Alanine substitution of conserved residues in the cytoplasmic tail of herpes simplex virus gB can enhance or abolish cell fusion activity and viral entry

Ruel, Nancy; Zago, Anna; Spear, Patricia G.

doi:10.1016/j.virol.2005.11.002

Cited by 37 publications

(50 citation statements)

References 43 publications

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“…This was found also for certain amino acid substitutions into conserved positions in the membrane-proximal region of the cytoplasmic tail of HSV-2 gB, although adjacent substitutions permitted cell surface expression and enhanced cell fusion activity (9). Amino acid substitutions at various positions in the cytoplasmic tails of HSV-1 and HSV-2 gB Only two of the insertions that map to the crystallized portion of the gB ectodomain (Fig.…”

Section: Locations Of Insertions On Gb Structure In Relation To Effecmentioning

confidence: 67%

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Random linker-insertion mutagenesis to identify functional domains of herpes simplex virus type 1 glycoprotein B

Lin

Spear

2007

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

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Herpes simplex virus glycoprotein B (gB) is one of four glycoproteins essential for viral entry and cell fusion. Recently, an x-ray structure of the nearly full-length trimeric gB ectodomain was determined. Five structural domains and two linker regions were identified in what is probably a postfusion conformation. To identify functional domains of gB, we performed random linkerinsertion mutagenesis. Analyses of 81 mutants revealed that only 27 could fold to permit processing and transport of gB to the cell surface. These 27 mutants fell into three categories. Insertions into two regions excluded from the solved structure (the N terminus and the C-terminal cytoplasmic tail) had no negative effect on cell fusion and viral entry activity, identifying regions that can tolerate altered structure without loss of function. Insertions into a disordered region in domain II and the adjacent linker region also permitted partial cell fusion and viral entry activity. Insertions at 16 other positions resulted in loss of cell fusion and viral entry activity, despite detectable levels of cell surface expression. Four of these insertion sites were not included in the solved structure. Two were between residues exposed to a cavity that is too small to accommodate the 5-amino acid insertions, consistent with the solved structure being different from the native prefusion structure. Ten were between residues exposed to the surface of the trimer, identifying regions that may be critical for interactions with other viral proteins or cellular components or for transitions from the prefusion to postfusion state.cell fusion ͉ glycoprotein B ͉ viral entry ͉ mutation H erpes simplex virus (HSV) is a neurotropic virus that can cause recurrent mucocutaneous lesions of the oral or genital epithlelium, lesions on the cornea and, rarely, encephalitis. Infection of host cells occurs through virus attachment to the cell surface and subsequent membrane fusion to deliver the nucleocapsid containing the viral genome into the host cell. Virus attachment is mediated by binding of glycoproteins C (gC) or B (gB) to cell surface glycosaminoglycans, primarily heparan sulfate (1). Subsequent fusion between the virion envelope and a host cell membrane requires gB, glycoprotein D (gD), and the heterodimer glycoprotein H (gH)-glycoprotein L (gL) and one of the cellular receptors for gD. These receptors include herpesvirus entry mediator (HVEM), a member of the TNF receptor family; nectin-1 and nectin-2, cell adhesion molecules of the Ig superfamily; and specific sites in heparan sulfate generated by 3-O-sulfotransferases (2). It has been proposed that binding of gD to one of its cellular receptors induces gD to undergo a conformational change, resulting in interactions with gB and/or the gH-gL complex to trigger membrane fusion (3, 4).The exact roles for gB and gH-gL in the membrane fusion process have yet to be elucidated. Recently, it was shown (5) that gH-gL, along with gD and a gD receptor, were sufficient to induce hemifusion, mixing of the outer leafle...

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Section: Locations Of Insertions On Gb Structure In Relation To Effecmentioning

confidence: 67%

“…9), as has also been observed with the class I viral fusion proteins, HIV gp41 (10) and paramyxovirus F protein (11). An x-ray structure of a portion of the HSV type 1 (HSV-1) gB ectodomain exhibits characteristics of both class I and class II viral membrane fusion proteins (12).…”

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confidence: 67%

Random linker-insertion mutagenesis to identify functional domains of herpes simplex virus type 1 glycoprotein B

Lin

Spear

2007

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

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“…In this model, fusion activity would be inhibited when tegument proteins are bound to the tails of viral glycoproteins, but interactions with proteoglycans or other receptors would trigger rearrangements that allow the subsequent events of fusion to proceed. In support of this model, mutations in the cytoplasmic tails of gB and gH have been reported to enhance or abolish fusion activity in cell-cell and virus-cell fusion assays (19,33,39,46). Therefore, it seems likely that the interaction of tegument proteins with the tails of glycoproteins could also influence fusion activity.…”

Section: Discussionmentioning

confidence: 91%

Structural Rearrangement within an Enveloped Virus upon Binding to the Host Cell

Meckes

Wills

2008

J Virol

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We have made the surprising discovery that the interactions of herpes simplex virus with its initial cell attachment receptor induce a rapid and highly efficient structural change in the tegument, the region of the virion situated between the membrane and the capsid. It has been known for nearly a decade that viruses can trigger host signaling pathways when they bind to receptors on the cell surface; however, until now there has been no evidence that a signal can be sent in reverse-from the "outside in"-across a viral membrane. Evidence for this signaling event was found during studies of UL16, a tegument protein that is conserved among all the herpesviruses. Previous work has demonstrated that UL16 is bound to capsids isolated from the cytoplasm of infected cells, but this interaction is destabilized during subsequent egress steps, leading to release of the extracellular virion. Pretreatment with N-ethylmaleimide, a small, membrane-permeating compound that covalently modifies free cysteines, restabilizes the interaction, thereby permitting the capsid-UL16 complex to be isolated following disruption of virions with NP-40. In the experiments described here, we found that the natural signal for release of UL16 from capsids is sent when virions merely bind to cells at 4°C. The internal change was also observed upon binding to immobilized heparin in a manner that requires viral glycoprotein C. This represents the first example of signaling across a viral envelope following receptor binding.It is well established that viruses can trigger cellular signaling pathways when they bind to their receptors on the plasma membrane (reviewed in reference 26). For example, binding can induce virion movement across the cell surface, trigger polymerization of cortical actin filaments needed for endocytic uptake, and modify host gene expression to make the cellular environment more conducive for replication. It is also quite clear that binding triggers changes in the virion proteins that directly interact with the receptor, namely, the capsids of nonenveloped viruses (44) and the glycoproteins found on the surfaces of enveloped viruses (10). In contrast, there has been no evidence to suggest that signals are transmitted in reverseacross the membrane to the interior of any enveloped virusupon receptor binding. Here we report that the interaction of herpes simplex virus (HSV) with its initial cell attachment protein (heparan sulfate) induces a rapid and highly efficient structural change in the tegument, the large proteinaceous region situated between the viral membrane and the DNAcontaining capsid.Our evidence for receptor-induced changes in the tegument was found during studies of UL16, a tegument protein that is conserved among all herpesviruses (47). The function of this protein is poorly understood, but based on its stable association with cytoplasmic capsids (27,35) and its ability to interact with a membrane-bound tegument protein named UL11 (24), we proposed that UL16 might provide a "bridging" function during virion budding ...

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“…Since gB is essential for HSV replication and the gB CT is required for function (47), these HSVs were propagated on complementing cells that express gB but were analyzed for expression of gB and phosphorylation on noncomplementing cells. gB825stop displayed a smaller gB polypeptide labeled with [ 35 S]Met-Cys, as expected (Fig.…”

Section: Vol 83 2009 Hsv Gb Phosphorylation In Egress Fusion 3119mentioning

confidence: 99%

Herpesvirus gB-Induced Fusion between the Virion Envelope and Outer Nuclear Membrane during Virus Egress Is Regulated by the Viral US3 Kinase

Wisner

Wright

Kato

et al. 2009

J Virol

126

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Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.Most enveloped viruses assemble their capsids in the cytoplasm and then become enveloped by budding from the plasma membrane or into cytoplasmic (CT) membranes. Viruses that assemble capsids in the nucleus face the fundamental problem of transporting capsids across the nuclear envelope (NE). Nonenveloped viruses, such as polyomaviruses, that are relatively small can move through nuclear pores, and larger nonenveloped viruses, such as adenoviruses (Ad), disrupt the NE (12, 55). Herpesvirus capsids are too large to move through nuclear pores (18), and, instead, herpesviruses encode machinery which promotes envelopment at the inner nuclear membrane (NM) followed by fusion with the outer NM (29, 52). There appears to be very rapid fusion at the NE because few enveloped herpesvirus particles are normally observed within the perinuclear space. Once present in the cytoplasm, nonenveloped capsids acquire a secondary envelope, producing virions that bud into the Golgi apparatus or trans-Golgi network (13, 29, 52). These mature virions are then secreted from cells.Some aspects of the envelopment of herpesviruses at the inner NM are known. Here, the viruses encounter a major obstacle, the nuclear lamina, a rigid network of lamin proteins lining the inner NM. Herpesviruses disrupt the nuclear lamina in order to assemble along the inner surface of the NM...

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Alanine substitution of conserved residues in the cytoplasmic tail of herpes simplex virus gB can enhance or abolish cell fusion activity and viral entry

Cited by 37 publications

References 43 publications

Random linker-insertion mutagenesis to identify functional domains of herpes simplex virus type 1 glycoprotein B

Random linker-insertion mutagenesis to identify functional domains of herpes simplex virus type 1 glycoprotein B

Structural Rearrangement within an Enveloped Virus upon Binding to the Host Cell

Herpesvirus gB-Induced Fusion between the Virion Envelope and Outer Nuclear Membrane during Virus Egress Is Regulated by the Viral US3 Kinase

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