Influence of N-glycosylation on Expression and Function of Pseudorabies Virus Glycoprotein gB

• Published in: Pathogens (Basel) 2021-01, Vol.10 (1), p.61
• Main Authors: Vallbracht, Melina, Klupp, Barbara G, Mettenleiter, Thomas C
• Format: Article
• Language: English
• Subjects: Amino acids; Asparagine; Cell fusion; Cell membranes; Crystal structure; Deactivation; Furin; glycoprotein B; Glycoproteins; Glycosylation; Herpes viruses; herpesvirus; Inactivation; Localization; membrane fusion; Mutants; Mutation; N-glycans; N-linked glycosylation; Occupancy; Pathogens; Plasmids; Polypeptides; Polysaccharides; Protein transport; Proteins; Pseudorabies; pseudorabies virus; Structure-function relationships; virus entry; Viruses
• ISSN: 2076-0817; 2076-0817
• DOI: 10.3390/pathogens10010061

Envelope glycoprotein (g)B is conserved throughout the and mediates fusion of the viral envelope with cellular membranes for infectious entry and spread. Like all viral envelope fusion proteins, gB is modified by asparagine (N)-linked glycosylation. Glycans can contribute to protein function, intracellular transport, trafficking, structure and immune evasion. gB of the alphaherpesvirus pseudorabies virus (PrV) contains six consensus sites for N-linked glycosylation, but their functional relevance is unknown. Here, we investigated the occupancy and functional relevance of N-glycosylation sites in PrV gB. To this end, all predicted N-glycosylation sites were inactivated either singly or in combination by the introduction of conservative mutations (N➔Q). The resulting proteins were tested for expression, fusion activity in cell-cell fusion assays and complementation of a gB-deficient PrV mutant. Our results indicate that all six sites are indeed modified. However, while glycosylation at most sites was dispensable for gB expression and fusogenicity, inactivation of N154 and N700 affected gB processing by furin cleavage and surface localization. Although all single mutants were functional in cell-cell fusion and viral entry, simultaneous inactivation of all six N-glycosylation sites severely impaired fusion activity and viral entry, suggesting a critical role of N-glycans for maintaining gB structure and function.