Cysteines and N-Glycosylation Sites Conserved among All Alphaherpesviruses Regulate Membrane Fusion in Herpes Simplex Virus 1 Infection

Abstract

Neurotropism is a defining characteristic of alphaherpesvirus pathogenicity. Glycoprotein K (gK) is a conserved virion glycoprotein of all alphaherpesviruses that is not found in other herpesvirus subfamilies. The extracellular amino terminus of gK has been shown to be important to the ability of the prototypic alphaherpesvirus herpes simplex virus 1 (HSV-1) to enter neurons via axonal termini. Here, we determined the role of the two conserved N-linked glycosylation (N48 and N58) sites of gK in virus-induced cell fusion and replication. We found that N-linked glycosylation is important to the regulation of HSV-1-induced membrane fusion since mutating N58 to alanine (N58A) caused extensive virus-induced cell fusion. Due to the known contributions of N-linked glycosylation to protein processing and correct disulfide bond formation, we investigated whether the conserved extracellular cysteine residues within the amino terminus of gK contributed to the regulation of HSV-1-induced membrane fusion. We found that mutation of C37 and C114 residues led to a gK-null phenotype characterized by very small plaque formation and drastic reduction in infectious virus production, while mutation of C82 and C243 caused extensive virus-induced cell fusion. Comparison of N-linked glycosylation and cysteine mutant replication kinetics identified disparate effects on infectious virion egress from infected cells. Specifically, cysteine mutations caused defects in the accumulation of infectious virus in both the cellular and supernatant fractions, while glycosylation site mutants did not adversely affect virion egress from infected cells. These results demonstrate a critical role for the N glycosylation sites and cysteines for the structure and function of the amino terminus of gK. We have previously identified important entry and neurotropic determinants in the amino terminus of HSV-1 glycoprotein K (gK). Alphaherpesvirus-mediated membrane fusion is a complex and highly regulated process that is not clearly understood. gK and UL20, which are highly conserved across all alphaherpesviruses, play important roles in the regulation of HSV-1 fusion in the context of infection. A greater understanding of mechanisms governing alphaherpesvirus membrane fusion is expected to inform the rational design of therapeutic and prevention strategies to combat herpesviral infection and pathogenesis. This work adds to the growing reports regarding the importance of gK to alphaherpesvirus pathogenesis and details important structural features of gK that are involved in gK-mediated regulation of virus-induced membrane fusion.

Novel Oncolytic Herpes Simplex Virus 1 VC2 Promotes Long-Lasting, Systemic Anti-melanoma Tumor Immune Responses and Increased Survival in an Immunocompetent B16F10-Derived Mouse Melanoma Model

“…The VC2 virus harbors mutations in two of its viral envelope proteins: glycoprotein K (gK) and UL20 (36). VC2 virus was grown and titrated in Vero cells (ATCC), as described previously (65). and B16F10n-1 cells were engrafted orthotopically in the dermis of the dorsal left dorsal pinna, as previously reported (66).…”

Role of Herpes Simplex Virus Type 1 (HSV-1) Glycoprotein K (gK) Pathogenic CD8+ T Cells in Exacerbation of Eye Disease

“…Studies by the same group showed that both gK and PlLRα (paired immunoglobulin–like type 2 receptor α) bound gB in infected cells and that the association between gB-PILRα protein complex regulates membrane fusion of virus and the host cell which aids in virus penetration (75). Along with the role of amino terminus of gK in virus entry, a recent study described the role of two conserved N-linked glycosylation sites (N48 and N58) of gK in virus-induced cell fusion and replication (76). Mutation at N58 to alanine (N58A) resulted in extensive virus-induced cell fusion.…”

“…Mutation at N58 to alanine (N58A) resulted in extensive virus-induced cell fusion. The same group showed that mutation of cysteine residues within the amino terminus of gK, C37, and C114, led to significant reduction in virus production (76). In addition, gK plays a vital part in the recruitment of other viral glycoproteins into intracellular virus assembly.…”

Targeting STT3A‐oligosaccharyltransferase with NGI‐1 causes herpes simplex virus 1 dysfunction

“…Alternatively, it is feasible that hypoglycosylated particles adhere or fuse with Vero cells, but that a downstream step necessary for viral replication or distribution fails. For example, HSV-1 induced cell fusion and syncytia formation increase with gK hypoglycosylation because of point mutation (13). NGI-1 treatment of HEK293 lines might similarly yield hypoglycosylated HSV-1 particles that infect Vero cells and replicate, but instead of causing cell death, the resultant nascent particles might lead to syncytia that do not result in typical plaques.…”

“…In contrast with gC and gD, an N-glycan on envelope glycoprotein gK is important for HSV-1 pathogenesis because gK must be properly N-glycosylated for cytopathic effects associated with plaque formation. Mutation of gK, to prevent glycosylation of Asn 58 , leads to obscured plaques and greater syncytia formation on Vero cell monolayers (13). In summary, there is a substantial body of evidence supporting the importance of N-glycans of envelope proteins in HSV-1 function, but potential roles may be host cell and context dependent.…”