scholarly journals The Neutralizing Linear Epitope of Human Herpesvirus 6A Glycoprotein B Does Not Affect Virus Infectivity

2017 ◽  
Vol 92 (5) ◽  
Author(s):  
Aika Wakata ◽  
Satoshi Kanemoto ◽  
Huamin Tang ◽  
Akiko Kawabata ◽  
Mitsuhiro Nishimura ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Cell Fusion ◽  
Virus Entry ◽  
Human Herpesvirus ◽  
Homology Model ◽  
Glycoprotein B ◽  
Linear Epitope ◽  
Epitope Residue ◽  
Cell Cell

ABSTRACTHuman herpesvirus 6A (HHV-6A) glycoprotein B (gB) is a glycoprotein consisting of 830 amino acids and is essential for the growth of the virus. Previously, we reported that a neutralizing monoclonal antibody (MAb) called 87-y-13 specifically reacts with HHV-6A gB, and we identified its epitope residue at asparagine (Asn) 347 on gB. In this study, we examined whether the epitope recognized by the neutralizing MAb is essential for HHV-6A infection. We constructed HHV-6A bacterial artificial chromosome (BAC) genomes harboring substitutions at Asn347, namely, HHV-6A BACgB(N347K) and HHV-6A BACgB(N347A). These mutant viruses could be reconstituted and propagated in the same manner as the wild type and their revertants, and MAb 87-y-13 could not inhibit infection by either mutant. In a cell-cell fusion assay, Asn at position 347 on gB was found to be nonessential for cell-cell fusion. In addition, in building an HHV-6A gB homology model, we found that the epitope of the neutralizing MAb is located on domain II of gB and is accessible to solvents. These results indicate that Asn at position 347, the linear epitope of the neutralizing MAb, does not affect HHV-6A infectivity.IMPORTANCEGlycoprotein B (gB) is one of the most conserved glycoproteins among all herpesviruses and is a key factor for virus entry. Therefore, antibodies targeted to gB may neutralize virus entry. Human herpesvirus 6A (HHV-6A) encodes gB, which is translated to a protein of about 830 amino acids (aa). Using a monoclonal antibody (MAb) for HHV-6A gB, which has a neutralizing linear epitope, we analyzed the role of its epitope residue, N347, in HHV-6A infectivity. Interestingly, this gB linear epitope residue, N347, was not essential for HHV-6A growth. By constructing a homology model of HHV-6A gB, we found that N347 was located in the region corresponding to domain II. Therefore, with regard to its neutralizing activity against HHV-6A infection, the epitope on gB might be exposed to solvents, suggesting that it might be a target of the immune system.

Herpes simplex virus glycoprotein B (gB) mutations define structural sites in domain I, the membrane proximal region, and the cytodomain that regulate entry.

2021 ◽  
Author(s):  
Qing Fan ◽  
Richard Longnecker ◽  
Sarah A. Connolly
Keyword(s):  
Cell Fusion ◽  
Virus Entry ◽  
Proximal Region ◽  
Glycoprotein B ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Membrane Proximal Region ◽  
Domain V ◽  
Domain I ◽  
Cell Cell

The viral fusion protein glycoprotein B (gB) is conserved in all herpesviruses and is essential for virus entry. During entry, gB fuses viral and host cell membranes by refolding from a prefusion to a postfusion form. We previously introduced three structure-based mutations (gB-I671A/H681A/F683A) into the domain V arm of the gB ectodomain that resulted in reduced cell-cell fusion. A virus carrying these three mutations (called gB3A) displayed a small plaque phenotype and remarkably delayed entry into cells. To identify mutations that could counteract this phenotype, we serially passaged the gB3A virus and selected for revertant viruses with increased plaque size. Genomic sequencing revealed that the revertant viruses had second-site mutations in gB, including E187A, M742T, and S383F/G645R/V705I/V880G. Using expression constructs encoding these mutations, only gB-V880G was shown to enhance cell-cell fusion. In contrast, all of the revertant viruses showed enhanced entry kinetics, underscoring the fact that cell-cell fusion and virus-cell fusion are different. The results indicate that mutations in three different regions of gB (domain I, the membrane proximal region, and the cytoplasmic tail domain) can counteract the slow entry phenotype of gB3A virus. Mapping these compensatory mutations to prefusion and postfusion structural models suggests sites of intramolecular functional interactions with the gB domain V arm that may contribute to the gB fusion function. Importance The nine human herpesviruses are ubiquitous and cause a range of disease in humans. Glycoprotein B (gB) is an essential viral fusion protein that is conserved in all herpesviruses. During host cell entry, gB mediates virus-cell membrane fusion by undergoing a conformational change. Structural models for the prefusion and postfusion form of gB exist, but the details of how the protein converts from one to the other are unclear. We previously introduced structure-based mutations into gB that inhibited virus entry and fusion. By passaging this entry-deficient virus over time, we selected second-site mutations that partially restore virus entry. The location of these mutations suggest regulatory sites that contribute to fusion and gB refolding during entry. gB is a target of neutralizing antibodies and defining how gB refolds during entry could provide a basis for the development of fusion inhibitors for future research or clinical use.

scholarly journals Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

2008 ◽  
Vol 82 (6) ◽  
pp. 2883-2894 ◽  
Author(s):  
Megan W. Howard ◽  
Emily A. Travanty ◽  
Scott A. Jeffers ◽  
M. K. Smith ◽  
Sonia T. Wennier ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Fusion ◽  
Transmembrane Domain ◽  
Virus Entry ◽  
Aromatic Amino Acids ◽  
Wild Type ◽  
Aromatic Residues ◽  
Wild Type Level ◽  
S Proteins ◽  
Cell Cell

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein (S) is a class I viral fusion protein that binds to its receptor glycoprotein, human angiotensin converting enzyme 2 (hACE2), and mediates virus entry and cell-cell fusion. The juxtamembrane domain (JMD) of S is an aromatic amino acid-rich region proximal to the transmembrane domain that is highly conserved in all coronaviruses. Alanine substitutions for one or two of the six aromatic residues in the JMD did not alter the surface expression of the SARS-CoV S proteins with a deletion of the C-terminal 19 amino acids (S Δ19) or reduce binding to soluble human ACE2 (hACE2). However, hACE2-dependent entry of trypsin-treated retrovirus pseudotyped viruses expressing JMD mutant S Δ19 proteins was greatly reduced. Single alanine substitutions for aromatic residues reduced entry to 10 to 60% of the wild-type level. The greatest reduction was caused by residues nearest the transmembrane domain. Four double alanine substitutions reduced entry to 5 to 10% of the wild-type level. Rapid hACE2-dependent S-mediated cell-cell fusion was reduced to 60 to 70% of the wild-type level for all single alanine substitutions and the Y1188A/Y1191A protein. S Δ19 proteins with other double alanine substitutions reduced cell-cell fusion further, from 40% to less than 20% of wild-type levels. The aromatic amino acids in the JMD of the SARS-CoV S glycoprotein play critical roles in receptor-dependent virus-cell and cell-cell fusion. Because the JMD is so highly conserved in all coronavirus S proteins, it is a potential target for development of drugs that may inhibit virus entry and/or cell-cell fusion mediated by S proteins of all coronaviruses.

scholarly journals Using Antibodies and Mutants To Localize the Presumptive gH/gL Binding Site on Herpes Simplex Virus gD

2018 ◽  
Vol 92 (24) ◽  
Author(s):  
Doina Atanasiu ◽  
Wan Ting Saw ◽  
Eric Lazear ◽  
J. Charles Whitbeck ◽  
Tina M. Cairns ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Protein Interactions ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Epitope Mapping ◽  
Virus Entry ◽  
Cellular Receptor ◽  
Functional Sites ◽  
Cell Cell

ABSTRACTHSV virus-cell and cell-cell fusion requires multiple interactions between four essential virion envelope glycoproteins, gD, gB, gH, and gL, and between gD and a cellular receptor, nectin-1 or herpesvirus entry mediator (HVEM). Current models suggest that binding of gD to receptors induces a conformational change that leads to activation of gH/gL and consequent triggering of the prefusion form of gB to promote membrane fusion. Since protein-protein interactions guide each step of fusion, identifying the sites of interaction may lead to the identification of potential therapeutic targets that block this process. We have previously identified two “faces” on gD: one for receptor binding and the other for its presumed interaction with gH/gL. We previously separated the gD monoclonal antibodies (MAbs) into five competition communities. MAbs from two communities (MC2 and MC5) neutralize virus infection and block cell-cell fusion but do not block receptor binding, suggesting that they block binding of gD to gH/gL. Using a combination of classical epitope mapping of gD mutants with fusion and entry assays, we identified two residues (R67 and P54) on the presumed gH/gL interaction face of gD that allowed for fusion and viral entry but were no longer sensitive to inhibition by MC2 or MC5, yet both were blocked by other MAbs. As neutralizing antibodies interfere with essential steps in the fusion pathway, our studies strongly suggest that these key residues block the interaction of gD with gH/gL.IMPORTANCEVirus entry and cell-cell fusion mediated by HSV require gD, gH/gL, gB, and a gD receptor. Neutralizing antibodies directed against any of these proteins bind to residues within key functional sites and interfere with an essential step in the fusion pathway. Thus, the epitopes of these MAbs identify critical, functional sites on their target proteins. Unlike many anti-gD MAbs, which block binding of gD to a cellular receptor, two, MC2 and MC5, block a separate, downstream step in the fusion pathway which is presumed to be the activation of the modulator of fusion, gH/gL. By combining epitope mapping of a panel of gD mutants with fusion and virus entry assays, we have identified residues that are critical in the binding and function of these two MAbs. This new information helps to define the site of the presumptive interaction of gD with gH/gL, of which we have limited knowledge.

Evidence for involvement of equine herpesvirus 1 glycoprotein B in cell-cell fusion

1996 ◽  
Vol 141 (1) ◽  
pp. 167-175 ◽  
Author(s):  
J. E. Wellington ◽  
D. N. Love ◽  
J. M. Whalley
Keyword(s):  
Cell Fusion ◽  
Glycoprotein B ◽  
Equine Herpesvirus ◽  
Equine Herpesvirus 1 ◽  
Herpesvirus 1 ◽  
Cell Cell

EHV-1 glycoprotein D (EHV-1 gD) is required for virus entry and cell-cell fusion, and an EHV-1 gD deletion mutant induces a protective immune response in mice

2000 ◽  
Vol 145 (11) ◽  
pp. 2371-2385 ◽  
Author(s):  
H. Csellner ◽  
C. Walker ◽  
J. E. Wellington ◽  
L. E. McLure ◽  
D. N. Love ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Fusion ◽  
Deletion Mutant ◽  
Virus Entry ◽  
Protective Immune Response ◽  
Glycoprotein D ◽  
Cell Cell

scholarly journals Cleavage of Epstein–Barr virus glycoprotein B is required for full function in cell–cell fusion with both epithelial and B cells

2009 ◽  
Vol 90 (3) ◽  
pp. 591-595 ◽  
Author(s):  
Jessica Sorem ◽  
Richard Longnecker
Keyword(s):  
B Cells ◽  
Cell Fusion ◽  
Epstein Barr Virus ◽  
Glycoprotein B ◽  
Barr Virus ◽  
High Sequence Conservation ◽  
A Cell ◽  
Epstein Barr ◽  
Cleavage Motif ◽  
Cell Cell

Glycoprotein B (gB) homologues within the herpesvirus family display high sequence conservation, and a number of gB homologues contain a cleavage motif R-X-K/R-R recognized by the cellular protease furin. Epstein–Barr virus (EBV) gB contains this motif and cleaved gB is found in EBV virions. To determine the functional significance of this cleavage motif in EBV gB, a deletion mutant (gB Δfurin) was created lacking the motif. This cleavage mutant was expressed well in cell culture but was not cleaved. Experiments examining gB Δfurin in a cell-fusion assay revealed that fusion was reduced by 52 % in epithelial and 28 % in B cells when compared with wild-type EBV gB. This decrease in cell–cell fusion is similar to that observed with multiple alphaherpesvirus gB cleavage mutants and supports a conserved function for cleaved gB.

scholarly journals Human herpesvirus 6 envelope cholesterol is required for virus entry

2006 ◽  
Vol 87 (2) ◽  
pp. 277-285 ◽  
Author(s):  
Honglan Huang ◽  
Yongmei Li ◽  
Tomohiko Sadaoka ◽  
Huanmin Tang ◽  
Takahito Yamamoto ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Viral Entry ◽  
Virus Entry ◽  
Human Herpesvirus ◽  
Fusion Process ◽  
Cholesterol Depletion ◽  
Human Herpesvirus 6 ◽  
Exogenous Cholesterol ◽  
Herpesvirus 6

In this study, the role of cholesterol in the envelope of human herpesvirus 6 (HHV-6) was examined by using methyl-β-cyclodextrin (MβCD) depletion. When cholesterol was removed from HHV-6 virions with MβCD, infectivity was abolished, but it could be rescued by the addition of exogenous cholesterol. HHV-6 binding was affected slightly by MβCD treatment. In contrast, envelope cholesterol depletion markedly affected HHV-6 infectivity and HHV-6-induced cell fusion. These results suggest that the cholesterol present in the HHV-6 envelope plays a prominent role in the fusion process and is a key component in viral entry.

scholarly journals The Human Herpesvirus 6 G Protein-Coupled Receptor Homolog U51 Positively Regulates Virus Replication and Enhances Cell-Cell Fusion In Vitro

2005 ◽  
Vol 79 (18) ◽  
pp. 11914-11924 ◽  
Author(s):  
Zhu Zhen ◽  
Birgit Bradel-Tretheway ◽  
Sarah Sumagin ◽  
Jean M. Bidlack ◽  
Stephen Dewhurst
Keyword(s):  
Viral Replication ◽  
G Protein ◽  
Cell Fusion ◽  
Virus Replication ◽  
Human Herpesvirus ◽  
Human Herpesvirus 6 ◽  
G Protein Coupled Receptor ◽  
G Protein Coupled ◽  
Cell Cell

ABSTRACT Human herpesvirus 6 (HHV-6) is a ubiquitous T-lymphotropic betaherpesvirus that encodes two G protein-coupled receptor homologs, U12 and U51. HHV-6A U51 has been reported to bind to CC chemokines including RANTES, but the biological function of U51 remains uncertain. In this report, we stably expressed short interfering RNAs (siRNAs) specific for U51 in human T cells and then infected these cells with HHV-6. Viral DNA replication was reduced 50-fold by the U51 siRNA, and virally induced cytopathic effects were also inhibited. In contrast, viral replication and syncytium formation were unaltered in cells that expressed a scrambled derivative of the siRNA or an irrelevant siRNA and were restored to normal when a human codon-optimized derivative of U51 was introduced into cells containing the U51 siRNA. To examine the mechanism whereby U51 might contribute to viral replication, we explored the signaling characteristics of U51. None of the chemokines and opioids tested was able to induce G protein coupling by U51, and no evidence for opioid ligand binding by U51 was obtained. The effect of U51 on cell-cell fusion was also evaluated; these studies showed that U51 enhanced cell fusion mediated by the G protein of vesicular stomatitis virus. However, a U51-specific antiserum had no virus-neutralizing activity, suggesting that U51 may not be involved in the initial interaction between the virus particle and host cell. Overall, these studies suggest that HHV-6 U51 is a positive regulator of virus replication in vitro, perhaps because it may promote membrane fusion and facilitates cell-cell spread of this highly cell-associated virus.

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