scholarly journals Regulation of Herpes Simplex Virus Glycoprotein-Induced Cascade of Events Governing Cell-Cell Fusion

2016 ◽  
Vol 90 (23) ◽  
pp. 10535-10544 ◽  
Author(s):  
Doina Atanasiu ◽  
Wan Ting Saw ◽  
Roselyn J. Eisenberg ◽  
Gary H. Cohen
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Fusion ◽  
Critical Role ◽  
Fusion Process ◽  
Simplex Virus ◽  
The Impact ◽  
Hsv 1 ◽  
Cell Cell

ABSTRACTReceptor-dependent herpes simplex virus (HSV)-induced cell-cell fusion requires glycoproteins gD, gH/gL, and gB. Our current model posits that during fusion, receptor-activated conformational changes in gD activate gH/gL, which subsequently triggers the transformation of the prefusion form of gB into a fusogenic state. To examine the role of each glycoprotein in receptor-dependent cell-cell fusion, we took advantage of our discovery that fusion by wild-type herpes simplex virus 2 (HSV-2) glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we established that fusion speed was governed by gH/gL, with gH being the main contributor. While the mutant forms of gB fuse at distinct rates that are dictated by their molecular structure, these restrictions can be overcome by gH/gL of HSV-2 (gH2/gL2), thereby enhancing their activity. We also found that deregulated forms of gD of HSV-1 (gD1) and gH2/gL2can alter the fusogenic potential of gB, promoting cell fusion in the absence of a cellular receptor, and that deregulated forms of gB can drive the fusion machinery to even higher levels. Low pH enhanced fusion by affecting the structure of both gB and gH/gL mutants. Together, our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process, and the critical role of gH/gL in regulating HSV-induced fusion.IMPORTANCECell-cell fusion mediated by HSV glycoproteins requires gD, gH/gL, gB, and a gD receptor. Here, we show that fusion by wild-type HSV-2 glycoproteins occurs twice as fast as that achieved by HSV-1 glycoproteins. By sequentially swapping each glycoprotein between the two serotypes, we found that the fusion process was controlled by gH/gL. Restrictions imposed on the gB structure by mutations could be overcome by gH2/gL2, enhancing the activity of the mutants. Under low-pH conditions or when using deregulated forms of gD1and gH2/gL2, the fusogenic potential of gB could only be increased in the absence of receptor, underlining the exquisite regulation that occurs in the presence of receptor. Our data highlight the complexity of the fusion machinery, the impact of the activation state of each glycoprotein on the fusion process, and the critical role of gH/gL in regulating HSV-induced fusion.

scholarly journals Structure-Function Analysis of Herpes Simplex Virus Type 1 gD and gH-gL: Clues from gDgH Chimeras

2003 ◽  
Vol 77 (12) ◽  
pp. 6731-6742 ◽  
Author(s):  
Tina M. Cairns ◽  
Richard S. B. Milne ◽  
Manuel Ponce-de-Leon ◽  
Deanna K. Tobin ◽  
Gary H. Cohen ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Surface ◽  
Cell Fusion ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Simplex Virus ◽  
Hsv 1 ◽  
Cell Cell

ABSTRACT In alphaherpesviruses, glycoprotein B (gB), gD, gH, and gL are essential for virus entry. A replication-competent gL-null pseudorabies virus (PrV) (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014-3022, 1999) was shown to express a gDgH hybrid protein that could replace gD, gH, and gL in cell-cell fusion and null virus complementation assays. To study this phenomenon in herpes simplex virus type 1 (HSV-1), we constructed four gDgH chimeras, joining the first 308 gD amino acids to various gH N-terminal truncations. The chimeras were named for the first amino acid of gH at which each was truncated: 22, 259, 388, and 432. All chimeras were immunoprecipitated with both gD and gH antibodies to conformational epitopes. Normally, transport of gH to the cell surface requires gH-gL complex formation. Chimera 22 contains full-length gH fused to gD308. Unlike PrV gDgH, chimera 22 required gL for transport to the surface of transfected Vero cells. Interestingly, although chimera 259 failed to reach the cell surface, chimeras 388 and 432 exhibited gL-independent transport. To examine gD and gH domain function, each chimera was tested in cell-cell fusion and null virus complementation assays. Unlike PrV gDgH, none of the HSV-1 chimeras substituted for gL for fusion. Only chimera 22 was able to replace gH for fusion and could also replace either gH or gD in the complementation assay. Surprisingly, this chimera performed very poorly as a substitute for gD in the fusion assay despite its ability to complement gD-null virus and bind HSV entry receptors (HveA and nectin-1). Chimeras 388 and 432, which contain the same portion of gD as that in chimera 22, substituted for gD for fusion at 25 to 50% of wild-type levels. However, these chimeras functioned poorly in gD-null virus complementation assays. The results highlight the fact that these two functional assays are measuring two related but distinct processes.

scholarly journals HveA (Herpesvirus Entry Mediator A), a Coreceptor for Herpes Simplex Virus Entry, also Participates in Virus-Induced Cell Fusion

1998 ◽  
Vol 72 (7) ◽  
pp. 5802-5810 ◽  
Author(s):  
Tracy Terry-Allison ◽  
Rebecca I. Montgomery ◽  
J. Charles Whitbeck ◽  
Ruliang Xu ◽  
Gary H. Cohen ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Fusion ◽  
Viral Entry ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Simplex Virus ◽  
Herpesvirus Entry Mediator ◽  
Hsv 1 ◽  
Cell Cell

ABSTRACT The purpose of this study was to determine whether a cell surface protein that can serve as coreceptor for herpes simplex virus type 1 (HSV-1) entry, herpesvirus entry mediator (previously designated HVEM but renamed HveA), also mediates HSV-1-induced cell-cell fusion. We found that transfection of DNA from KOS-804, a previously described HSV-1 syncytial (Syn) strain whose Syn mutation was mapped to an amino acid substitution in gK, induced numerous large syncytia on HveA-expressing Chinese hamster ovary cells (CHO-HVEM12) but not on control cells (CHO-C8). Antibodies specific for gD as well as for HveA were effective inhibitors of KOS-804-induced fusion, consistent with previously described direct interactions between gD and HveA. Since mutations in gD determine the ability of HSV-1 to utilize HveA for entry, we examined whether the form of virally expressed gD also influenced the ability of HveA to mediate fusion. We produced a recombinant virus carrying the KOS-804 Syn mutation and the KOS-Rid1 gD mutation, which significantly reduces viral entry via HveA, and designated it KOS-SR1. KOS-SR1 DNA had a markedly reduced ability to induce syncytia on CHO-HVEM12 cells and a somewhat enhanced ability to induce syncytia on CHO-C8 cells. These results support previous findings concerning the relative abilities of KOS and KOS-Rid1 to infect CHO-HVEM12 and CHO-C8 cells. Thus, HveA mediates cell-cell fusion as well as viral entry and both activities of HveA are contingent upon the form of gD expressed by the virus.

scholarly journals Herpes Simplex Virus Glycoprotein K, but Not Its Syncytial Allele, Inhibits Cell-Cell Fusion Mediated by the Four Fusogenic Glycoproteins, gD, gB, gH, and gL

2003 ◽  
Vol 77 (12) ◽  
pp. 6836-6844 ◽  
Author(s):  
Elisa Avitabile ◽  
Giulia Lombardi ◽  
Gabriella Campadelli-Fiume
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Cell Fusion ◽  
Vero Cells ◽  
Giant Cells ◽  
Glycoprotein K ◽  
Herpes Simplex Virus Glycoprotein ◽  
Simplex Virus ◽  
Hsv 1 ◽  
Cell Cell

ABSTRACT A Myc epitope was inserted at residue 283 of herpes simplex virus type 1 (HSV-1) glycoprotein K (gK), a position previously shown not to interfere with gK activity. The Myc-tagged gK localized predominantly to the endoplasmic reticulum, both in uninfected and in HSV-infected cells. gK, coexpressed with the four HSV fusogenic glycoproteins, gD, gB, gH, and gL, inhibited cell-cell fusion. The effect was partially dose dependent and was observed both in baby hamster kidney (BHK) and in Vero cells, indicating that the antifusion activity of gK may be cell line independent. The antifusion activity of gK did not require viral proteins other than the four fusogenic glycoproteins. A syncytial (syn) allele of gK (syn-gK) carrying the A40V substitution present in HSV-1(MP) did not block fusion to the extent seen with the wild-type (wt) gK, indicating that the syn mutation ablated, at least in part, the antifusogenic activity of wt gK. We conclude that gK is part of the mechanism whereby HSV negatively regulates its own fusion activity. Its effect accounts for the notion that cells infected with wt HSV do not fuse with adjacent, uninfected cells into multinucleated giant cells or syncytia. gK may also function to preclude fusion between virion envelope and the virion-encasing vesicles during virus transport to the extracellular compartment, thus preventing nucleocapsid de-envelopment in the cytoplasm.

scholarly journals Control of Herpes Simplex Virus Replication Is Mediated through an Interferon Regulatory Factor 3-Dependent Pathway

2009 ◽  
Vol 83 (23) ◽  
pp. 12399-12406 ◽  
Author(s):  
Vineet D. Menachery ◽  
David A. Leib
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Replication ◽  
Early Recognition ◽  
Critical Role ◽  
Type I ◽  
Type I Ifn ◽  
Regulatory Factor ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The type I interferon (IFN) cascade is critical in controlling viral replication and pathogenesis. Recognition pathways triggered by viral infection rapidly induce the type I IFN cascade, often in an IFN regulatory factor 3 (IRF-3)-dependent fashion. This dependence predicts that loss of IRF-3 would render early recognition pathways inoperative and thereby impact virus replication, but this has not been observed previously with herpes simplex virus type 1 (HSV-1) in vitro. In this study, HSV-1-infected IRF-3−/− bone marrow-derived dendritic cells (BMDCs) and macrophages supported increased HSV replication compared to control cells. In addition, IRF-3-deficient BMDCs exhibited delayed type I IFN synthesis compared to control cells. However, while IFN pretreatment of IRF-3−/− BMDCs resulted in reduced virus titers, a far greater reduction was seen after IFN treatment of wild-type cells. This suggests that even in the presence of exogenously supplied IFN, IRF-3−/− BMDCs are inherently defective in the control of HSV-1 replication. Together, these results demonstrate a critical role for IRF-3-mediated pathways in controlling HSV-1 replication in cells of the murine immune system.

scholarly journals MORC3, a Component of PML Nuclear Bodies, Has a Role in Restricting Herpes Simplex Virus 1 and Human Cytomegalovirus

2016 ◽  
Vol 90 (19) ◽  
pp. 8621-8633 ◽  
Author(s):  
Elizabeth Sloan ◽  
Anne Orr ◽  
Roger D. Everett
Keyword(s):  
Immune Response ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Hcmv Infection ◽  
Nuclear Bodies ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Antiviral Role ◽  
Hsv 1

ABSTRACTWe previously reported that MORC3, a protein associated with promyelocytic leukemia nuclear bodies (PML NBs), is a target of herpes simplex virus 1 (HSV-1) ICP0-mediated degradation (E. Sloan, et al., PLoS Pathog11:e1005059, 2015,http://dx.doi.org/10.1371/journal.ppat.1005059). Since it is well known that certain other components of the PML NB complex play an important role during an intrinsic immune response to HSV-1 and are also degraded or inactivated by ICP0, here we further investigate the role of MORC3 during HSV-1 infection. We demonstrate that MORC3 has antiviral activity during HSV-1 infection and that this antiviral role is counteracted by ICP0. In addition, MORC3's antiviral role extends to wild-type (wt) human cytomegalovirus (HCMV) infection, as its plaque-forming efficiency increased in MORC3-depleted cells. We found that MORC3 is recruited to sites associated with HSV-1 genomes after their entry into the nucleus of an infected cell, and in wt infections this is followed by its association with ICP0 foci prior to its degradation. The RING finger domain of ICP0 was required for degradation of MORC3, and we confirmed that no other HSV-1 protein is required for the loss of MORC3. We also found that MORC3 is required for fully efficient recruitment of PML, Sp100, hDaxx, and γH2AX to sites associated with HSV-1 genomes entering the host cell nucleus. This study further unravels the intricate ways in which HSV-1 has evolved to counteract the host immune response and reveals a novel function for MORC3 during the host intrinsic immune response.IMPORTANCEHerpesviruses have devised ways to manipulate the host intrinsic immune response to promote their own survival and persistence within the human population. One way in which this is achieved is through degradation or functional inactivation of PML NB proteins, which are recruited to viral genomes in order to repress viral transcription. Because MORC3 associates with PML NBs in uninfected cells and is a target for HSV-1-mediated degradation, we investigated the role of MORC3 during HSV-1 infection. We found that MORC3 is also recruited to viral HSV-1 genomes, and importantly it contributes to the fully efficient recruitment of PML, hDaxx, Sp100, and γH2AX to these sites. Depletion of MORC3 resulted in an increase in ICP0-null HSV-1 and wt HCMV replication and plaque formation; therefore, this study reveals that MORC3 is an antiviral factor which plays an important role during HSV-1 and HCMV infection.

Construction and optimization of herpes simplex virus vectors for central nervous system gene delivery based on CRISPR/Cas9-mediated genome editing

2021 ◽  
Vol 21 ◽  
Author(s):  
Xinwei Huang ◽  
Xiuqing Li ◽  
Lijuan Yang ◽  
Pengfei Wang ◽  
Jingyuan Yan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Transgene Expression ◽  
Mouse Hippocampus ◽  
Simplex Virus ◽  
Hsv 1

Aims: We aim to define parameters affecting the safety and long-term transgene expression of attenuated HSV-1 vectors and optimize the expression cassettes to achieve robust and sustained expression in CNS. Background: Engineered, attenuated Herpes simplex virus (HSV) vectors are promising vehicles for gene delivery to the peripheral and central nervous systems. The virus latent promoter (LAP) is commonly used to drive exogenous gene expression; however, parameters affecting the safety and long-term transgene expression of attenuated HSV-1 vectors have not been fully understood. Objective: This study aimed to construct attenuated HSV-1 vectors using the CRISPR-Cas9 system and examine the influence of transgene cassette construction and insertion site on transgene expression and vector safety. Method: In this study, we used a CRISPR-Cas9 system to accurately and efficiently edit attenuated HSV-1 strain 1716, and constructed two series of recombinant virus LMR and LMRx with different sets of gene cassettes insertion in Exon1(LAP2) and 2.0 kb intron downstream of LAP, respectively. The transgene expression and viral gene transcriptional kinetics were compared in in-vitro cell lines. The reporter gene expression and safety profiles of each vector were further evaluated in the mouse hippocampus gene transduction model. Result: The in-vitro cell line analysis indicated that the insertion of a gene expression cassette would disrupt virus gene transcription. Mouse hippocampus transducing analysis suggested that complete expression cassette insertion at 2.0 kb intron could achieve robust and longtime gene expression than the other constructs. Recombinants with gene expression cassettes lacked Poly (A), which induced significant neuronal inflammation due to persistent viral antigen expression and microglia activation. Conclusion: Our results indicated that the integrity of LAT transcripts was not necessary for the establishment of long-term latent expression. Exogenous strong promoters (like cBh promoter) could remain active during latency when placed in Exon1 or 2.0 Kb Intron of LAT locus, although their transcriptional activity declined with time. Consistent with previous research, the foreign gene expression would last much longer when the gene cassette was located downstream of Exon1, which suggested a role of LAP2 in maintaining promoter activity during latency. Besides, over-transcription of the downstream part of LAT may induce continuous activation of the attenuated vectors, suggesting an important role of LAT in maintaining viral reactivation potential.

scholarly journals Cell-to-Cell Spread Blocking Activity Is Extremely Limited in the Sera of Herpes Simplex Virus 1 (HSV-1)- and HSV-2-Infected Subjects

2019 ◽  
Vol 93 (11) ◽  
Author(s):  
Elena Criscuolo ◽  
Matteo Castelli ◽  
Roberta A. Diotti ◽  
Virginia Amato ◽  
Roberto Burioni ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Humoral Immunity ◽  
Virus Entry ◽  
Serum Antibody ◽  
Herpes Simplex Virus 1 ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACTHerpes simplex virus 1 (HSV-1) and HSV-2 can evade serum antibody-mediated neutralization through cell-to-cell transmission mechanisms, which represent one of the central steps in disease reactivation. To address the role of humoral immunity in controlling HSV-1 and HSV-2 replication, we analyzed serum samples from 44 HSV-1 and HSV-2 seropositive subjects by evaluating (i) their efficiency in binding both the purified viral particles and recombinant gD and gB viral glycoproteins, (ii) their neutralizing activity, and (iii) their capacity to inhibit the cell-to-cell virus passagein vitro. All of the sera were capable of binding gD, gB, and whole virions, and all sera significantly neutralized cell-free virus. However, neither whole sera nor purified serum IgG fraction was able to inhibit significantly cell-to-cell virus spreading inin vitropost-virus-entry infectious assays. Conversely, when spiked with an already described anti-gD human monoclonal neutralizing antibody capable of inhibiting HSV-1 and -2 cell-to-cell transmission, each serum boosted both its neutralizing and post-virus-entry inhibitory activity, with no interference exerted by serum antibody subpopulations.IMPORTANCEDespite its importance in the physiopathology of HSV-1 and -2 infections, the cell-to-cell spreading mechanism is still poorly understood. The data shown here suggest that infection-elicited neutralizing antibodies capable of inhibiting cell-to-cell virus spread can be underrepresented in most infected subjects. These observations can be of great help in better understanding the role of humoral immunity in controlling virus reactivation and in the perspective of developing novel therapeutic strategies, studying novel correlates of protection, and designing effective vaccines.

scholarly journals Early herpes simplex virus type 1 infection is dependent on regulated Rac1/Cdc42 signalling in epithelial MDCKII cells

2006 ◽  
Vol 87 (12) ◽  
pp. 3483-3494 ◽  
Author(s):  
Sven Hoppe ◽  
Mario Schelhaas ◽  
Verena Jaeger ◽  
Timo Liebig ◽  
Philipp Petermann ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Dominant Negative ◽  
Actin Dynamics ◽  
Simplex Virus ◽  
Hsv 1

The aim of this study was to understand how molecular determinants of epithelial cells influence initial infection by herpes simplex virus type 1 (HSV-1). Upon infection of the epithelial MDCKII cell line, enhanced association of virus particles with cells forming actin protrusions was observed, suggesting a putative role of actin dynamics in HSV-1 infection. Thus, the impact of the small Rho-like GTPases Rac1, Cdc42 and RhoA acting as key regulators of actin dynamics was addressed. Endogenous Rac1 and Cdc42 were temporarily activated at 15 and 30 min after HSV-1 infection. When constitutively active Cdc42 or Rac1 mutants were expressed transiently, a significant decrease in infectivity was observed, whereas expression of RhoA mutants had no influence. Furthermore, dominant-negative Cdc42 led to decreased infectivity, whereas dominant-negative Rac1 had no effect. So far, the study of potential effectors indicated that Rac1/Cdc42 mutants inhibited infectivity independently of p21-activated kinase (Pak1). The inhibitory effect of Rac1/Cdc42 mutant expression on HSV-1 infection was characterized further and it was found that binding, internalization and transport of HSV-1 were not affected by expression of Rac1/Cdc42 mutants. Thus, these results provide the first evidence for a role of Rac1/Cdc42 signalling during early HSV-1 infection and suggest a mechanism relying on virus-induced regulation of Rac1/Cdc42 activities.

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