Functional role of N-linked glycosylation in pseudorabies virus glycoprotein gH

Vallbracht, Melina GND; Rehwaldt, Sascha; Klupp, Barbara GND; Mettenleiter, Thomas C. GND; Fuchs, Walter GND

Many viral envelope proteins are modified by asparagine(N)-linked glycosylation, which can influence their structure, physio-chemical 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 in vitro fusion activity, delayed penetration kinetics and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells resulting in ER retention and reduced surface expression. In contrast, mutation of N604, conserved in the varicellovirus genus, resulted in enhanced in vitro 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.

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Vallbracht, Melina / Rehwaldt, Sascha / Klupp, Barbara / et al: Functional role of N-linked glycosylation in pseudorabies virus glycoprotein gH. 2018.

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