Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH

Abstract

Many viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reduced fusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in the genus, resulted in enhanced fusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. Herpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide fusion protein gB depends on the presence of the gH/gL complex for activation. Viral envelope glycoproteins, such as gH, usually contain N-glycans, which can have a strong impact on their folding, transport, and functions. Here, we systematically analyzed the functional relevance of all five predicted N-linked glycosylation sites in the alphaherpesvirus pseudorabies virus (PrV) gH. Despite the fact that mutation of specific sites affected gH transport, fusion activity, and cell-to-cell spread and resulted in delayed penetration kinetics, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. Thus, our results demonstrate a modulatory but nonessential role of N-glycans for gH function.

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

“…We demonstrate that the individual N-glycosylation sites play a modulatory but dispensable role for gB function, whereas simultaneous inactivation of all sites severely affects gB function in fusion, highlighting an important role of these glycans for correct folding, structure and/or stability of gB. Although mutation of most of the sites had no notable effect on gB processing and function during in vitro fusion and entry, which is largely congruent to our results on the role of N-glycans on PrV gH [30], we found that N-glycosylation of PrV gB at the highly conserved positions N154 and N700 is important for proper processing of gB by cellular furin. Taken together, this study expands our knowledge on the role of N-linked glycans on the herpesvirus fusion protein gB.…”

“…Host-cell-derived N-linked glycans on viral envelope proteins can influence protein folding and stability, transport, viral entry and spread, and immune evasion [ 49 ]. Recently, we investigated the functional relevance of N-linked glycosylation in PrV gH [ 30 ]. In the present study, we extended our analyses to N-linked glycans on PrV gB.…”

“…The majority of viral envelope proteins, including herpesvirus gB, gH and gD, are modified by asparagine (N)-linked glycosylation, which can play crucial roles in correct folding, trafficking, function and immune evasion [ 28 , 29 , 30 ]. N-glycosylation starts in the endoplasmic reticulum (ER) with the en bloc attachment of oligosaccharides to the asparagine side chain in N-X-threonine (T) or -serine (S) sequons of the nascent polypeptide chain (X represents any amino acid except proline) [ 31 ].…”

“…Recently, we have investigated the occupancy and functional relevance of N-glycosylation sites in PrV gH, highlighting a modulatory but non-essential role of N-linked glycans for gH function during membrane fusion [ 30 ]. In contrast to gH, there is only limited information on the role of N-linked glycans on gB.…”

Effects of gE/gI deletions on the miRNA expression of PRV-infected PK-15 cells

“…Sows infected with PRV show clinical symptoms of abortion, and infected newborn piglets have severe neurological symptoms, with morbidity and mortality near 100% for those younger than 2 weeks old. PRV mutant strains also cause severe respiratory symptoms in adult pigs, and cause reproductive failure in boar [7][8][9][10][11][12][13].…”