over time.…”

The role of single N-glycans in proteolytic processing and cell surface transport of the Lassa virus glycoprotein GP-C

“…N-glycosylation has long been known to be essential for correct folding and intracellular transport of viral glyco-proteins, as shown for instances for influenza hemagglutinin [19]. In order to investigate the importance of the individual N-glycosylation sites of Lassa virus GP-C for intracellular trafficking we analysed cell surface expression of the mutated glycoproteins using a biotinylation approach.…”

“…Fusiogenic glyc-oproteins of most other enveloped viruses are proteolytically cleaved C-terminally of a single arginine residue or at a multibasic recognition motif by the subtilase furin [11]. N-glycans play not only an important role in folding and intracellular transport of viral glycoproteins but also are known to modulate their antigenicity and activity [12][13][14][15][16][17][18][19][20][21]. Glycoproteins of arenaviruses vary considerably in number and position of potential N-glycosylation sites as shown by an N-glycosylation prediction program ( Table 1).…”

N-Linked Glycosylation Attenuates H3N2 Influenza Viruses

“…Both of these regions undergo posttranslational modifications in the Golgi, where glycoconjugates are added to certain sites of N-linked glycosylation. Some of these glycosylation sites, primarily in the stalk region, are indispensable to the proper folding and conformation of the HA molecule (9,33). During the last 4 decades of circulation in humans, N-linked glycosylation in and around the globular head has gradually increased in H3N2 subtype viruses (36,48).…”