pathogens Article Article InfluenceInfluence of N-glycosylation of N-glycosylation on Expression on Expression and andFunction Function of of PseudorabiesPseudorabies Virus Virus Glycoprotein Glycoprotein gB gB Melina Vallbracht,Melina Vallbracht Barbara G., Barbara Klupp and G. Klupp Thomas and C. Thomas Mettenleiter C. Mettenleiter * * Institute ofInstitute Molecular of MolecularVirology and Virology Cell Biology, and Cell Friedrich-Loeffler-Institut, Biology, Friedrich-Loeffler-Institut, 17493 Greifswald-Insel 17493 Greifswald-Insel Riems, Riems, Germany; [email protected]; Melina.Vallbracht@fli.de (M.V.); [email protected] (M.V.); barbara.klupp@fli.de (B.G.K.) (B.G.K.) * Correspondence:* Correspondence: [email protected]; thomas.mettenleiter@fli.de; Tel.: +49-383-517-1250 Tel.: +49-383-517-1250 Abstract: Abstract:Envelope Envelopeglycoprotein glycoprotein (g)B is conserved (g)B is conserved throughout throughout the Herpesviridae the Herpesviridae and mediatesand mediatesfusion fusion of the viralof envelope the viral with envelope cellular with membranes cellular membranes for infectious for infectious entry and entry spread. and Like spread. all viral Like envelope all viral envelope fusion proteins,fusion proteins,gB is modified gB is modifiedby asparagine by asparagine (N)-linked (N)-linked glycosylation. glycosylation. Glycans can Glycans contribute can contribute to to protein function,protein function,intracellular intracellular transport, transport, trafficking, trafficking, structure structure and immune and immune evasion. evasion. gB of the gB ofal- the alpha- phaherpesvirusherpesvirus pseudorabies pseudorabies virus virus(PrV) (PrV)contains contains six consensus six consensus sites for sites N-linked for N-linked glycosylation, glycosylation, but but their functionaltheir functional relevance relevance is unknown. is unknown. Here, Here, we investigated we investigated the occupancy the occupancy and andfunctional functional rel- relevance evance ofof N-glycosylation N-glycosylation sites sites in inPrV PrV gB. gB. To To this this end, end, all all predicted predicted N-glycosylation N-glycosylation sites sites were were in- inactivated activated either singly or in combination by the introduction of conservative mutations (N Q). The resulting resulting proteins were tested for expression,expression, fusionfusion activityactivity inin cell–cellcell–cell fusionfusion assaysassays andand complementationcomple- mentationof of a a gB-deficient gB-deficient PrV PrV mutant. mutant. Our Our results results indicate indicate that that all all six six sites sites are are indeed indeed modified. modified. However, However,while while glycosylation glycosylation at at most most sites sites was was dispensable dispensable for for gB gB expression expression and and fusogenicity, fusogenicity, inactivation inactivationof N154of N154 and and N700 N700 affected affected gB processinggB processing by furinby furin cleavage cleavage and and surface surface localization. localization. Although all Althoughsingle all single mutants mutants were were functional functional in cell–cell in cell– fusioncell fusion and and viral viral entry, entry, simultaneous simultaneous inactivation inacti- of all six vation of N-glycosylationall six N-glycosylation sites severely sites severely impaired impaired fusion fusion activity activity and viral and entry, viral suggestingentry, suggesting a critical role of a critical roleN-glycans of N-glycans for maintaining for maintaining gB structure gB structure and function. and function. Citation: Vallbracht, M.; Klupp, Keywords:Keywords: herpesvirus;herpesvirus; pseudorabies pseudorabies virus; glycoprotein virus; glycoprotein B; membrane B; membrane fusion; N-linked fusion; N-linked glycosyl- glycosyla- Citation: Vallbracht, M.; Klupp, B.G.; B.G.; Mettenleiter, T.C. Influence of Mettenleiter, T.C. Influence of ation; virustion; entry virus entry N-glycosylation on Expression and N-glycosylation on Expression and Function of Pseudorabies Virus Function of Pseudorabies Virus Glycoprotein gB. Pathogens 2021, 10, Glycoprotein gB. Pathogens 2021, 10, x. https://doi.org/10.3390/xxxxx 61. https://doi.org/10.3390/ 1. Introduction1. Introduction pathogens10010061 Received: 14 December 2020 HerpesvirusesHerpesviruses are large-enveloped are large-enveloped double-stranded double-stranded DNA viruses DNA that viruses include that many include many Accepted: 7 January 2021 importantimportant human and human animal and pathogens. animal pathogens. Members Members of the Alphaherpesvirinae of the Alphaherpesvirinae subfamilysubfamily Received: 14 December 2020 Published: 12 January 2021 have thehave broadest the broadest host range, host are range, neurotropic are neurotropic and can and persist can persist in the ininfected the infected hosts hostsfor life for life [1]. Accepted: 7 January 2021 [1]. In additionIn addition to the to thehuman human herpes herpes simplex simplex viruses viruses 1 and 1 and 2 (HSV-1, 2 (HSV-1, -2) -2) and and Varicella Varicella zoster Published: 12 January 2021 Publisher’s Note: MDPI stays zoster virusvirus (VZV), (VZV), this this subfamily containscontains manymany economically economically important important veterinary veterinary pathogens neutral with regard to jurisdictional such as the Varicellovirus Pseudorabies virus (PrV, Suid alphaherpesvirus 1). PrV is the Publisher’s Note: MDPI stayspathogens neu- such as the Varicellovirus Pseudorabies virus (PrV, Suid alphaherpesvirus 1). PrV claims in published maps and causative agent of Aujeszky’s disease in swine and is capable of causing lethal disease in a tral with regard to jurisdictionalis clai-the causative agent of Aujeszky’s disease in swine and is capable of causing lethal dis- institutional affiliations. variety of other mammalian species [2,3]. ms in published maps and institutio-ease in a variety of other mammalian species [2,3]. Infectious entry and spread of herpesviruses require the fusion of the viral envelope nal affiliations. Infectious entry and spread of herpesviruses require the fusion of the viral envelope with thewith host cell the membrane. host cell membrane. Whereas many Whereas enveloped many viruses enveloped encode viruses a single encode but mul- a single but multifunctional glycoprotein (g) for host–cell receptor-binding and membrane fusion [4], Copyright: © 2021 by the authors. tifunctional glycoprotein (g) for host–cell receptor-binding and membrane fusion [4], her- pesvirusesherpesviruses utilize a multiprotein utilize a multiprotein machinery machinery to mediate to these mediate processes. these processes. The so-called The so-called Submitted forCopyright: possible open© 2021 access by the authors. Li- “core-fusion“core-fusion machinery” machinery” which is which conserved is conserved in all herpesviruses in all herpesviruses is formed is by formed gB, the by gB, the publication undercensee the MDPI, terms Basel,and Switzerland. bona fide fusion protein, and the heterodimeric complex of the membrane-bound gH and bona fide fusion protein, and the heterodimeric complex of the membrane-bound gH and conditions ofThis the articleCreative is anCommons open access article anchorless gL (gH/gL) [5]. In addition to the core-fusion machinery, herpesviruses require Attribution (CC BY) license anchorless gL (gH/gL) [5]. In addition to the core-fusion machinery, herpesviruses require distributed under the terms and con- the assistance of nonconserved subgroup-specific receptor-binding proteins such as gD of (http://creativecommons.org/licenses the assistance of nonconserved subgroup-specific receptor-binding proteins such as gD of ditions of the Creative Commons At- the Alphaherpesvirinae for entry [6–8]. /by/4.0/). the Alphaherpesvirinae for entry [6–8]. tribution (CC BY) license (https:// Herpesvirus-mediated membrane fusion is a highly regulated process whose molec- creativecommons.org/licenses/by/ ular details remain incompletely understood. The current model for alphaherpesvirus 4.0/). Pathogens 2021, 10, x. https://doi.org/10.3390/xxxxx www.mdpi.com/journal/pathogens Pathogens 2021, 10, 61. https://doi.org/10.3390/pathogens10010061 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 61 2 of 15 entry proposes that gD, gH/gL and gB drive membrane fusion in a cascade-like, pH- independent fashion [9–11]. Binding of gD to an appropriate host cell receptor, such as nectin-1 or herpesvirus entry mediator (HVEM), serves as the initial fusion trigger [12,13]. A conformational change in receptor-bound gD is proposed to allow its crosstalk with gH/gL, possibly by direct interaction between their ectodomains [14]. The gH/gL com- plex is believed to function as a fusion regulator and, upon triggering, is thought to activate gB, the sole fusion executor [5,9,11,15]. Although the exact molecular mechanism of gB fusion activation by gH/gL is unclear, direct interactions between the respective ectodomains and/or cytoplasmic domains have been reported to be essential for fusion activation [16,17]. Activated gB is proposed to facilitate fusion by refolding from a trimeric metastable high-energy pre-fusion conformation to a stable post-fusion state. During the fusogenic conformational change, gB exposes two internal fusion loops that interact with the target membrane [18]. The subsequent fold-back process into the energetically more favorable post-fusion conformation juxtaposes the viral and cellular membranes, ultimately leading to their fusion [5]. gB is the most highly conserved