Extracellular filaments revealed by affinity capture cryo-electron tomography of lymphocytes.

Cryogenic-electron tomography (cryo-ET) has provided an un-precedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron tomography. Micropatterning of extracellular matrix proteins is increasingly used as a method to prepare adherent cell types for cryo-ET as it promotes optimal positioning of cells and subcellular regions of interest for vitrification, cryo-focused ion beam (cryo-FIB) milling, and data acquisition. Here we demonstrate a micropatterning workflow for capturing minimally adherent cell types, human T-cells and Jurkat cells, for cryo-FIB and cryo-ET. Our affinity capture system facilitated the nanoscale imaging of Jurkat cells, revealing extracellular filamentous structures. It improved workflow efficiency by consistently producing grids with a sufficient number of well-positioned cells for an entire cryo-FIB session. Affinity capture can be extended to facilitate high resolution imaging of other adherent and non-adherent cell types with cryo-ET.

Targeted mutagenesis of the herpesvirus fusogen central helix captures transition states.

Herpesviruses remain a burden for animal and human health, including the medically important varicella-zoster virus (VZV). Membrane fusion mediated by conserved core glycoproteins, the fusogen gB and the heterodimer gH-gL, enables herpesvirus cell entry. The ectodomain of gB orthologs has five domains and is proposed to transition from a prefusion to postfusion conformation but the functional relevance of the domains for this transition remains poorly defined. Here we describe structure-function studies of the VZV gB DIII central helix targeting residues 526EHV528. Critically, a H527P mutation captures gB in a prefusion conformation as determined by cryo-EM, a loss of membrane fusion in a virus free assay, and failure of recombinant VZV to spread in cell monolayers. Importantly, two predominant cryo-EM structures of gB[H527P] are identified by 3D classification and focused refinement, suggesting they represented gB conformations in transition. These studies reveal gB DIII as a critical element for herpesvirus gB fusion function.

The Structures and Functions of VZV Glycoproteins.

The virions of all enveloped viruses, including those of the Herpesviridae, must bind to the cell surface then undergo a process of membrane fusion between the cell plasma membrane and the virus particle envelope. As for all herpesviruses, glycoproteins in the virion envelope are the modus operandi of these events.

Target highlights in CASP14: Analysis of models by structure providers.

The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.

The N-terminus of varicella-zoster virus glycoprotein B has a functional role in fusion.

Varicella-zoster virus (VZV) is a medically important alphaherpesvirus that induces fusion of the virion envelope and the cell membrane during entry, and between cells to form polykaryocytes within infected tissues during pathogenesis. All members of the Herpesviridae, including VZV, have a conserved core fusion complex composed of glycoproteins, gB, gH and gL. The ectodomain of the primary fusogen, gB, has five domains, DI-V, of which DI contains the fusion loops needed for fusion function. We recently demonstrated that DIV is critical for fusion initiation, which was revealed by a 2.8Å structure of a VZV neutralizing mAb, 93k, bound to gB and mutagenesis of the gB-93k interface. To further assess the mechanism of mAb 93k neutralization, the binding site of a non-neutralizing mAb to gB, SG2, was compared to mAb 93k using single particle cryogenic electron microscopy (cryo-EM). The gB-SG2 interface partially overlapped with that of gB-93k but, unlike mAb 93k, mAb SG2 did not interact with the gB N-terminus, suggesting a potential role for the gB N-terminus in membrane fusion. The gB ectodomain structure in the absence of antibody was defined at near atomic resolution by single particle cryo-EM (3.9Å) of native, full-length gB purified from infected cells and by X-ray crystallography (2.4Å) of the transiently expressed ectodomain. Both structures revealed that the VZV gB N-terminus (aa72-114) was flexible based on the absence of visible structures in the cryo-EM or X-ray crystallography data but the presence of gB N-terminal peptides were confirmed by mass spectrometry. Notably, N-terminal residues 109KSQD112 were predicted to form a small α-helix and alanine substitution of these residues abolished cell-cell fusion in a virus-free assay. Importantly, transferring the 109AAAA112 mutation into the VZV genome significantly impaired viral propagation. These data establish a functional role for the gB N-terminus in membrane fusion broadly relevant to the Herpesviridae.

Varicella-zoster virus: molecular controls of cell fusion-dependent pathogenesis.

Varicella-zoster virus (VZV) is the causative agent of chicken pox (varicella) and shingles (zoster). Although considered benign diseases, both varicella and zoster can cause complications. Zoster is painful and can lead to post herpetic neuralgia. VZV has also been linked to stroke, related to giant cell arteritis in some cases. Vaccines are available but the attenuated vaccine is not recommended in immunocompromised individuals and the efficacy of the glycoprotein E (gE) based subunit vaccine has not been evaluated for the prevention of varicella. A hallmark of VZV pathology is the formation of multinucleated cells termed polykaryocytes in skin lesions. This cell-cell fusion (abbreviated as cell fusion) is mediated by the VZV glycoproteins gB, gH and gL, which constitute the fusion complex of VZV, also needed for virion entry. Expression of gB, gH and gL during VZV infection and trafficking to the cell surface enables cell fusion. Recent evidence supports the concept that cellular processes are required for regulating cell fusion induced by gB/gH-gL. Mutations within the carboxyl domains of either gB or gH have profound effects on fusion regulation and dramatically restrict the ability of VZV to replicate in human skin. This loss of regulation modifies the transcriptome of VZV infected cells. Furthermore, cellular proteins have significant effects on the regulation of gB/gH-gL-mediated cell fusion and the replication of VZV, exemplified by the cellular phosphatase, calcineurin. This review provides the current state-of-the-art knowledge about the molecular controls of cell fusion-dependent pathogenesis caused by VZV.

Calcineurin phosphatase activity regulates Varicella-Zoster Virus induced cell-cell fusion.

Cell-cell fusion (abbreviated as cell fusion) is a characteristic pathology of medically important viruses, including varicella-zoster virus (VZV), the causative agent of chickenpox and shingles. Cell fusion is mediated by a complex of VZV glycoproteins, gB and gH-gL, and must be tightly regulated to enable skin pathogenesis based on studies with gB and gH hyperfusogenic VZV mutants. Although the function of gB and gH-gL in the regulation of cell fusion has been explored, whether host factors are directly involved in this regulation process is unknown. Here, we discovered host factors that modulated VZV gB/gH-gL mediated cell fusion via high-throughput screening of bioactive compounds with known cellular targets. Two structurally related non-antibiotic macrolides, tacrolimus and pimecrolimus, both significantly increased VZV gB/gH-gL mediated cell fusion. These compounds form a drug-protein complex with FKBP1A, which binds to calcineurin and specifically inhibits calcineurin phosphatase activity. Inhibition of calcineurin phosphatase activity also enhanced both herpes simplex virus-1 fusion complex and syncytin-1 mediated cell fusion, indicating a broad role of calcineurin in modulating this process. To characterize the role of calcineurin phosphatase activity in VZV gB/gH-gL mediated fusion, a series of biochemical, biological and infectivity assays was performed. Pimecrolimus-induced, enhanced cell fusion was significantly reduced by shRNA knockdown of FKBP1A, further supporting the role of calcineurin phosphatase activity in fusion regulation. Importantly, inhibition of calcineurin phosphatase activity during VZV infection caused exaggerated syncytia formation and suppressed virus propagation, which was consistent with the previously reported phenotypes of gB and gH hyperfusogenic VZV mutants. Seven host cell proteins that remained uniquely phosphorylated when calcineurin phosphatase activity was inhibited were identified as potential downstream factors involved in fusion regulation. These findings demonstrate that calcineurin is a critical host cell factor pivotal in the regulation of VZV induced cell fusion, which is essential for VZV pathogenesis.

Publisher Correction: A glycoprotein B-neutralizing antibody structure at 2.8 Å uncovers a critical domain for herpesvirus fusion initiation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A glycoprotein B-neutralizing antibody structure at 2.8 Å uncovers a critical domain for herpesvirus fusion initiation.

Members of the Herpesviridae, including the medically important alphaherpesvirus varicella-zoster virus (VZV), induce fusion of the virion envelope with cell membranes during entry, and between cells to form polykaryocytes in infected tissues. The conserved glycoproteins, gB, gH and gL, are the core functional proteins of the herpesvirus fusion complex. gB serves as the primary fusogen via its fusion loops, but functions for the remaining gB domains remain unexplained. As a pathway for biological discovery of domain function, our approach used structure-based analysis of the viral fusogen together with a neutralizing antibody. We report here a 2.8 Å cryogenic-electron microscopy structure of native gB recovered from VZV-infected cells, in complex with a human monoclonal antibody, 93k. This high-resolution structure guided targeted mutagenesis at the gB-93k interface, providing compelling evidence that a domain spatially distant from the gB fusion loops is critical for herpesvirus fusion, revealing a potential new target for antiviral therapies.

HIV-1 inhibitory properties of eCD4-Igmim2 determined using an Env-mediated membrane fusion assay.

Human Immunodeficiency Virus-1 (HIV-1) entry is dependent on the envelope glycoprotein (Env) that is present on the virion and facilitates fusion between the envelope and the cellular membrane. The protein consists of two subunits, gp120 and gp41, with the former required for binding the CD4 receptor and either the CXCR4 or CCR5 coreceptor, and the latter for mediating fusion. The requirement of fusion for infection has made Env an attractive target for HIV therapy development and led to the FDA approval of enfuvirtide, a fusion inhibitor. Continued development of entry inhibitors is warranted because enfuvirtide resistant HIV-1 strains have emerged. In this study, a novel HIV-1 fusion assay was validated using neutralizing antibodies and then used to investigate the mechanism of action of eCD4-Igmim2, an HIV-1 inhibitor proposed to cooperatively bind the CD4 binding site and the sulfotyrosine-binding pocket of gp120. Greater reduction in fusion levels was observed with eCD4-Igmim2 in the fusion assay than all of the gp120 antibodies evaluated. Lab adapted isolates, HIV-1HXB2 and HIV-1YU2, were sensitive to eCD4-Igmim2 in the fusion assay, while primary isolates, HIV-1BG505 and HIV-1ZM651 were resistant. These results correlated with greater IC50 values for primary isolates compared to the lab adapted isolates observed in a virus neutralization assay. Analysis of gp120 models identified differences in the V1 and V2 domains that are associated with eCD4-Igmim2 sensitivity. This study highlights the use of a fusion assay to identify key areas for improving the potency of eCD4-Igmim2.

Dysregulated Glycoprotein B-Mediated Cell-Cell Fusion Disrupts Varicella-Zoster Virus and Host Gene Transcription during Infection.

The highly conserved herpesvirus glycoprotein complex gB/gH-gL mediates membrane fusion during virion entry and cell-cell fusion. Varicella-zoster virus (VZV) characteristically forms multinucleated cells, or syncytia, during the infection of human tissues, but little is known about this process. The cytoplasmic domain of VZV gB (gBcyt) has been implicated in cell-cell fusion regulation because a gB[Y881F] substitution causes hyperfusion. gBcyt regulation is necessary for VZV pathogenesis, as the hyperfusogenic mutant gB[Y881F] is severely attenuated in human skin xenografts. In this study, gBcyt-regulated fusion was investigated by comparing melanoma cells infected with wild-type-like VZV or hyperfusogenic mutants. The gB[Y881F] mutant exhibited dramatically accelerated syncytium formation in melanoma cells caused by fusion of infected cells with many uninfected cells, increased cytoskeleton reorganization, and rapid displacement of nuclei to dense central structures compared to pOka using live-cell confocal microscopy. VZV and human transcriptomes were concurrently investigated using whole transcriptome sequencing (RNA-seq) to identify viral and cellular responses induced when gBcyt regulation was disrupted by the gB[Y881F] substitution. The expression of four vital VZV genes, ORF61 and the genes for glycoproteins gC, gE, and gI, was significantly reduced at 36 h postinfection for the hyperfusogenic mutants. Importantly, hierarchical clustering demonstrated an association of differential gene expression with dysregulated gBcyt-mediated fusion. A subset of Ras GTPase genes linked to membrane remodeling were upregulated in cells infected with the hyperfusogenic mutants. These data implicate gBcyt in the regulation of gB fusion function that, if unmodulated, triggers cellular processes leading to hyperfusion that attenuates VZV infection. IMPORTANCE: The highly infectious, human-restricted pathogen varicella-zoster virus (VZV) causes chickenpox and shingles. Postherpetic neuralgia (PHN) is a common complication of shingles that manifests as prolonged excruciating pain, which has proven difficult to treat. The formation of fused multinucleated cells in ganglia might be associated with this condition. An effective vaccine against VZV is available but not recommended for immunocompromised individuals, highlighting the need for new therapies. This study investigated the viral and cellular responses to hyperfusion, a condition where the usual constraints of cell membranes are overcome and cells form multinucleated cells. This process hinders VZV and is regulated by a viral glycoprotein, gB. A combination of live-cell imaging and next-generation genomics revealed an alteration in viral and cellular responses during hyperfusion that was caused by the loss of gB regulation. These studies reveal mechanisms central to VZV pathogenesis, potentially leading to improved therapies.

The Glycoprotein B Cytoplasmic Domain Lysine Cluster Is Critical for Varicella-Zoster Virus Cell-Cell Fusion Regulation and Infection.

The conserved glycoproteins gB and gH-gL are essential for herpesvirus entry and cell-cell fusion induced syncytium formation, a characteristic of varicella-zoster virus (VZV) pathology in skin and sensory ganglia. VZV syncytium formation, which has been implicated in the painful condition of postherpetic neuralgia, is regulated by the cytoplasmic domains of gB (gBcyt) via an immunoreceptor tyrosine-based inhibition motif (ITIM) and gH (gHcyt). A lysine cluster (K894, K897, K898, and K900) in the VZV gBcyt was identified by sequence alignment to be conserved among alphaherpesviruses, suggesting a functional role. Alanine and arginine substitutions were used to determine if the positive charge and susceptibility to posttranslational modifications of these lysines contributed to gB/gH-gL cell-cell fusion. Critically, the positive charge of the lysine residues was necessary for fusion regulation, as alanine substitutions induced a 440% increase in fusion compared to that of the wild-type gBcyt while arginine substitutions had wild-type-like fusion levels in an in vitro gB/gH-gL cell fusion assay. Consistent with these results, the alanine substitutions in the viral genome caused exaggerated syncytium formation, reduced VZV titers (-1.5 log10), and smaller plaques than with the parental Oka (pOka) strain. In contrast, arginine substitutions resulted in syncytia with only 2-fold more nuclei, a -0.5-log10 reduction in titers, and pOka-like plaques. VZV mutants with both an ITIM mutation and either alanine or arginine substitutions had reduced titers and small plaques but differed in syncytium morphology. Thus, effective VZV propagation is dependent on cell-cell fusion regulation by the conserved gBcyt lysine cluster, in addition to the gBcyt ITIM and the gHcyt. IMPORTANCE: Varicella-zoster virus (VZV) is a ubiquitous pathogen that causes chickenpox and shingles. Individuals afflicted with shingles risk developing the painful condition of postherpetic neuralgia (PHN), which has been difficult to treat because the underlying cause is not well understood. Additional therapies are needed, as the current vaccine is not recommended for immunocompromised individuals and its efficacy decreases with the age of the recipient. VZV is known to induce the formation of multinuclear cells in neuronal tissue, which has been proposed to be a factor contributing to PHN. This study examines the role of a lysine cluster in the cytoplasmic domain of the VZV fusion protein, gB, in the formation of VZV induced multinuclear cells and in virus replication kinetics and spread. The findings further elucidate how VZV self-regulates multinuclear cell formation and may provide insight into the development of new PHN therapies.

Role for the αV Integrin Subunit in Varicella-Zoster Virus-Mediated Fusion and Infection.

UNLABELLED: Varicella-zoster virus (VZV) is an alphaherpesvirus that causes varicella and herpes zoster. Membrane fusion is essential for VZV entry and the distinctive syncytium formation in VZV-infected skin and neuronal tissue. Herpesvirus fusion is mediated by a complex of glycoproteins gB and gH-gL, which are necessary and sufficient for VZV to induce membrane fusion. However, the cellular requirements of fusion are poorly understood. Integrins have been implicated to facilitate entry of several human herpesviruses, but their role in VZV entry has not yet been explored. To determine the involvement of integrins in VZV fusion, a quantitative cell-cell fusion assay was developed using a VZV-permissive melanoma cell line. The cells constitutively expressed a reporter protein and short hairpin RNAs (shRNAs) to knock down the expression of integrin subunits shown to be expressed in these cells by RNA sequencing. The αV integrin subunit was identified as mediating VZV gB/gH-gL fusion, as its knockdown by shRNAs reduced fusion levels to 60% of that of control cells. A comparable reduction in fusion levels was observed when an anti-αV antibody specific to its extracellular domain was tested in the fusion assay, confirming that the domain was important for VZV fusion. In addition, reduced spread was observed in αV knockdown cells infected with the VZV pOka strain relative to that of the control cells. This was demonstrated by reductions in plaque size, replication kinetics, and virion entry in the αV subunit knockdown cells. Thus, the αV integrin subunit is important for VZV gB/gH-gL fusion and infection. IMPORTANCE: Varicella-zoster virus (VZV) is a highly infectious pathogen that causes chickenpox and shingles. A common complication of shingles is the excruciating condition called postherpetic neuralgia, which has proven difficult to treat. While a vaccine is now available, it is not recommended for immunocompromised individuals and its efficacy decreases with the recipient's age. These limitations highlight the need for new therapies. This study examines the role of integrins in membrane fusion mediated by VZV glycoproteins gB and gH-gL, a required process for VZV infection. This knowledge will further the understanding of VZV entry and provide insight into the development of better therapies.

Varicella-Zoster Virus Glycoproteins: Entry, Replication, and Pathogenesis.

Varicella-zoster virus (VZV), an alphaherpesvirus that causes chicken pox (varicella) and shingles (herpes zoster), is a medically important pathogen that causes considerable morbidity and, on occasion, mortality in immunocompromised patients. Herpes zoster can afflict the elderly with a debilitating condition, postherpetic neuralgia, triggering severe, untreatable pain for months or years. The lipid envelope of VZV, similar to all herpesviruses, contains numerous glycoproteins required for replication and pathogenesis. PURPOSE OF REVIEW: To summarize the current knowledge about VZV glycoproteins and their roles in cell entry, replication and pathogenesis. RECENT FINDINGS: The functions for some VZV glycoproteins are known, such as gB, gH and gL in membrane fusion, cell-cell fusion regulation, and receptor binding properties. However, the molecular mechanisms that trigger or mediate VZV glycoproteins remains poorly understood. SUMMARY: VZV glycoproteins are central to successful replication but their modus operandi during replication and pathogenesis remain elusive requiring further mechanistic based studies.

A site of varicella-zoster virus vulnerability identified by structural studies of neutralizing antibodies bound to the glycoprotein complex gHgL.

Varicella-zoster virus (VZV), of the family Alphaherpesvirinae, causes varicella in children and young adults, potentially leading to herpes zoster later in life on reactivation from latency. The conserved herpesvirus glycoprotein gB and the heterodimer gHgL mediate virion envelope fusion with cell membranes during virus entry. Naturally occurring neutralizing antibodies against herpesviruses target these entry proteins. To determine the molecular basis for VZV neutralization, crystal structures of gHgL were determined in complex with fragments of antigen binding (Fabs) from two human monoclonal antibodies, IgG-94 and IgG-RC, isolated from seropositive subjects. These structures reveal that the antibodies target the same site, composed of residues from both gH and gL, distinct from two other neutralizing epitopes identified by negative-stain electron microscopy and mutational analysis. Inhibition of gB/gHgL-mediated membrane fusion and structural comparisons with herpesvirus homologs suggest that the IgG-RC/94 epitope is in proximity to the site on VZV gHgL that activates gB. Immunization studies proved that the anti-gHgL IgG-RC/94 epitope is a critical target for antibodies that neutralize VZV. Thus, the gHgL/Fab structures delineate a site of herpesvirus vulnerability targeted by natural immunity.

The cytoplasmic domain of varicella-zoster virus glycoprotein H regulates syncytia formation and skin pathogenesis.

The conserved herpesvirus fusion complex consists of glycoproteins gB, gH, and gL which is critical for virion envelope fusion with the cell membrane during entry. For Varicella Zoster Virus (VZV), the complex is necessary for cell-cell fusion and presumed to mediate entry. VZV causes syncytia formation via cell-cell fusion in skin and in sensory ganglia during VZV reactivation, leading to neuronal damage, a potential contributory factor for the debilitating condition of postherpetic neuralgia. The gH cytoplasmic domain (gHcyt) is linked to the regulation of gB/gH-gL-mediated cell fusion as demonstrated by increased cell fusion in vitro by an eight amino acid (aa834-841) truncation of the gHcyt. The gHcyt regulation was identified to be dependent on the physical presence of the domain, and not of specific motifs or biochemical properties as substitution of aa834-841 with V5, cMyc, and hydrophobic or hydrophilic sequences did not affect fusion. The importance of the gHcyt length was corroborated by stepwise deletions of aa834-841 causing incremental increases in cell fusion, independent of gH surface expression and endocytosis. Consistent with the fusion assay, truncating the gHcyt in the viral genome caused exaggerated syncytia formation and significant reduction in viral titers. Importantly, infection of human skin xenografts in SCID mice was severely impaired by the truncation while maintaining the gHcyt length with the V5 substitution preserved typical replication in vitro and in skin. A role for the gHcyt in modulating the functions of the gB cytoplasmic domain (gBcyt) is proposed as the gHcyt truncation substantially enhanced cell fusion in the presence of the gB[Y881F] mutation. The significant reduction in skin infection caused by hyperfusogenic mutations in either the gHcyt or gBcyt demonstrates that both domains are critical for regulating syncytia formation and failure to control cell fusion, rather than enhancing viral spread, is severely detrimental to VZV pathogenesis.

Molecular mechanisms of varicella zoster virus pathogenesis.

Varicella zoster virus (VZV) is the causative agent of varicella (chickenpox) and zoster (shingles). Investigating VZV pathogenesis is challenging as VZV is a human-specific virus and infection does not occur, or is highly restricted, in other species. However, the use of human tissue xenografts in mice with severe combined immunodeficiency (SCID) enables the analysis of VZV infection in differentiated human cells in their typical tissue microenvironment. Xenografts of human skin, dorsal root ganglia or foetal thymus that contains T cells can be infected with mutant viruses or in the presence of inhibitors of viral or cellular functions to assess the molecular mechanisms of VZV-host interactions. In this Review, we discuss how these models have improved our understanding of VZV pathogenesis.

ORF11 protein interacts with the ORF9 essential tegument protein in varicella-zoster virus infection.

The tegument proteins encoded by ORF11 and ORF9 of varicella-zoster virus (VZV) are conserved among all alphaherpesvirus. We previously demonstrated that the ORF9 gene is essential, whereas ORF11 is dispensable in vitro but its deletion severely impairs VZV infection of skin xenografts in the SCID mouse model in vivo. Here we report that ORF11 protein interacts with ORF9 protein in infected cells as well as in the absence of other viral proteins, and we have mapped the ORF11 protein domain involved in their interaction. Although ORF11 is an RNA binding protein, the interaction between ORF11 and ORF9 proteins was not mediated by RNA or DNA bridging. VZV recombinants with mutations preventing ORF11 protein binding to ORF9 protein had no effect on 6-day growth kinetics based on plaque numbers, but plaque sizes were reduced in vitro. However, disruption of the ORF11 and ORF9 protein interaction was associated with failure to replicate in skin xenografts in vivo. Further, we demonstrate that in the absence of their interaction, the ORF9 protein displays an identical cellular localization, accumulating in the trans-Golgi region, whereas the ORF11 protein exhibits aberrant localization, dispersing throughout the cytoplasm. Overall, our observations suggest that while complete tegument assembly may not be necessary for VZV replication in vitro, the interaction between the ORF11 and ORF9 proteins appears to be critical for the proper localization of ORF11 protein to the assembly complex and for production of infectious virus during VZV pathogenesis in skin.

An immunoreceptor tyrosine-based inhibition motif in varicella-zoster virus glycoprotein B regulates cell fusion and skin pathogenesis.

Herpesvirus entry functions of the conserved glycoproteins gB and gH-gL have been delineated, but their role in regulating cell-cell fusion is poorly understood. Varicella-zoster virus (VZV) infection provides a valuable model for investigating cell-cell fusion because of the importance of this process for pathogenesis in human skin and sensory ganglia. The present study identifies a canonical immunoreceptor tyrosine-based inhibition motif (ITIM) in the gB cytoplasmic domain (gBcyt) and demonstrates that the gBcyt is a tyrosine kinase substrate. Orbitrap mass spectrometry confirmed that Y881, central to the ITIM, is phosphorylated. To determine whether the gBcyt ITIM regulates gB/gH-gL-induced cell-cell fusion in vitro, tyrosine residues Y881 and Y920 in the gBcyt were substituted with phenylalanine separately or together. Recombinant viruses with these substitutions were generated to establish their effects on syncytia formation in replication in vitro and in the human skin xenograft model of VZV pathogenesis. The Y881F substitution caused significantly increased cell-cell fusion despite reduced cell-surface gB. Importantly, the Y881F or Y881/920F substitutions in VZV caused aggressive syncytia formation, reducing cell-cell spread. These in vitro effects of aggressive syncytia formation translated to severely impaired skin infection in vivo. In contrast, the Y920F substitution did not affect virus replication in vitro or in vivo. These observations suggest that gB modulates cell-cell fusion via an ITIM-mediated Y881 phosphorylation-dependent mechanism, supporting a unique concept that intracellular signaling through this gBcyt motif regulates VZV syncytia formation and is essential for skin pathogenesis.

Structure-function analysis of varicella-zoster virus glycoprotein H identifies domain-specific roles for fusion and skin tropism.

Enveloped viruses require membrane fusion for cell entry and replication. For herpesviruses, this event is governed by the multiprotein core complex of conserved glycoproteins (g)B and gH/gL. The recent crystal structures of gH/gL from herpes simplex virus 2, pseudorabies virus, and Epstein-Barr virus revealed distinct domains that, surprisingly, do not resemble known viral fusogens. Varicella-zoster virus (VZV) causes chicken pox and shingles. VZV is an α-herpesvirus closely related to herpes simplex virus 2, enabling prediction of the VZV gH structure by homology modeling. We have defined specific roles for each gH domain in VZV replication and pathogenesis using structure-based site-directed mutagenesis of gH. The distal tip of domain (D)I was important for skin tropism, entry, and fusion. DII helices and a conserved disulfide bond were essential for gH structure and VZV replication. An essential (724)CXXC(727) motif was critical for DIII structural stability and membrane fusion. This assignment of domain-dependent mechanisms to VZV gH links elements of the glycoprotein structure to function in herpesvirus replication and virulence.