scholarly journals Mildly Acidic pH Triggers an Irreversible Conformational Change in the Fusion Domain of Herpes Simplex Virus 1 Glycoprotein B and Inactivation of Viral Entry

2016 ◽  
Vol 91 (5) ◽  
Author(s):  
Darin J. Weed ◽  
Suzanne M. Pritchard ◽  
Floricel Gonzalez ◽  
Hector C. Aguilar ◽  
Anthony V. Nicola
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Viral Entry ◽  
Low Ph ◽  
Fusion Activity ◽  
Ph Inactivation ◽  
Target Membrane ◽  
Fusion Domain ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Herpes simplex virus (HSV) entry into a subset of cells requires endocytosis and endosomal low pH. Preexposure of isolated virions to mildly acidic pH of 5 to 6 partially inactivates HSV infectivity in an irreversible manner. Acid inactivation is a hallmark of viruses that enter via low-pH pathways; this occurs by pretriggering conformational changes essential for fusion. The target and mechanism(s) of low-pH inactivation of HSV are unclear. Here, low-pH-treated HSV-1 was defective in fusion activity and yet retained normal levels of attachment to cell surface heparan sulfate and binding to nectin-1 receptor. Low-pH-triggered conformational changes in gB reported to date are reversible, despite irreversible low-pH inactivation. gB conformational changes and their reversibility were measured by antigenic analysis with a panel of monoclonal antibodies and by detecting changes in oligomeric conformation. Three-hour treatment of HSV-1 virions with pH 5 or multiple sequential treatments at pH 5 followed by neutral pH caused an irreversible >2.5 log infectivity reduction. While changes in several gB antigenic sites were reversible, alteration of the H126 epitope was irreversible. gB oligomeric conformational change remained reversible under all conditions tested. Altogether, our results reveal that oligomeric alterations and fusion domain changes represent distinct conformational changes in gB, and the latter correlates with irreversible low-pH inactivation of HSV. We propose that conformational change in the gB fusion domain is important for activation of membrane fusion during viral entry and that in the absence of a host target membrane, this change results in irreversible inactivation of virions. IMPORTANCE HSV-1 is an important pathogen with a high seroprevalence throughout the human population. HSV infects cells via multiple pathways, including a low-pH route into epithelial cells, the primary portal into the host. HSV is inactivated by low-pH preexposure, and gB, a class III fusion protein, undergoes reversible conformational changes in response to low-pH exposure. Here, we show that low-pH inactivation of HSV is irreversible and due to a defect in virion fusion activity. We identified an irreversible change in the fusion domain of gB following multiple sequential low-pH exposures or following prolonged low-pH treatment. This change appears to be separable from the alteration in gB quaternary structure. Together, the results are consistent with a model by which low pH can have an activating or inactivating effect on HSV depending on the presence of a target membrane.

scholarly journals Acidic pH Mediates Changes in Antigenic and Oligomeric Conformation of Herpes Simplex Virus gB and Is a Determinant of Cell-Specific Entry

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Darin J. Weed ◽  
Stephen J. Dollery ◽  
Tri Komala Sari ◽  
Anthony V. Nicola
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Conformational Changes ◽  
Viral Entry ◽  
Ph Dependence ◽  
Low Ph ◽  
Acidic Ph ◽  
Class Iii ◽  
Simplex Virus

ABSTRACTHerpes simplex virus (HSV) is an important human pathogen with a high worldwide seroprevalence. HSV enters epithelial cells, the primary site of infection, by a low-pH pathway. HSV glycoprotein B (gB) undergoes low pH-induced conformational changes, which are thought to drive membrane fusion. When neutralized back to physiological pH, these changes become reversible. Here, HSV-infected cells were subjected to short pulses of radiolabeling, followed by immunoprecipitation with a panel of gB monoclonal antibodies (MAbs), demonstrating that gB folds and oligomerizes rapidly and cotranslationally in the endoplasmic reticulum. Full-length gB from transfected cells underwent low-pH-triggered changes in oligomeric conformation in the absence of other viral proteins. MAbs to gB neutralized HSV entry into cells regardless of the pH dependence of the entry pathway, suggesting a conservation of gB function in distinct fusion mechanisms. The combination of heat and acidic pH triggered irreversible changes in the antigenic conformation of the gB fusion domain, while changes in the gB oligomer remained reversible. An elevated temperature alone was not sufficient to induce gB conformational change. Together, these results shed light on the conformation and function of the HSV-1 gB oligomer, which serves as part of the core fusion machinery during viral entry.IMPORTANCEHerpes simplex virus (HSV) causes infection of the mouth, skin, eyes, and genitals and establishes lifelong latency in humans. gB is conserved among all herpesviruses. HSV gB undergoes reversible conformational changes following exposure to acidic pH which are thought to mediate fusion and entry into epithelial cells. Here, we identified cotranslational folding and oligomerization of newly synthesized gB. A panel of antibodies to gB blocked both low-pH and pH-neutral entry of HSV, suggesting conserved conformational changes in gB regardless of cell entry route. Changes in HSV gB conformation were not triggered by increased temperature alone, in contrast to results with EBV gB. Acid pH-induced changes in the oligomeric conformation of gB are related but distinct from pH-triggered changes in gB antigenic conformation. These results highlight critical aspects of the class III fusion protein, gB, and inform strategies to block HSV infection at the level of fusion and entry.

scholarly journals Differential Effects on Cell Fusion Activity of Mutations in Herpes Simplex Virus 1 Glycoprotein B (gB) Dependent on Whether a gD Receptor or a gB Receptor Is Overexpressed

2009 ◽  
Vol 83 (15) ◽  
pp. 7384-7390 ◽  
Author(s):  
Qing Fan ◽  
Erick Lin ◽  
Takeshi Satoh ◽  
Hisashi Arase ◽  
Patricia G. Spear
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Fusion ◽  
Viral Entry ◽  
Selective Reduction ◽  
Glycoprotein B ◽  
Fusion Activity ◽  
Differential Effects ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Glycoprotein B (gB) of herpes simplex virus (HSV) is one of four glycoproteins essential for viral entry and cell fusion. Recently, paired immunoglobulin-like type 2 receptor (PILRα) was identified as a receptor for HSV type 1 (HSV-1) gB. Both PILRα and a gD receptor were shown to participate in HSV-1 entry into certain cell types. The purpose of this study was to determine whether insertional mutations in gB had differential effects on its function with PILRα and the gD receptor, nectin-1. Previously described gB mutants and additional newly characterized mutants were used in this study. We found that insertional mutations near the N terminus and C terminus of gB and especially in the central region of the ectodomain reduced cell fusion activity when PILRα was overexpressed much more than when nectin-1 was overexpressed. Most of the insertions reduced the binding of gB to PILRα, for at least some forms of gB, but this reduction did not necessarily correlate with the selective reduction in cell fusion activity with PILRα. These results suggest that the regions targeted by the relevant mutations are critical for functional activity with PILRα. They also suggest that, although both the binding of gB to a gB receptor and the binding of gD to a gD receptor may be required for HSV-induced cell fusion, the two receptor-binding activities may have unequal weights in triggering fusogenic activity, depending on the ratios of gB and gD receptors or other factors.

scholarly journals New Insights into the Spring-Loaded Conformational Change of Influenza Virus Hemagglutinin

2002 ◽  
Vol 76 (9) ◽  
pp. 4456-4466 ◽  
Author(s):  
Jennifer A. Gruenke ◽  
R. Todd Armstrong ◽  
William W. Newcomb ◽  
Jay C. Brown ◽  
Judith M. White
Keyword(s):  
Influenza Virus ◽  
Conformational Change ◽  
Conformational Changes ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Fusion Peptide ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Target Membrane ◽  
Mutant Forms

ABSTRACT Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix “spring-loaded” conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only ∼50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.

scholarly journals TRPC1 participates in the HSV-1 infection process by facilitating viral entry

2020 ◽  
Vol 6 (12) ◽  
pp. eaaz3367 ◽  
Author(s):  
DongXu He ◽  
AiQin Mao ◽  
YouRan Li ◽  
SiuCheung Tam ◽  
YongTang Zheng ◽  
...  
Keyword(s):  
Viral Entry ◽  
Transient Receptor Potential ◽  
Trp Channels ◽  
Critical Role ◽  
Receptor Potential ◽  
Infection Process ◽  
Ocular Abnormality ◽  
Simplex Virus ◽  
Hsv 1

Mammalian transient receptor potential (TRP) channels are major components of Ca2+ signaling pathways and control a diversity of physiological functions. Here, we report a specific role for TRPC1 in the entry of herpes simplex virus type 1 (HSV-1) into cells. HSV-1–induced Ca2+ release and entry were dependent on Orai1, STIM1, and TRPC1. Inhibition of Ca2+ entry or knockdown of these proteins attenuated viral entry and infection. HSV-1 glycoprotein D interacted with the third ectodomain of TRPC1, and this interaction facilitated viral entry. Knockout of TRPC1 attenuated HSV-1–induced ocular abnormality and morbidity in vivo in TRPC1−/− mice. There was a strong correlation between HSV-1 infection and plasma membrane localization of TRPC1 in epithelial cells within oral lesions in buccal biopsies from HSV-1–infected patients. Together, our findings demonstrate a critical role for TRPC1 in HSV-1 infection and suggest the channel as a potential target for anti-HSV therapy.

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 Soluble V Domain of Nectin-1/HveC Enables Entry of Herpes Simplex Virus Type 1 (HSV-1) into HSV-Resistant Cells by Binding to Viral Glycoprotein D

2006 ◽  
Vol 80 (1) ◽  
pp. 138-148 ◽  
Author(s):  
Heechung Kwon ◽  
Qing Bai ◽  
Hyun-Jung Baek ◽  
Kelly Felmet ◽  
Edward A. Burton ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Surface ◽  
Viral Entry ◽  
Glycoprotein D ◽  
Viral Glycoprotein ◽  
Virus Attachment ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Interaction of herpes simplex virus (HSV) glycoprotein D (gD) with specific cellular receptors is essential for HSV infection of susceptible cells. Virus mutants that lack gD can bind to the cell surface (attachment) but do not enter, implying that interaction of gD with its receptor(s) initiates the postattachment (entry) phase of HSV infection. In this report, we have studied HSV entry in the presence of the gD-binding variable (V) domain of the common gD receptor nectin-1/HveC to determine whether cell association of the gD receptor is required for HSV infection. In the presence of increasing amounts of the soluble nectin-1 V domain (sNec1123), increasing viral entry into HSV-resistant CHO-K1 cells was observed. At a multiplicity of 3 in the presence of optimal amounts of sNec1123, approximately 90% of the cells were infected. The soluble V domain of nectin-2, a strain-specific HSV entry receptor, promoted entry of the HSV type 1 (HSV-1) Rid-1 mutant strain, but not of wild-type HSV-1. Preincubation and immunofluorescence studies indicated that free or gD-bound sNec1123 did not associate with the cell surface. sNec1123-mediated entry was highly impaired by interference with the cell-binding activities of viral glycoproteins B and C. While gD has at least two functions, virus attachment to the cell and initiation of the virus entry process, our results demonstrate that the attachment function of gD is dispensable for entry provided that other means of attachment are available, such as gB and gC binding to cell surface glycosaminoglycans.

scholarly journals HIV-1 Replication in HIV-Infected Individuals Is Significantly Reduced When Peripheral Blood Mononuclear Cells Are Superinfected with HSV-1

2012 ◽  
Vol 2012 ◽  
pp. 1-6
Author(s):  
Taneth Yamsuwan ◽  
Chintana Chirathaworn ◽  
Pokrath Hansasuta ◽  
Parvapan Bhattarakosol
Keyword(s):  
T Lymphocytes ◽  
Viral Entry ◽  
Mononuclear Cells ◽  
Multiplicity Of Infection ◽  
Peripheral Blood Mononuclear ◽  
Healthy Donors ◽  
Immunodeficiency Virus ◽  
Simplex Virus ◽  
Hiv 1 ◽  
Hsv 1

Herpes simplex virus (HSV) can cause generalized infection in human immunodeficiency virus- (HIV-) infected patients leading to death. This study investigated HSV-1 replication in PBMCs from 25 HIV-infected individuals and 15 healthy donors and the effects of HSV-1 superinfection on HIV-1 production. Herpes viral entry mediator (HVEM) receptor on T lymphocytes was also evaluated. Our results confirmed that the number of activated (CD3+ and CD38+) T lymphocytes in HIV-infected individuals (46.51±17.54%) was significantly higher than in healthy donors (27.54±14.12%,Pvalue = 0.001) without any significant differences in HVEM expression. Even though the percentages of HSV-1 infected T lymphocytes between HIV-infected individuals (79.25±14.63%) and healthy donors (80.76±7.13%) were not different (Pvalue = 0.922), yet HSV-1 production in HIV-infected individuals (47.34±11.14×103 PFU/ml) was significantly greater than that of healthy donors (34.17±8.48×103 PFU/ml,Pvalue = 0.001). Moreover, HSV-1 virions were released extracellularly rather than being associated with the cells, and superinfection of HSV-1 at a multiplicity of infection (MOI) of 5 significantly decreased HIV production (Pvalue < 0.001).

scholarly journals Multiple Peptides Homologous to Herpes Simplex Virus Type 1 Glycoprotein B Inhibit Viral Infection

2008 ◽  
Vol 53 (3) ◽  
pp. 987-996 ◽  
Author(s):  
Radeekorn Akkarawongsa ◽  
Nina E. Pocaro ◽  
Gary Case ◽  
Aaron W. Kolb ◽  
Curtis R. Brandt
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Viral Entry ◽  
Herpes Simplex Virus Type ◽  
Glycoprotein B ◽  
Induced Mutations ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The 773-residue ectodomain of the herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) has been resistant to the use of mutagenic strategies because the majority of the induced mutations result in defective proteins. As an alternative strategy for the identification of functionally important regions and novel inhibitors of infection, we prepared a library of overlapping peptides homologous to the ectodomain of gB and screened for the ability of the peptides to block infection. Seven of 138 15-mer peptides inhibited infection by more than 50% at a concentration of 100 μM. Three peptides (gB94, gB122, and gB131) with 50% effective concentrations (EC50s) below 20 μM were selected for further studies. The gB131 peptide (residues 681 to 695 in HSV-1 gB [gB-1]) was a specific entry inhibitor (EC50, ∼12 μM). The gB122 peptide (residues 636 to 650 in gB-1) blocked viral entry (EC50, ∼18 μM), protected cells from infection (EC50, ∼72 μM), and inactivated virions in solution (EC50, ∼138 μM). We were unable to discern the step or steps inhibited by the gB94 peptide, which is homologous to residues 496 to 510 in gB-1. Substitution of a tyrosine in the gB122 peptide (Y640 in full-length gB-1) reduced the antiviral activity eightfold, suggesting that this residue is critical for inhibition. This peptide-based strategy could lead to the identification of functionally important regions of gB or other membrane proteins and identify novel inhibitors of HSV-1 entry.

scholarly journals Herpesvirus Entry Mediator on Radiation-Resistant Cell Lineages Promotes Ocular Herpes Simplex Virus 1 Pathogenesis in an Entry-Independent Manner

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Rebecca G. Edwards ◽  
Sarah J. Kopp ◽  
Andrew H. Karaba ◽  
Douglas R. Wilcox ◽  
Richard Longnecker
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Viral Entry ◽  
Resistant Cell ◽  
Herpes Simplex Virus 1 ◽  
Ocular Herpes ◽  
Radiation Resistant ◽  
Stromal Keratitis ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTOcular herpes simplex virus 1 (HSV-1) infection leads to a potentially blinding immunoinflammatory syndrome, herpes stromal keratitis (HSK). Herpesvirus entry mediator (HVEM), a widely expressed tumor necrosis factor (TNF) receptor superfamily member with diverse roles in immune signaling, facilitates viral entry through interactions with viral glycoprotein D (gD) and is important for HSV-1 pathogenesis. We subjected mice to corneal infection with an HSV-1 mutant in which HVEM-mediated entry was specifically abolished and found that the HVEM-entry mutant produced clinical disease comparable to that produced by the control virus. HVEM-mediated induction of corneal cytokines, which correlated with an HVEM-dependent increase in levels of corneal immune cell infiltrates, was also gD independent. Given the complexity of HVEM immune signaling, we used hematopoietic chimeric mice to determine which HVEM-expressing cells mediate HSV-1 pathogenesis in the eye. Regardless of whether the donor was a wild-type (WT) or HVEM knockout (KO) strain, HVEM KO recipients were protected from ocular HSV-1, suggesting that HVEM on radiation-resistant cell types, likely resident cells of the cornea, confers wild-type-like susceptibility to disease. Together, these data indicate that HVEM contributes to ocular pathogenesis independently of entry and point to an immunomodulatory role for this protein specifically on radiation-resistant cells.IMPORTANCEImmune privilege is maintained in the eye in order to protect specialized ocular tissues, such as the translucent cornea, from vision-reducing damage. Ocular herpes simplex virus 1 (HSV-1) infection can disrupt this immune privilege, provoking a host response that ultimately brings about the majority of the damage seen with the immunoinflammatory syndrome herpes stromal keratitis (HSK). Our previous work has shown that HVEM, a host TNF receptor superfamily member that also serves as a viral entry receptor, is a critical component contributing to ocular HSV-1 pathogenesis, although its precise role in this process remains unclear. We hypothesized that HVEM promotes an inflammatory microenvironment in the eye through immunomodulatory actions, enhancing disease after ocular inoculation of HSV-1. Investigating the mechanisms responsible for orchestrating this aberrant immune response shed light on the initiation and maintenance of HSK, one of the leading causes of infectious blindness in the developed world.

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