Role of Individual Oligosaccharide Chains in Antigenic Properties, Intracellular Transport, and Biological Activities of Influenza C Virus Hemagglutinin-Esterase Protein

Abstract

The hemagglutinin-esterase (HE) glycoprotein of influenza C virus is composed of three domains: a stem domain active in membrane fusion (F), an acetylesterase domain (E), and a receptor-binding domain (R). The protein contains eight N-linked glycosylation sites, four (positions 26, 395, 552, and 603) in the F domain, three (positions 61, 131, and 144) in the E domain, and one (position 189) in the R domain. Here, we investigated the role of the individual oligosaccharide chains in antigenic properties, intracellular transport, and biological activities of the HE protein by eliminating each of the glycosylation sites by site-specific mutagenesis. Comparison of electrophoretic mobility between the wild-type and the mutant proteins showed that while seven of the glycosylation sites are used, one (position 131) is not. Analysis of reactivity of the mutants with anti-HE monoclonal antibodies demonstrated that glycosylation at position 144 is essential for the formation of conformation-dependent epitopes. It was also evident that glycosylation at the two sites in the F domain (positions 26 and 603), in addition to that in the E domain (position 144), is required for the HE molecule to be transported from the endoplasmic reticulum and that mutant HEs lacking one of these three sites failed to undergo the trimer assembly. Removal of an oligosaccharide chain at position 144 or 189 resulted in a decrease in the esterase activity. By contrast, two mutants lacking an oligosaccharide chain at position 26 or 603, which were defective not only in cell surface expression but in trimerization, possessed full-enzyme activity, suggesting that the HE monomers present within the cell have acetylesterase activity. Fusion activity of cells expressing each of mutant HEs was found to be comparable with the ability of the protein to be transported to the cell surface, suggesting that there is no specific oligosaccharide chain that plays a critical role in promoting membrane fusion.

Bioinformatic Analysis of the Genome of Infectious Salmon Anemia Virus Associated with Outbreaks with High Mortality in Chile

“…It has been shown that in other myxoviruses, N glycosylation of the F domain in Sendai and influenza C viruses is important for protein fusion activity. The absence of N glycosylation decreases the transport from the endoplasmic reticulum to the cytoplasmic membrane and alters protein trimerization (70,71,74). A glycosylated form of fusion protein has been reported (50 kDa) (4,19) with an approximate mass difference of 2 kDa compared to its immature isoform.…”

Intrinsic Temperature Sensitivity of Influenza C Virus Hemagglutinin-Esterase-Fusion Protein

“…To determine the mechanism by which temperature influences events involving HEF cell surface expression, we examined HEF oligomerization by using sucrose velocity sedimentation, as previously described (16). HEF expression plasmid-transfected COS-1 cells were cultured at 37°C for 24 h, followed by either further incubation at 37°C or a temperature shift to 33°C.…”

Molecular characterization of the pseudorabies virus UL2 gene

“…N-linked glycosylation is an important post-translation modification that profoundly affects protein folding, oligomerization, and stability (Mitra et al, 2006). In addition, it is involved in trafficking modifications, ligand interactions (Meng et al, 2008), and enzyme activity (Hausmann et al, 1997;Sugahara et al, 2001). Because the potential N-linked glycosylation of UL2 occurs near the predicted beta-strand and within a potential strongly hydrophobic region, it might be associated with UDG activity.…”