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 Low pH-Induced Conformational Change in Herpes Simplex Virus Glycoprotein B

2010 ◽  
Vol 84 (8) ◽  
pp. 3759-3766 ◽  
Author(s):  
Stephen J. Dollery ◽  
Mark G. Delboy ◽  
Anthony V. Nicola
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Conformational Changes ◽  
Low Ph ◽  
Host Cells ◽  
Glycoprotein B ◽  
Antigenic Structure ◽  
Acidic Ph ◽  
Herpes Simplex Virus Glycoprotein ◽  
Simplex Virus

ABSTRACT Herpesviruses can enter host cells using pH-dependent endocytosis pathways in a cell-specific manner. Envelope glycoprotein B (gB) is conserved among all herpesviruses and is a critical component of the complex that mediates membrane fusion and entry. Here we demonstrate that mildly acidic pH triggers specific conformational changes in herpes simplex virus (HSV) gB. The antigenic structure of gB was specifically altered by exposure to low pH both in vitro and during entry into host cells. The oligomeric conformation of gB was altered at a similar pH range. Exposure to acid pH appeared to convert virion gB into a lower-order oligomer. The detected conformational changes were reversible, similar to those in other class III fusion proteins. Exposure of purified, recombinant gB to mildly acidic pH resulted in similar changes in conformation and caused gB to become more hydrophobic, suggesting that low pH directly affects gB. We propose that intracellular low pH induces alterations in gB conformation that, together with additional triggers such as receptor binding, are essential for virion-cell fusion during herpesviral entry by endocytosis.

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 Herpes Simplex Virus Glycoprotein C Regulates Low pH Entry

2019 ◽  
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

AbstractHerpes simplex viruses (HSVs) cause significant morbidity and mortality in humans worldwide. Herpesviruses mediate entry by a multi-component, virus-encoded machinery. Herpesviruses enter cells by endosomal low pH and pH-neutral mechanisms in a cell-specific manner. HSV mediates cell entry via envelope glycoproteins gB, gD, and the heterodimer gH/gL regardless of pH or endocytosis requirements. 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 to 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.ImportanceHerpesviruses are ubiquitous pathogens that cause lifelong latent infections and are characterized by multiple entry pathways. We propose that herpes simplex virus (HSV) gC plays a selective role in modulating HSV entry by a low pH pathway, such as into epithelial cells. gC facilitates 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 study identifies a new function for gC and provides novel insight into the complex mechanism of HSV entry and fusion.

scholarly journals Herpes simplex virus type 2 virion host shutoff protein suppresses innate dsRNA antiviral pathways in human vaginal epithelial cells

2011 ◽  
Vol 92 (9) ◽  
pp. 1981-1993 ◽  
Author(s):  
Xiao-Dan Yao ◽  
Kenneth Lee Rosenthal
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Herpes Simplex Virus Type ◽  
Innate Immune ◽  
Antiviral Responses ◽  
Virion Host Shutoff ◽  
Vaginal Epithelial Cells ◽  
Simplex Virus

Viruses that establish persistent infections have evolved numerous strategies to evade host innate antiviral responses. We functionally assessed the role of herpes simplex virus type 2 (HSV-2) virion host shutoff (vhs) protein on innate immune sensing pathways in human vaginal epithelial cells (VK2 ECs). Infection of cells with wild-type (WT) HSV-2 significantly decreased expression of innate immune sensors of viral infection, Toll-like receptor (TLR)2, TLR3, retinoic acid inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (Mda-5), relative to cells infected with a mutant that lacks vhs (vhsB) or mock-infected cells. Transfection with HSV-2 vhs similarly decreased expression of TLR2, TLR3, RIG-I and Mda-5, which was also confirmed in human embryonic kidney (HEK) 293 cells. vhsB infection of VK2 cells caused robust increases in the active form of interferon regulatory factor (IRF)3 and its translocation to the nucleus compared with the WT. Additionally, IRF3 activation by Sendai virus and polyinosinic : polycytidylic acid-induced stimulation of beta interferon (IFN-β) was significantly inhibited in vhs-transfected cells. Overall, our findings provide the first evidence that HSV-2 vhs plays roles in selectively inhibiting TLR3 and RIG-I/Mda-5, as well as TLR2-mediated antiviral pathways for sensing dsRNA and effectively suppresses IFN-β antiviral responses in human vaginal ECs.

scholarly journals Histidine-Rich Glycoprotein Inhibits HIV-1 Infection in a pH-Dependent Manner

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Ezequiel Dantas ◽  
Fernando Erra Díaz ◽  
Pehuén Pereyra Gerber ◽  
Augusto Varese ◽  
Diana Alicia Jerusalinsky ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Respiratory Syncytial Virus ◽  
Herpes Simplex ◽  
Low Ph ◽  
Vaginal Mucosa ◽  
Ph Values ◽  
Syncytial Virus ◽  
Simplex Virus ◽  
Herpes Simplex Virus 2 ◽  
Hiv 1

ABSTRACTHistidine-rich glycoprotein (HRG) is an abundant plasma protein with a multidomain structure, allowing its interaction with many ligands, including phospholipids, plasminogen, fibrinogen, IgG antibodies, and heparan sulfate. HRG has been shown to regulate different biological responses, such as angiogenesis, coagulation, and fibrinolysis. Here, we found that HRG almost completely abrogated the infection of Ghost cells, Jurkat cells, CD4+T cells, and macrophages by HIV-1 at a low pH (range, 6.5 to 5.5) but not at a neutral pH. HRG was shown to interact with the heparan sulfate expressed by target cells, inhibiting an early postbinding step associated with HIV-1 infection. More importantly, by acting on the viral particle itself, HRG induced a deleterious effect, which reduces viral infectivity. Because cervicovaginal secretions in healthy women show low pH values, even after semen deposition, our observations suggest that HRG might represent a constitutive defense mechanism in the vaginal mucosa. Of note, low pH also enabled HRG to inhibit the infection of HEp-2 cells and Vero cells by respiratory syncytial virus (RSV) and herpes simplex virus 2 (HSV-2), respectively, suggesting that HRG might display broad antiviral activity under acidic conditions.IMPORTANCEVaginal intercourse represents a high-risk route for HIV-1 transmission. The efficiency of male-to-female HIV-1 transmission has been estimated to be 1 in every 1,000 episodes of sexual intercourse, reflecting the high degree of protection conferred by the genital mucosa. However, the contribution of different host factors to the protection against HIV-1 at mucosal surfaces remains poorly defined. Here, we report for the first time that acidic values of pH enable the plasma protein histidine-rich glycoprotein (HRG) to strongly inhibit HIV-1 infection. Because cervicovaginal secretions usually show low pH values, our observations suggest that HRG might represent a constitutive antiviral mechanism in the vaginal mucosa. Interestingly, infection by other viruses, such as respiratory syncytial virus and herpes simplex virus 2, was also markedly inhibited by HRG at low pH values, suggesting that extracellular acidosis enables HRG to display broad antiviral activity.

scholarly journals Acute Infection and Subsequent Subclinical Reactivation of Herpes Simplex Virus 2 after Vaginal Inoculation of Rhesus Macaques

2018 ◽  
Vol 93 (2) ◽  
Author(s):  
Ming Lo ◽  
Jia Zhu ◽  
Scott G. Hansen ◽  
Timothy Carroll ◽  
Christina Farr Zuend ◽  
...  
Keyword(s):  
Animal Model ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Rhesus Macaques ◽  
Sexually Transmitted ◽  
Key Features ◽  
Simplex Virus ◽  
Herpes Simplex Virus 2 ◽  
Incident Cases

ABSTRACTHerpes simplex virus 2 (HSV-2) is a common sexually transmitted infection with a highly variable clinical course. Many infections quickly become subclinical, with episodes of spontaneous virus reactivation. To study host–HSV-2 interactions, an animal model of subclinical HSV-2 infection is needed. In an effort to develop a relevant model, rhesus macaques (RM) were inoculated intravaginally with two or three HSV-2 strains (186, 333, and/or G) at a total dose of 1 × 107PFU of HSV-2 per animal. Infectious HSV-2 and HSV-2 DNA were consistently shed in vaginal swabs for the first 7 to 14 days after each inoculation. Proteins associated with wound healing, innate immunity, and inflammation were significantly increased in cervical secretions immediately after HSV-2 inoculation. There was histologic evidence of acute herpesvirus pathology, including acantholysis in the squamous epithelium and ballooning degeneration of and intranuclear inclusion bodies in epithelial cells, with HSV antigen in mucosal epithelial cells and keratinocytes. Further, an intense inflammatory infiltrate was found in the cervix and vulva. Evidence of latent infection and reactivation was demonstrated by the detection of spontaneous HSV-2 shedding post-acute inoculation (102to 103DNA copies/swab) in 80% of RM. Further, HSV-2 DNA was detected in ganglia in most necropsied animals. HSV-2-specifc T-cell responses were detected in all animals, although antibodies to HSV-2 were detected in only 30% of the animals. Thus, HSV-2 infection of RM recapitulates many of the key features of subclinical HSV-2 infection in women but seems to be more limited, as virus shedding was undetectable more than 40 days after the last virus inoculation.IMPORTANCEHerpes simplex virus 2 (HSV-2) infects nearly 500 million persons globally, with an estimated 21 million incident cases each year, making it one of the most common sexually transmitted infections (STIs). HSV-2 is associated with increased human immunodeficiency virus type 1 (HIV-1) acquisition, and this risk does not decline with the use of antiherpes drugs. As initial acquisition of both HIV and HSV-2 infections is subclinical, study of the initial molecular interactions of the two agents requires an animal model. We found that HSV-2 can infect RM after vaginal inoculation, establish latency in the nervous system, and spontaneously reactivate; these features mimic some of the key features of HSV-2 infection in women. RM may provide an animal model to develop strategies to prevent HSV-2 acquisition and reactivation.

scholarly journals Herpes Simplex Virus Entry by a Nonconventional Endocytic Pathway

2020 ◽  
Vol 94 (24) ◽  
Author(s):  
Giulia Tebaldi ◽  
Suzanne M. Pritchard ◽  
Anthony V. Nicola
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Epithelial Cells ◽  
Retrograde Transport ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Golgi Network ◽  
Hsv 1

ABSTRACT Herpes simplex virus 1 (HSV-1) causes significant morbidity and mortality in humans worldwide. HSV-1 enters epithelial cells via an endocytosis mechanism that is low-pH dependent. However, the precise intracellular pathway has not been identified, including the compartment where fusion occurs. In this study, we utilized a combination of molecular and pharmacological approaches to better characterize HSV entry by endocytosis. HSV-1 entry was unaltered in both cells treated with small interfering RNA (siRNA) to Rab5 or Rab7 and cells expressing dominant negative forms of these GTPases, suggesting entry is independent of the conventional endo-lysosomal network. The fungal metabolite brefeldin A (BFA) and the quinoline compound Golgicide A (GCA) inhibited HSV-1 entry via beta-galactosidase reporter assay and impaired incoming virus transport to the nuclear periphery, suggesting a role for trans-Golgi network (TGN) functions and retrograde transport in HSV entry. Silencing of Rab9 or Rab11 GTPases, which are involved in the retrograde transport pathway, resulted in only a slight reduction in HSV infection. Together, these results suggest that HSV enters host cells by an intracellular route independent of the lysosome-terminal endocytic pathway. IMPORTANCE Herpes simplex virus 1 (HSV-1), the prototype alphaherpesvirus, is ubiquitous in the human population and causes lifelong infection that can be fatal in neonatal and immunocompromised individuals. HSV enters many cell types by endocytosis, including epithelial cells, the site of primary infection in the host. The intracellular itinerary for HSV entry remains unclear. We probed the potential involvement of several Rab GTPases in HSV-1 entry and suggest that endocytic entry of HSV-1 is independent of the canonical lysosome-terminal pathway. A nontraditional endocytic route may be employed, such as one that intersects with the trans-Golgi network (TGN). These results may lead to novel targets for intervention.

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.

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