Abstract
Envelope glycoprotein (g)B is conserved throughout the Herpesviridae 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.
Highlights
Envelope Envelope glycoprotein (g)B is conserved throughout the Herpesviridae and mediates fusion fusion glycoprotein (g)B is conserved throughout the Herpesviridae and mediates of the viralofenvelope with cellular membranes for infectious entry and spread
High-resolution crystal structures of the stable post-fusion state have been determined for gB ectodomains of five different herpesviruses, including pseudorabies virus (PrV) [18,19], HSV-1 [20], and variants showed zoster virus (VZV) [21], all revealing rod-shaped trimers which are composed of five domains (DI-V)
These results indicate that gB positions N264, N444, N519 and N636 are ocand gD to Mutation mediate cell–cell fusion in the absence of other components, cupied by N-glycans
Summary
DNA viruses include important human and animal pathogens. Members of the Alphaherpesvirinae subfamily important human and animal pathogens. High-resolution crystal structures of the stable post-fusion state have been determined for gB ectodomains of five different herpesviruses, including PrV [18,19], HSV-1 [20], and VZV [21], all revealing rod-shaped trimers which are composed of five domains (DI-V). Functional and structural studies on N-linked glycans from several viral fusion proteins, including influenza hemagglutinin [33,34], coronavirus spike protein [35,36,37,38], Ebola virus glycoprotein GP [39], envelope glycoprotein (Env) of human immunodeficiency virus-1 (HIV-1) [40,41], or envelope (E). Occupation of three predicted sites by N-glycans (N264 in DI, N444 in DII, N636 in DIV) was confirmed by crystal structure analysis of the PrV gB post-fusion ectodomain, but their functional relevance is unknown
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