Structural Domains Involved in Human Cytomegalovirus Glycoprotein B-Mediated Cell-cell Fusion

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.

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

Archives of Virology ◽

10.1007/bf01718598 ◽

1996 ◽

Vol 141 (1) ◽

pp. 167-175 ◽

Cited By ~ 19

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

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Specific Inhibition of Human Cytomegalovirus Glycoprotein B-Mediated Fusion by a Novel Thiourea Small Molecule

Journal of Virology ◽

10.1128/jvi.78.3.1289-1300.2004 ◽

2004 ◽

Vol 78 (3) ◽

pp. 1289-1300 ◽

Cited By ~ 23

Author(s):

Thomas R. Jones ◽

Shi-Wu Lee ◽

Stephen V. Johann ◽

Vladimir Razinkov ◽

Robert J. Visalli ◽

...

Keyword(s):

Small Molecule ◽

Human Cytomegalovirus ◽

Small Molecule Inhibitor ◽

Glycoprotein B ◽

Chemical Library ◽

Cell Plasma ◽

Molecule Inhibitor ◽

Virion Envelope ◽

Cell Spread ◽

Cell Cell

ABSTRACT A novel small molecule inhibitor of human cytomegalovirus (HCMV) was identified as the result of screening a chemical library by using a whole-virus infected-cell assay. Synthetic chemistry efforts yielded the analog designated CFI02, a compound whose potency had been increased about 100-fold over an initial inhibitor. The inhibitory concentration of CFI02 in various assays is in the low nanomolar range. CFI02 is a selective and potent inhibitor of HCMV; it has no activity against other CMVs, alphaherpesviruses, or unrelated viruses. Mechanism-of-action studies indicate that CFI02 acts very early in the replication cycle, inhibiting virion envelope fusion with the cell plasma membrane. Mutants resistant to CFI02 have mutations in the abundant virion envelope glycoprotein B that are sufficient to confer resistance. Taken together, the data suggest that CFI02 inhibits glycoprotein B-mediated HCMV virion fusion. Furthermore, CFI02 inhibits the cell-cell spread of HCMV. This is the first study of a potent and selective small molecule inhibitor of CMV fusion and cell-cell spread.

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Human cytomegalovirus glycoprotein B variants affect viral entry, cell fusion, and genome stability

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1907447116 ◽

2019 ◽

Vol 116 (36) ◽

pp. 18021-18030 ◽

Cited By ~ 5

Author(s):

Jiajia Tang ◽

Giada Frascaroli ◽

Robert J. Lebbink ◽

Eleonore Ostermann ◽

Wolfram Brune

Keyword(s):

Dna Damage ◽

Cell Fusion ◽

Human Cytomegalovirus ◽

Viral Entry ◽

Genome Stability ◽

Genome Instability ◽

Glycoprotein B ◽

Dna Breaks ◽

Signaling Axis ◽

Caspase 2

Human cytomegalovirus (HCMV), like many other DNA viruses, can cause genome instability and activate a DNA damage response (DDR). Activation of ataxia-telangiectasia mutated (ATM), a kinase activated by DNA breaks, is a hallmark of the HCMV-induced DDR. Here we investigated the activation of caspase-2, an initiator caspase activated in response to DNA damage and supernumerary centrosomes. Of 7 HCMV strains tested, only strain AD169 activated caspase-2 in infected fibroblasts. Treatment with an ATM inhibitor or inactivation of PIDD or RAIDD inhibited caspase-2 activation, indicating that caspase-2 was activated by the PIDDosome. A set of chimeric HCMV strains was used to identify the genetic basis of this phenotype. Surprisingly, we found a single nucleotide polymorphism within the AD169 UL55 ORF, resulting in a D275Y amino acid exchange within glycoprotein B (gB), to be responsible for caspase-2 activation. As gB is an envelope glycoprotein required for fusion with host cell membranes, we tested whether gB(275Y) altered viral entry into fibroblasts. While entry of AD169 expressing gB(275D) proceeded slowly and could be blocked by a macropinocytosis inhibitor, entry of wild-type AD169 expressing gB(275Y) proceeded more rapidly, presumably by envelope fusion with the plasma membrane. Moreover, gB(275Y) caused the formation of syncytia with numerous centrosomes, suggesting that cell fusion triggered caspase-2 activation. These results suggest that gB variants with increased fusogenicity accelerate viral entry, cause cell fusion, and thereby compromise genome stability. They further suggest the ATM-PIDDosome-caspase-2 signaling axis alerts the cell of potentially dangerous cell fusion.

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

Journal of General Virology ◽

10.1099/vir.0.007237-0 ◽

2009 ◽

Vol 90 (3) ◽

pp. 591-595 ◽

Cited By ~ 24

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.

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Human Cytomegalovirus Glycoproteins gB and gH/gL Mediate Epithelial Cell-Cell Fusion When Expressed either in cis or in trans

Journal of Virology ◽

10.1128/jvi.01623-08 ◽

2008 ◽

Vol 82 (23) ◽

pp. 11837-11850 ◽

Cited By ~ 105

Author(s):

Adam L. Vanarsdall ◽

Brent J. Ryckman ◽

Marie C. Chase ◽

David C. Johnson

Keyword(s):

Cell Surface ◽

Cell Fusion ◽

Human Cytomegalovirus ◽

Cell Types ◽

Surface Proteins ◽

Viral Fusion ◽

Surface Molecules ◽

Necessary And Sufficient ◽

Cell Cell

ABSTRACT Herpesviruses use a cascade of interactions with different cell surface molecules to gain entry into cells. In many cases, this involves binding to abundant glycosaminoglycans or integrins followed by interactions with more limited cell surface proteins, leading to fusion with cellular membranes. Human cytomegalovirus (HCMV) has the ability to infect a wide variety of human cell types in vivo. However, very little is known about which HCMV glycoproteins mediate entry into various cell types, including relevant epithelial and endothelial cells. For other herpesviruses, studies of cell-cell fusion induced by viral proteins have provided substantial information about late stages of entry. In this report, we describe the fusion of epithelial, endothelial, microglial, and fibroblast cells in which HCMV gB and gH/gL were expressed from nonreplicating adenovirus vectors. Fusion frequently involved the majority of cells, and gB and gH/gL were both necessary and sufficient for fusion, whereas no fusion occurred when either glycoprotein was omitted. Coexpression of UL128, UL130, and UL131 did not enhance fusion. We concluded that the HCMV core fusion machinery consists of gB and gH/gL. Coimmunoprecipitation indicated that HCMV gB and gH/gL can interact. Importantly, expression of gB and gH/gL in trans (gB-expressing cells mixed with other gH/gL-expressing cells) resulted in substantial fusion. We believe that this is the first description of a multicomponent viral fusion machine that can be split between cells. If gB and gH/gL must interact for fusion, then these molecules must reach across the space between apposing cells. Expression of gB and gH/gL in trans with different cell types revealed surface molecules that are required for fusion on HCMV-permissive cells but not on nonpermissive cells.

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

Journal of Virology ◽

10.1128/jvi.02074-17 ◽

2017 ◽

Vol 92 (5) ◽

Cited By ~ 2

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.

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