Cross Talk among the Glycoproteins Involved in Herpes Simplex Virus Entry and Fusion: the Interaction between gB and gH/gL Does Not Necessarily Require gD

Avitabile, Elisa; Forghieri, Cristina; Campadelli-Fiume, Gabriella

doi:10.1128/jvi.01287-09

Cited by 58 publications

(52 citation statements)

References 43 publications

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“…Some confounding data. A recent study, also employing BiMC, reported that gB and gH/gL can interact in the absence of gD (3). One of the problems with that study was the overexpression of the glycoproteins, and a second was the use of a mutant form of gB designed to send the protein to an abnormal cell compartment.…”

Section: Discussionmentioning

confidence: 99%

Bimolecular Complementation Defines Functional Regions of Herpes Simplex Virus gB That Are Involved with gH/gL as a Necessary Step Leading to Cell Fusion

Atanasiu

Whitbeck

Leon

et al. 2010

J Virol

117

153

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Herpes simplex virus (HSV) entry into cells requires four membrane glycoproteins: gD is the receptor binding protein, and gB and gH/gL constitute the core fusion machinery. Crystal structures of gD and its receptors have provided a basis for understanding the initial triggering steps, but how the core fusion proteins function remains unknown. The gB crystal structure shows that it is a class III fusion protein, yet unlike other class members, gB itself does not cause fusion. Bimolecular complementation (BiMC) studies have shown that gD-receptor binding triggers an interaction between gB and gH/gL and concurrently triggers fusion. Left unanswered was whether BiMC led to fusion or was a by-product of it. We used gB monoclonal antibodies (MAbs) to block different aspects of these events. Non-virus-neutralizing MAbs to gB failed to block BiMC or fusion. In contrast, gB MAbs that neutralize virus blocked fusion. These MAbs map to three functional regions (FR) of gB. MAbs to FR1, which contains the fusion loops, and FR2 blocked both BiMC and fusion. In contrast, MAbs to FR3, a region involved in receptor binding, blocked fusion but not BiMC. Thus, FR3 MAbs separate the BiMC interaction from fusion, suggesting that BiMC occurs prior to fusion. When substituted for wild-type (wt) gB, fusion loop mutants blocked fusion and BiMC, suggesting that loop insertion precedes BiMC. Thus, we postulate that each of the gB FRs are involved in different aspects of the path leading to fusion. Upon triggering by gD, gB fusion loops are inserted into target lipid membranes. gB then interacts with gH/gL, and this interaction is eventually followed by fusion.

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Section: Discussionmentioning

confidence: 99%

Bimolecular Complementation Defines Functional Regions of Herpes Simplex Virus gB That Are Involved with gH/gL as a Necessary Step Leading to Cell Fusion

Atanasiu

Whitbeck

Leon

et al. 2010

J Virol

117

153

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“…These data suggest that, at least in HSV-1, gH/gL facilitates the activation and not the repression of gB. Complexes of gB:gH/gL have been detected from both HSV and CMV (13,14). CMV lacks gD and requires the minimal complex of gB:gH/gL for entry into fibroblasts.…”

mentioning

confidence: 92%

Mechanism for neutralizing activity by the anti-CMV gH/gL monoclonal antibody MSL-109

Fouts¹,

Comps-Agrar²,

Stengel

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Cytomegalovirus (CMV) is a widespread opportunistic pathogen that causes birth defects when transmitted transplacentally and severe systemic illness in immunocompromised individuals. MSL-109, a human monoclonal IgG isolated from a CMV seropositive individual, binds to the essential CMV entry glycoprotein H (gH) and prevents infection of cells. Here, we suggest a mechanism for neutralization activity by MSL-109. We define a genetic basis for resistance to MSL-109 and have generated a structural model of gH that reveals the epitope of this neutralizing antibody. Using surface-based, time-resolved FRET, we demonstrate that gH/gL interacts with glycoprotein B (gB). Additionally, we detect homodimers of soluble gH/gL heterodimers and confirm this novel oligomeric assembly on full-length gH/gL expressed on the cell surface. We show that MSL-109 perturbs the dimerization of gH/gL:gH/gL, suggesting that dimerization of gH/gL may be required for infectivity. gH/gL homodimerization may be conserved between alpha-and betaherpesviruses, because both CMV and HSV gH/gL demonstrate self-association in the FRET system. This study provides evidence for a novel mechanism of action for MSL-109 and reveals a previously undescribed aspect of viral entry that may be susceptible to therapeutic intervention.H uman CMV is a β-group herpesvirus that causes severe complications in immunocompromised individuals. CMV infects between 60% and 80% of the adult population worldwide (1). As with other herpesviruses, CMV establishes a lifelong latency in the host but is largely asymptomatic among infected immunocompetent individuals (2). However, during severe immunosuppression (e.g., in the setting of hematopoietic stem cell transplantation and solid organ transplantation, or advanced HIV/AIDS), CMV reactivation or primary infection can result in life-threatening disease. In addition, the acquisition of primary CMV infection during pregnancy, although of little consequence to the mother, can have severe clinical consequences in the developing fetus (3, 4). The current therapy for CMV disease is treatment with either ganciclovir or valganciclovir, which are associated with significant toxicity and not approved for use in pregnant women or for congenitally damaged infants (5). CMV hyperimmunoglobulin (CMV-HIG; pooled human IgG from CMV-positive individuals) has demonstrated efficacy in certain solid organ transplant recipients and more recently found to show limited success in protecting infants from congenital CMV disease (1, 6, 7). These findings suggest that more potent or differently targeted antibody therapy may prove to be an effective and safe alternative to the current forms of CMV therapy.Like other herpesviruses, CMV uses multiprotein entry complexes to initiate infection of host cells. Three glycoproteins, gB, gH, and gL, known as the "core fusion machinery," are conserved in all herpesviruses and are required for entry (8,9). gB, the most conserved of these glycoproteins, exists as a homotrimer and catalyzes membrane fusion during vira...

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“…Several lines of research suggest that the subsequent step in fusion is an interaction between gH-gL and gB, with the latter glycoprotein being required for a committed and expanding fusion pore (1-3, 16, 28, 41). However, it is still unclear whether the gB and gH-gL interaction requires that gD first bind a receptor (1,3), indicating that another viable model of HSV entry may be nonsequential gD-gB-gH-gL complex formation.…”

mentioning

confidence: 99%

Insertion Mutations in Herpes Simplex Virus 1 Glycoprotein H Reduce Cell Surface Expression, Slow the Rate of Cell Fusion, or Abrogate Functions in Cell Fusion and Viral Entry

Jackson

Lin

Spear

et al. 2010

J Virol

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Of the four required herpes simplex virus (HSV) entry glycoproteins, the precise role of gH-gL in fusion remains the most elusive. The heterodimer gH-gL has been proposed to mediate hemifusion after the interaction of another required glycoprotein, gD, with a receptor. To identify functional domains of HSV-1 gH, we generated 22 randomized linker-insertion mutants. Analyses of 22 gH mutants revealed that gH is relatively tolerant of insertion mutations, as 15 of 22 mutants permitted normal processing and transport of gH-gL to the cell surface. gH mutants that were not expressed well at the cell surface did not function in fusion or viral entry. The screening of gH mutants for function revealed the following: (i) for wild-type gH and some gH mutants, fusion with nectin-1-expressing target cells occurred more rapidly than with herpesvirus entry mediator (HVEM)-expressing target cells; (ii) some gH mutants reduced the rate of cell fusion without abrogating fusion completely, indicating that gH may play a role in governing the kinetics of fusion and may be responsible for a rate-limiting first stage in HSV-1 fusion; and (iii) only one gH mutant, located within the short cytoplasmic tail, completely abrogated function, indicating that the gH cytoplasmic tail is crucial for cell fusion and viral infectivity.Herpes simplex virus (HSV), an enveloped neurotropic virus, infects target cells via membrane fusion, a process executed by viral fusion proteins capable of inserting into target membranes. Unlike many enveloped viruses that induce fusion through the activity of a single viral fusion protein, HSV requires four glycoproteins, glycoprotein B (gB), glycoprotein D (gD), glycoprotein H (gH), and glycoprotein L (gL), to execute fusion (6,40,42). The focus of this study, gH, is expressed as a heterodimer with gL (gH-gL). HSV gH and gL rely on one another for proper folding, posttranslational processing, and transport to the cell and virion surface (5,23,35).A sequential model of entry is the prevailing working hypothesis of HSV entry (1-3, 28, 32, 41). Viral attachment is mediated by the binding of glycoprotein C (gC) or gB to cell surface glycosaminoglycans such as heparan sulfate (38). The subsequent fusion between the virion envelope and host cell membrane is thought to result from a series of concerted events. First, gD binds to one of its host cell receptors. These receptors include herpesvirus entry mediator (HVEM), a member of the tumor necrosis factor (TNF) receptor family; nectin-1 and nectin-2, cell adhesion molecules of the Ig superfamily; and heparan sulfate modified by specific 3-O-sulfotransferases (39).It was previously proposed that gD binding a receptor induces a conformational change that allows for interactions between gD, gB, and/or gH-gL (1,2,8,10,16,25,32). It is thought that while gD functions primarily in receptor binding, gB and gH-gL function as the core fusion machinery of HSV.Based on its crystal structure, gB has structural features typical of viral fusion proteins in general and is structurall...

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Cross Talk among the Glycoproteins Involved in Herpes Simplex Virus Entry and Fusion: the Interaction between gB and gH/gL Does Not Necessarily Require gD

Cited by 58 publications

References 43 publications

Bimolecular Complementation Defines Functional Regions of Herpes Simplex Virus gB That Are Involved with gH/gL as a Necessary Step Leading to Cell Fusion

Bimolecular Complementation Defines Functional Regions of Herpes Simplex Virus gB That Are Involved with gH/gL as a Necessary Step Leading to Cell Fusion

Mechanism for neutralizing activity by the anti-CMV gH/gL monoclonal antibody MSL-109

Insertion Mutations in Herpes Simplex Virus 1 Glycoprotein H Reduce Cell Surface Expression, Slow the Rate of Cell Fusion, or Abrogate Functions in Cell Fusion and Viral Entry

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