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

2015 ◽  
Vol 89 (24) ◽  
pp. 12262-12272 ◽  
Author(s):  
Henry B. Rogalin ◽  
Ekaterina E. Heldwein
Keyword(s):  
Herpes Simplex ◽  
Cell Fusion ◽  
Conformational Changes ◽  
Vaccine Development ◽  
Viral Envelope ◽  
Herpes Simplex Virus 1 ◽  
Host Cell Membrane ◽  
Herpes Simplex Viruses ◽  
Simplex Virus ◽  
Cell Cell

ABSTRACTHerpesvirus 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.

scholarly journals Characterization of Vesicular Stomatitis Virus Pseudotypes Bearing Essential Entry Glycoproteins gB, gD, gH, and gL of Herpes Simplex Virus 1

2016 ◽  
Vol 90 (22) ◽  
pp. 10321-10328 ◽  
Author(s):  
Henry B. Rogalin ◽  
Ekaterina E. Heldwein
Keyword(s):  
Herpes Simplex ◽  
Membrane Fusion ◽  
Viral Envelope ◽  
Herpes Simplex Virus 1 ◽  
Herpes Simplex Viruses ◽  
Systematic Exploration ◽  
Vesicular Stomatitis ◽  
Simplex Virus ◽  
First Time ◽  
Hsv 1

ABSTRACTHerpes simplex viruses (HSVs) are unusual in that unlike most enveloped viruses, they require at least four entry glycoproteins, gB, gD, gH, and gL, for entry into target cells in addition to a cellular receptor for gD. The dissection of the herpes simplex virus 1 (HSV-1) entry mechanism is complicated by the presence of more than a dozen proteins on the viral envelope. To investigate HSV-1 entry requirements in a simplified system, we generated vesicular stomatitis virus (VSV) virions pseudotyped with HSV-1 essential entry glycoproteins gB, gD, gH, and gL but lacking the native VSV fusogen G. These virions, referred to here as VSVΔG-BHLD virions, infected a cell line expressing a gD receptor, demonstrating for the first time that the four essential entry glycoproteins of HSV-1 are not only required but also sufficient for cell entry. To our knowledge, this is the first time the VSV pseudotyping system has been successfully extended beyond two proteins. Entry of pseudotyped virions required a gD receptor and was inhibited by HSV-1 specific anti-gB or anti-gH/gL neutralizing antibodies, which suggests that membrane fusion during the entry of the pseudotyped virions shares common requirements with the membrane fusion involved in HSV-1 entry and HSV-1-mediated syncytium formation. The HSV pseudotyping system established in this study presents a novel tool for systematic exploration of the HSV entry and membrane fusion mechanisms.IMPORTANCEHerpes simplex viruses (HSVs) are human pathogens that can cause cold sores, genital herpes, and blindness. No vaccines or preventatives are available. HSV entry into cells—a prerequisite for a successful infection—is a complex process that involves multiple viral and host proteins and occurs by different routes. Detailed mechanistic knowledge of the HSV entry is important for understanding its pathogenesis and would benefit antiviral and vaccine development, yet the presence of more than a dozen proteins on the viral envelope complicates the dissection of the HSV entry mechanisms. In this study, we generated heterologous virions displaying the four essential entry proteins of HSV-1 and showed that they are capable of cell entry and, like HSV-1, require all four entry glycoproteins along with a gD receptor. This HSV pseudotyping system pioneered in this work opens doors for future systematic exploration of the herpesvirus entry mechanisms.

scholarly journals The Domains of Glycoprotein D Required To Block Apoptosis Induced by Herpes Simplex Virus 1 Are Largely Distinct from Those Involved in Cell-Cell Fusion and Binding to Nectin1

2003 ◽  
Vol 77 (6) ◽  
pp. 3759-3767 ◽  
Author(s):  
Guoying Zhou ◽  
Elisa Avitabile ◽  
Gabriella Campadelli-Fiume ◽  
Bernard Roizman
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Fusion ◽  
Ectopic Expression ◽  
Full Length ◽  
Glycoprotein D ◽  
Herpes Simplex Virus 1 ◽  
Mannose 6 Phosphate ◽  
Simplex Virus ◽  
Cell Cell

ABSTRACT Glycoprotein D (gD) interacts with two alternative protein receptors, nectin1 and HveA, to mediate herpes simplex virus (HSV) entry into cells. Fusion of the envelope with the plasma membrane requires, in addition to gD, glycoproteins gB, gH, and gL. Coexpression of the four glycoproteins (gD, gB, gH, and gL) promotes cell-cell fusion. gD delivered in trans is also capable of blocking the apoptosis induced by gD deletion viruses grown either in noncomplementing cells (gD−/−) or in complementing cells (gD−/+). While ectopic expression of cation-independent mannose-6 phosphate receptor blocks apoptosis induced by both stocks, other requirements differ. Thus, apoptosis induced by gD−/− virus is blocked by full-length gD (or two gD fragments reconstituting a full-length molecule), whereas ectopic expression of the gD ectodomain is sufficient to block apoptosis induced by gD−/+ virus. In this report we took advantage of a set of gD insertion-deletion mutants to map the domains of gD required to block apoptosis by gD−/− and gD−/+ viruses and those involved in cell-cell fusion. The mutations that resulted in failure to block apoptosis were the same for gD−/− and gD−/+ viruses and were located in three sites, one within the immunoglobulin-type core region (residues 125, 126, and 151), one in the upstream connector region (residues 34 and 43), and one in the C-terminal portion of the ectodomain (residue 277). A mutant that carried amino acid substitutions at the three glycosylation sites failed to block apoptosis but behaved like wild-type gD in all other assays. The mutations that inhibited polykaryocyte formation were located in the upstream connector region (residues 34 and 43), at the α1 helix (residue 77), in the immunoglobulin core and downstream regions (residue 151 and 187), and at the α3 helix (residues 243 and 246). Binding of soluble nectin1-Fc to cells expressing the mutant gDs was generally affected by the same mutations that affected fusion, with one notable exception (Δ277-310), which affected fusion without hampering nectin1 binding. This deletion likely identifies a region of gD involved in fusion activity at a post-nectin1-binding step. We conclude that whereas mutations that affected all functions (e.g., upstream connector region and residue 151) may be detrimental to overall gD structure, the mutations that affect specific activities identify domains of gD involved in the interactions with entry receptors and fusogenic glycoproteins and with cellular proteins required to block apoptosis. The evidence that glycosylation of gD is required for blocking apoptosis supports the conclusion that the interacting protein is the mannose-6 phosphate receptor.

scholarly journals Cascade of Events Governing Cell-Cell Fusion Induced by Herpes Simplex Virus Glycoproteins gD, gH/gL, and gB

2010 ◽  
Vol 84 (23) ◽  
pp. 12292-12299 ◽  
Author(s):  
Doina Atanasiu ◽  
Wan Ting Saw ◽  
Gary H. Cohen ◽  
Roselyn J. Eisenberg
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Protein ◽  
Cell Fusion ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Structural Level ◽  
Viral Fusion Protein ◽  
Simplex Virus ◽  
Cell Cell

ABSTRACT Herpesviruses minimally require the envelope proteins gB and gH/gL for virus entry and cell-cell fusion; herpes simplex virus (HSV) additionally requires the receptor-binding protein gD. Although gB is a class III fusion protein, gH/gL does not resemble any documented viral fusion protein at a structural level. Based on those data, we proposed that gH/gL does not function as a cofusogen with gB but instead regulates the fusogenic activity of gB. Here, we present data to support that hypothesis. First, receptor-positive B78H1-C10 cells expressing gH/gL fused with receptor-negative B78H1 cells expressing gB and gD (fusion in trans). Second, fusion occurred when gH/gL-expressing C10 cells preexposed to soluble gD were subsequently cocultured with gB-expressing B78 cells. In contrast, prior exposure of gB-expressing C10 cells to soluble gD did not promote subsequent fusion with gH/gL-expressing B78 cells. These data suggest that fusion involves activation of gH/gL by receptor-bound gD. Most importantly, soluble gH/gL triggered a low level of fusion of C10 cells expressing gD and gB; a much higher level was achieved when gB-expressing C10 cells were exposed to a combination of soluble gH/gL and gD. These data clearly show that gB acts as the HSV fusogen following activation by gD and gH/gL. We suggest the following steps leading to fusion: (i) conformational changes to gD upon receptor binding, (ii) alteration of gH/gL by receptor-activated gD, and (iii) upregulation of the fusogenic potential of gB following its interaction with activated gH/gL. The third step may be common to other herpesviruses.

scholarly journals Characterization of cell–cell fusion mediated by herpes simplex virus 2 glycoproteins gB, gD, gH and gL in transfected cells

2000 ◽  
Vol 81 (8) ◽  
pp. 2017-2027 ◽  
Author(s):  
Martin I. Muggeridge
Keyword(s):  
Herpes Simplex ◽  
Cell Fusion ◽  
Plasma Membranes ◽  
Syncytium Formation ◽  
Giant Cells ◽  
Minimal Set ◽  
Herpes Simplex Viruses ◽  
Transfected Cells ◽  
Simplex Virus ◽  
Cell Cell

The mechanisms by which herpes simplex viruses (HSV) mediate fusion between their envelope and the plasma membrane during entry into cells, and between the plasma membranes of adjacent infected and uninfected cells to form multinucleated giant cells, are poorly understood. Four viral glycoproteins (gB, gD, gH and gL) are required for virus–cell fusion, whereas these plus several others are required for cell–cell fusion (syncytium formation). A better understanding would be aided by the availability of a model system, whereby fusion could be induced with a minimal set of proteins, in the absence of infection. A suitable system has now been developed for HSV-2, using transfected COS7, 293 or HEp-2 cells. Insofar as the minimal set of HSV-2 proteins required to cause cell–cell fusion in this system is gB, gD, gH and gL, it would appear to resemble virus–cell fusion rather than syncytium formation. However, the ability of a mutation in gB to enhance the fusion of both transfected cells and infected cells, while having no effect on virus–cell fusion, points to the opposite conclusion. The differential effects of a panel of anti-HSV antibodies, and of the fusion-inhibitor cyclosporin A, confirm that the fusion of transfected cells shares some properties with virus–cell fusion and others with syncytium formation. It may therefore prove useful for determining how these processes differ, and for testing the hypothesis that some viral proteins prevent membrane fusion until the appropriate point in the virus life-cycle, with other proteins then overcoming this block.

scholarly journals A Functional Interaction between Herpes Simplex Virus 1 Glycoprotein gH/gL Domains I and II and gD Is Defined by Using Alphaherpesvirus gH and gL Chimeras

2015 ◽  
Vol 89 (14) ◽  
pp. 7159-7169 ◽  
Author(s):  
Qing Fan ◽  
Richard Longnecker ◽  
Sarah A. Connolly
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Host Cell ◽  
Current Model ◽  
Viral Envelope ◽  
Functional Interaction ◽  
Herpes Simplex Virus 1 ◽  
Homotypic Interaction ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTWhereas most viruses require only a single protein to bind to and fuse with cells, herpesviruses use multiple glycoproteins to mediate virus entry, and thus communication among these proteins is required. For most alphaherpesviruses, the minimal set of viral proteins required for fusion with the host cell includes glycoproteins gD, gB, and a gH/gL heterodimer. In the current model of entry, gD binds to a cellular receptor and transmits a signal to gH/gL. This signal then triggers gB, the conserved fusion protein, to insert into the target membrane and refold to merge the viral and cellular membranes. We previously demonstrated that gB homologs from two alphaherpesviruses, herpes simplex virus 1 (HSV-1) and saimiriine herpesvirus 1 (SaHV-1), were interchangeable. In contrast, neither gD nor gH/gL functioned with heterotypic entry glycoproteins, indicating that gD and gH/gL exhibit an essential type-specific functional interaction. To map this homotypic interaction site on gH/gL, we generated HSV-1/SaHV-1 gH and gL chimeras. The functional interaction with HSV-1 gD mapped to the N-terminal domains I and II of the HSV-1 gH ectodomain. The core of HSV-1 gL that interacts with gH also was required for functional homotypic interaction. The N-terminal gH/gL domains I and II are the least conserved and may have evolved to support species-specific glycoprotein interactions.IMPORTANCEThe first step of the herpesvirus life cycle is entry into a host cell. A coordinated interaction among multiple viral glycoproteins is required to mediate fusion of the viral envelope with the cell membrane. The details of how these glycoproteins interact to trigger fusion are unclear. By swapping the entry glycoproteins of two alphaherpesviruses (HSV-1 and SaHV-1), we previously demonstrated a functional homotypic interaction between gD and gH/gL. To define the gH and gL requirements for homotypic interaction, we evaluated the function of a panel of HSV-1/SaHV-1 gH and gL chimeras. We demonstrate that domains I and II of HSV-1 gH are sufficient to promote a functional, albeit reduced, interaction with HSV-1 gD. These findings contribute to our model of how the entry glycoproteins cooperate to mediate herpesvirus entry into the cell.

scholarly journals Herpes Simplex Virus Glycoprotein C Regulates Low-pH Entry

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Tri Komala Sari ◽  
Katrina A. Gianopulos ◽  
Darin J. Weed ◽  
Seth M. Schneider ◽  
Suzanne M. Pritchard ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Fusion Protein ◽  
Conformational Changes ◽  
Low Ph ◽  
Cell Entry ◽  
Epidermal Keratinocytes ◽  
Herpes Simplex Viruses ◽  
Herpes Simplex Virus Glycoprotein ◽  
Simplex Virus

ABSTRACT Herpes simplex viruses (HSVs) cause significant morbidity and mortality in humans worldwide. Herpesviruses mediate entry by a multicomponent virus-encoded machinery. Herpesviruses enter cells by endosomal low-pH and pH-neutral mechanisms in a cell-specific manner. HSV mediates cell entry via the envelope glycoproteins gB and gD and the heterodimer gH/gL regardless of pH or endocytosis requirements. Specifics concerning HSV envelope proteins that function selectively in a given entry pathway have been elusive. Here, we demonstrate that gC regulates cell entry and infection by a low-pH pathway. Conformational changes in the core herpesviral fusogen gB are critical for membrane fusion. The presence of gC conferred a higher pH threshold for acid-induced antigenic changes in gB. Thus, gC may selectively facilitate low-pH entry by regulating conformational changes in the fusion protein gB. We propose that gC modulates the HSV fusion machinery during entry into pathophysiologically relevant cells, such as human epidermal keratinocytes. IMPORTANCE Herpesviruses are ubiquitous pathogens that cause lifelong latent infections and that are characterized by multiple entry pathways. We propose that herpes simplex virus (HSV) gC plays a selective role in modulating HSV entry, such as entry into epithelial cells, by a low-pH pathway. gC facilitates a conformational change of the main fusogen gB, a class III fusion protein. We propose a model whereby gC functions with gB, gD, and gH/gL to allow low-pH entry. In the absence of gC, HSV entry occurs at a lower pH, coincident with trafficking to a lower pH compartment where gB changes occur at more acidic pHs. This report identifies a new function for gC and provides novel insight into the complex mechanism of HSV entry and fusion.

scholarly journals Effects of Major Capsid Proteins, Capsid Assembly, and DNA Cleavage/Packaging on the pUL17/pUL25 Complex of Herpes Simplex Virus 1

2009 ◽  
Vol 83 (24) ◽  
pp. 12725-12737 ◽  
Author(s):  
Luella Scholtes ◽  
Joel D. Baines
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Localization ◽  
Conformational Changes ◽  
Viral Proteins ◽  
Dna Packaging ◽  
Capsid Proteins ◽  
Herpes Simplex Virus 1 ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT The UL17 and UL25 proteins (pUL17 and pUL25, respectively) of herpes simplex virus 1 are located at the external surface of capsids and are essential for DNA packaging and DNA retention in the capsid, respectively. The current studies were undertaken to determine whether DNA packaging or capsid assembly affected the pUL17/pUL25 interaction. We found that pUL17 and pUL25 coimmunoprecipitated from cells infected with wild-type virus, whereas the major capsid protein VP5 (encoded by the UL19 gene) did not coimmunoprecipitate with these proteins under stringent conditions. In addition, pUL17 (i) coimmunoprecipitated with pUL25 in the absence of other viral proteins, (ii) coimmunoprecipitated with pUL25 from lysates of infected cells in the presence or absence of VP5, (iii) did not coimmunoprecipitate efficiently with pUL25 in the absence of the triplex protein VP23 (encoded by the UL18 gene), (iv) required pUL25 for proper solubilization and localization within the viral replication compartment, (v) was essential for the sole nuclear localization of pUL25, and (vi) required capsid proteins VP5 and VP23 for nuclear localization and normal levels of immunoreactivity in an indirect immunofluorescence assay. Proper localization of pUL25 in infected cell nuclei required pUL17, pUL32, and the major capsid proteins VP5 and VP23, but not the DNA packaging protein pUL15. The data suggest that VP23 or triplexes augment the pUL17/pUL25 interaction and that VP23 and VP5 induce conformational changes in pUL17 and pUL25, exposing epitopes that are otherwise partially masked in infected cells. These conformational changes can occur in the absence of DNA packaging. The data indicate that the pUL17/pUL25 complex requires multiple viral proteins and functions for proper localization and biochemical behavior in the infected cell.

scholarly journals Contribution of Endocytic Motifs in the Cytoplasmic Tail of Herpes Simplex Virus Type 1 Glycoprotein B to Virus Replication and Cell-Cell Fusion

2007 ◽  
Vol 81 (24) ◽  
pp. 13889-13903 ◽  
Author(s):  
Igor Beitia Ortiz de Zarate ◽  
Lilia Cantero-Aguilar ◽  
Magalie Longo ◽  
Clarisse Berlioz-Torrent ◽  
Flore Rozenberg
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Surface ◽  
Cell Fusion ◽  
Virus Replication ◽  
Herpes Simplex Virus Type ◽  
Wild Type ◽  
Simplex Virus ◽  
Cell Cell

ABSTRACT The use of endocytic pathways by viral glycoproteins is thought to play various functions during viral infection. We previously showed in transfection assays that herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is transported from the cell surface back to the trans-Golgi network (TGN) and that two motifs of gB cytoplasmic tail, YTQV and LL, function distinctly in this process. To investigate the role of each of these gB trafficking signals in HSV-1 infection, we constructed recombinant viruses in which each motif was rendered nonfunctional by alanine mutagenesis. In infected cells, wild-type gB was internalized from the cell surface and concentrated in the TGN. Disruption of YTQV abolished internalization of gB during infection, whereas disruption of LL induced accumulation of internalized gB in early recycling endosomes and impaired its return to the TGN. The growth of both recombinants was moderately diminished. Moreover, the fusion phenotype of cells infected with the gB recombinants differed from that of cells infected with the wild-type virus. Cells infected with the YTQV-mutated virus displayed reduced cell-cell fusion, whereas giant syncytia were observed in cells infected with the LL-mutated virus. Furthermore, blocking gB internalization or impairing gB recycling to the cell surface, using drugs or a transdominant negative form of Rab11, significantly reduced cell-cell fusion. These results favor a role for endocytosis in virus replication and suggest that gB intracellular trafficking is involved in the regulation of cell-cell fusion.

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