The Epstein-Barr Virus (EBV) Glycoprotein B Cytoplasmic C-Terminal Tail Domain Regulates the Energy Requirement for EBV-Induced Membrane Fusion

Herpesvirus entry into cells is mediated by the viral fusogen gB, which is thought to refold from the prefusion to the postfusion form in a series of large conformational changes that energetically couple refolding to membrane fusion. In contrast to most viral fusogens, gB requires a conserved heterodimer, gH/gL, as well as other nonconserved proteins. In a further mechanistic twist, gB-mediated cell-cell fusion appears restricted by its intraviral or cytoplasmic domain (cytodomain) because mutations within it result in a hyperfusogenic phenotype. Here, we characterized a panel of hyperfusogenic HSV-1 gB cytodomain mutants and show that they are fully functional in cell-cell fusion at shorter coincubation times and at lower temperatures than those for wild-type (WT) gB, which suggests that these mutations reduce the kinetic energy barrier to fusion. Despite this, the mutants require both gH/gL and gD. We confirm previous observations that the gH cytotail is an essential component of the cell-cell fusion mechanism and show that the N-terminal portion of the gH cytotail is critical for this process. Moreover, the fusion levels achieved by all gB constructs, WT and mutant, were proportionate to the length of the gH cytotail. Putting these results together, we propose that the gH cytotail, in addition to the gH/gL ectodomain, plays an essential role in gB activation, potentially acting as a "wedge" to release the gB cytodomain "clamp" and enable gB activation. IMPORTANCEHerpesviruses infect their hosts for life and cause a substantial disease burden. Herpes simplex viruses cause oral and genital sores as well as rare yet severe encephalitis and a panoply of ocular ailments. Infection initiates when the viral envelope fuses with the host cell membrane in a process orchestrated by the viral fusogen gB, assisted by the viral glycoproteins gH, gL, and gD and a cellular gD receptor. This process is more complicated than that of most other viruses and is subject to multiple regulatory inputs. Antiviral and vaccine development would benefit from a detailed mechanistic knowledge of this process and how it is regulated. Herpesviruses, large, enveloped, double-stranded DNA (dsDNA) viruses, enter cells by the merger of the viral envelope and a host cell membrane, catalyzed by the conserved viral glycoprotein gB. As for other viral fusogens, gB is thought to refold from the prefusion to the postfusion form in a series of large conformational changes that provides the energy necessary to overcome the kinetic barrier associated with membrane fusion (1). However, unlike most viral fusogens, gB does not mediate fusion on its own and requires a conserved heterodimer, gH/gL (2), as well as other nonconserved proteins. For example, herpes simplex virus 1 (HSV-1) and HSV-2, members of the alphaherpesvirus subfamily, require the receptor-binding glycoprotein gD and a cellular gD receptor such as nectin-1 in addition to gB and gH/gL (3). These five proteins also mediate the fusion of transfected cells in the absence of any other ...

Section: Discussionsupporting

confidence: 93%

“…A hyperfusogenic truncation mutant of EBV gB has been shown to achieve the same fusion levels as the WT with less gH/gL (48). We tested whether hyperfusogenic mutants of HSV-1 gB had the same ability by measuring cell-cell fusion in the presence of different amounts of transfected WT gH.…”

Section: Resultsmentioning

confidence: 99%

Interplay between the Herpes Simplex Virus 1 gB Cytodomain and the gH Cytotail during Cell-Cell Fusion

Rogalin

Heldwein

2015

“…However, there is growing evidence that the gB cytoplasmic domain is critical for membrane fusion (22,28,36). The conserved gBcyt lysine cluster was first recognized to have a role in membrane fusion in an in vitro cell-free cell-cell fusion assay study involving HSV-1 gBcyt (28).…”

Section: Discussionmentioning

confidence: 99%

The Glycoprotein B Cytoplasmic Domain Lysine Cluster Is Critical for Varicella-Zoster Virus Cell-Cell Fusion Regulation and Infection

Yang

Arvin

Oliver

2017

The conserved glycoproteins gB and gH-gL are essential for herpesvirus entry and cell-cell fusion induced syncytium formation, a characteristic of varicellazoster virus (VZV) pathology in skin and sensory ganglia. VZV syncytium formation, which has been implicated in the painful condition of postherpetic neuralgia, is regulated by the cytoplasmic domains of gB (gBcyt) via an immunoreceptor tyrosinebased inhibition motif (ITIM) and gH (gHcyt). A lysine cluster (K894, K897, K898, and K900) in the VZV gBcyt was identified by sequence alignment to be conserved among alphaherpesviruses, suggesting a functional role. Alanine and arginine substitutions were used to determine if the positive charge and susceptibility to posttranslational modifications of these lysines contributed to gB/gH-gL cell-cell fusion. Critically, the positive charge of the lysine residues was necessary for fusion regulation, as alanine substitutions induced a 440% increase in fusion compared to that of the wild-type gBcyt while arginine substitutions had wild-type-like fusion levels in an in vitro gB/gH-gL cell fusion assay. Consistent with these results, the alanine substitutions in the viral genome caused exaggerated syncytium formation, reduced VZV titers (Ϫ1.5 log 10 ), and smaller plaques than with the parental Oka (pOka) strain. In contrast, arginine substitutions resulted in syncytia with only 2-fold more nuclei, a Ϫ0.5-log 10 reduction in titers, and pOka-like plaques. VZV mutants with both an ITIM mutation and either alanine or arginine substitutions had reduced titers and small plaques but differed in syncytium morphology. Thus, effective VZV propagation is dependent on cell-cell fusion regulation by the conserved gBcyt lysine cluster, in addition to the gBcyt ITIM and the gHcyt.IMPORTANCE Varicella-zoster virus (VZV) is a ubiquitous pathogen that causes chickenpox and shingles. Individuals afflicted with shingles risk developing the painful condition of postherpetic neuralgia (PHN), which has been difficult to treat because the underlying cause is not well understood. Additional therapies are needed, as the current vaccine is not recommended for immunocompromised individuals and its efficacy decreases with the age of the recipient. VZV is known to induce the formation of multinuclear cells in neuronal tissue, which has been proposed to be a factor contributing to PHN. This study examines the role of a lysine cluster in the cytoplasmic domain of the VZV fusion protein, gB, in the formation of VZV induced multinuclear cells and in virus replication kinetics and spread. The findings further elucidate how VZV self-regulates multinuclear cell formation and may provide insight into the development of new PHN therapies.KEYWORDS cell-cell fusion, glycoproteins, human herpesviruses, varicella-zoster virus Citation Yang E, Arvin AM, Oliver SL. 2017. The glycoprotein B cytoplasmic domain lysine cluster is critical for varicella-zoster virus cellcell fusion regulation and infection. J Virol

“…Cell-cell fusion in transfection-based assays could be significantly enhanced by deleting the most C-terminal amino acids, including the predicted endocytosis motifs (33,(52)(53)(54)(55)(56). Although it was originally speculated that enhanced presence of gB at the plasma membrane increases cell-cell fusion, recent data showed that the degree of surface localization is not necessarily causally related to increased fusion (52).…”

Section: Fig 4 Gbmentioning

confidence: 99%

Mutations in Pseudorabies Virus Glycoproteins gB, gD, and gH Functionally Compensate for the Absence of gL

Schröter

Vallbracht

Altenschmidt

et al. 2016

Entry of herpesviruses depends on the combined action of viral glycoprotein B (gB) and the heterodimeric gH/gL complex, which are activated by binding of the virion to specific cellular receptors. While gB carries signatures of a bona fide fusion protein, efficient membrane fusion requires gH/gL. However, although gB and gH/gL are essential for entry, the alphaherpesvirus pseudorabies virus (PrV) is capable of limited cell-to-cell spread in the absence of gL. To understand gH/gL function in more detail, the limited spread of PrV-⌬gL was used for reversion analyses by serial cell culture passages. In a first experiment, an infectious gL-negative mutant in which gL function was replaced by generation of a gD-gH hybrid protein was isolated (B. G. Klupp and T. C. Mettenleiter, J Virol 73:3014 -3022, 1999). In a second, independent experiment PrV-⌬gLPassB4.1, which also replicated productively without gL, was isolated. Sequence analysis revealed mutations in gH but also in gB and gD. In a transfection-based fusion assay, two amino acid substitutions in the N-terminal part of gH B4.1 (L 70 P and W 103 R) were found to be sufficient to compensate for lack of gL, while mutations present in gB B4.1 enhanced fusogenicity. Coexpression of gB B4.1 with the homologous gH B4.1 resulted in strongly increased syncytium formation, which was further augmented by truncation of the gB B4.1 C-terminal 29 amino acids. Nevertheless, gH was still required for membrane fusion. Surprisingly, coexpression of gD B4.1 blocked syncytium formation in the fusion assays, which could be attributed to a V 106 A substitution within the ectodomain of gD B4.1 . IMPORTANCEIn contrast to many other enveloped viruses, herpesviruses rely on the concerted action of four viral glycoproteins for membrane fusion during infectious entry. Although the highly conserved gB shows signatures of a fusion protein, for fusion induction it requires the gH/gL complex, whose role is still elusive. Here we demonstrated fusion activation by gH in the absence of gL after reversion analysis of gL-deleted pseudorabies virus. This gL-independent fusion activity depended on single amino acid exchanges affecting the gL-binding domain in gH, increasing fusogenicity in gB and allowing negative fusion regulation by gD. Thus, our results provide novel information on the interplay in the fusion machinery of herpesviruses.