Publications by authors named "Ann M Arvin"

132 Publications

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

PLoS Pathog 2021 Jan 7;17(1):e1008961. Epub 2021 Jan 7.

Department of Pediatrics, Stanford University School of Medicine, Stanford, California, United States of America.

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.
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http://dx.doi.org/10.1371/journal.ppat.1008961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817050PMC
January 2021

The latency-associated transcript locus of herpes simplex virus 1 is a virulence determinant in human skin.

PLoS Pathog 2020 12 28;16(12):e1009166. Epub 2020 Dec 28.

Departments of Pediatrics and Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

Herpes simplex virus 1 (HSV-1) infects skin and mucosal epithelial cells and then travels along axons to establish latency in the neurones of sensory ganglia. Although viral gene expression is restricted during latency, the latency-associated transcript (LAT) locus encodes many RNAs, including a 2 kb intron known as the hallmark of HSV-1 latency. Here, we studied HSV-1 infection and the role of the LAT locus in human skin xenografts in vivo and in cultured explants. We sequenced the genomes of our stock of HSV-1 strain 17syn+ and seven derived viruses and found nonsynonymous mutations in many viral proteins that had no impact on skin infection. In contrast, deletions in the LAT locus severely impaired HSV-1 replication and lesion formation in skin. However, skin replication was not affected by impaired intron splicing. Moreover, although the LAT locus has been implicated in regulating gene expression in neurones, we observed only small changes in transcript levels that were unrelated to the growth defect in skin, suggesting that its functions in skin may be different from those in neurones. Thus, although the LAT locus was previously thought to be dispensable for lytic infection, we show that it is a determinant of HSV-1 virulence during lytic infection of human skin.
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http://dx.doi.org/10.1371/journal.ppat.1009166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794027PMC
December 2020

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

Biochem Soc Trans 2020 Dec;48(6):2415-2435

Departments of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, U.S.A.

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.
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http://dx.doi.org/10.1042/BST20190511DOI Listing
December 2020

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

PLoS Pathog 2020 11 20;16(11):e1009022. Epub 2020 Nov 20.

Department of Pediatrics, Stanford University School of Medicine, Stanford, California, United States of America.

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.
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http://dx.doi.org/10.1371/journal.ppat.1009022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717522PMC
November 2020

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

Nat Commun 2020 08 28;11(1):4398. Epub 2020 Aug 28.

Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, 94305, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-18385-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455691PMC
August 2020

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

Nat Commun 2020 08 18;11(1):4141. Epub 2020 Aug 18.

Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, 94305, USA.

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.
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http://dx.doi.org/10.1038/s41467-020-17911-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435202PMC
August 2020

The Use of Single Cell Mass Cytometry to Define the Molecular Mechanisms of Varicella-Zoster Virus Lymphotropism.

Front Microbiol 2020 26;11:1224. Epub 2020 Jun 26.

Department of Pediatrics, Stanford University, Stanford, CA, United States.

Unraveling the heterogeneity in biological systems provides the key to understanding of the fundamental dynamics that regulate host pathogen relationships at the single cell level. While most studies have determined virus-host cell interactions using cultured cells in bulk, recent advances in deep protein profiling from single cells enable the understanding of the dynamic response equilibrium of single cells even within the same cell types. Mass cytometry allows the simultaneous detection of multiple proteins in single cells, which helps to evaluate alterations in multiple signaling networks that work in tandem in deciding the response of a cell to the presence of a pathogen or other stimulus. In applying this technique to studying varicella zoster virus (VZV), it was possible to better understand the molecular basis for lymphotropism of the virus and how virus-induced effects on T cells promoted skin tropism. While the ability of VZV to manifest itself in the skin is well established, how the virus is transported to the skin and causes the characteristic VZV skin lesions was not well elucidated. Through mass cytometry analysis of VZV-infected tonsil T cells, we were able to observe that VZV unleashes a "remodeling" program in the infected T cells that not only makes these T cells more skin tropic but also at the same time induces changes that make these T cells unlikely to respond to immune stimulation during the journey to the skin.
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http://dx.doi.org/10.3389/fmicb.2020.01224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7333520PMC
June 2020

Immunogenicity of Inactivated Varicella Zoster Vaccine in Autologous Hematopoietic Stem Cell Transplant Recipients and Patients With Solid or Hematologic Cancer.

Open Forum Infect Dis 2020 Jul 2;7(7):ofaa172. Epub 2020 Jun 2.

Merck & Co., Inc., Kenilworth, New Jersey, USA.

Background: In phase 3 trials, inactivated varicella zoster virus (VZV) vaccine (ZV) was well tolerated and efficacious against herpes zoster (HZ) in autologous hematopoietic stem cell transplant (auto-HSCT) recipients and patients with solid tumor malignancies receiving chemotherapy (STMc) but did not reduce HZ incidence in patients with hematologic malignancies (HMs). Here, we describe ZV immunogenicity from these studies.

Methods: Patients were randomized to ZV or placebo (4 doses). Immunogenicity was assessed by glycoprotein enzyme-linked immunosorbent assay (gpELISA) and VZV interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) assay in patients receiving all 4 doses without developing HZ at the time of blood sampling.

Results: Estimated geometric mean fold rise ratios (ZV/placebo) by gpELISA and IFN-y ELISPOT ~28 days post-dose 4 were 2.02 (95% confidence interval [CI], 1.53-2.67) and 5.41 (95% CI, 3.60-8.12) in auto-HSCT recipients; 1.88 (95% CI, 1.79-1.98) and 2.10 (95% CI, 1.69-2.62) in patients with STMc; and not assessed and 2.35 (95% CI, 1.81-3.05) in patients with HM.

Conclusions: ZV immunogenicity was directionally consistent with clinical efficacy in auto-HSCT recipients and patients with STMc even though HZ protection and VZV immunity were not statistically correlated. Despite a lack of clinical efficacy in patients with HM, ZV immunogenicity was observed in this population. Immunological results did not predict vaccine efficacy in these 3 populations.

Clinical Trial Registration: NCT01229267, NCT01254630.
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http://dx.doi.org/10.1093/ofid/ofaa172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336559PMC
July 2020

A perspective on potential antibody-dependent enhancement of SARS-CoV-2.

Nature 2020 08 13;584(7821):353-363. Epub 2020 Jul 13.

Vir Biotechnology, San Francisco, CA, USA.

Antibody-dependent enhancement (ADE) of disease is a general concern for the development of vaccines and antibody therapies because the mechanisms that underlie antibody protection against any virus have a theoretical potential to amplify the infection or trigger harmful immunopathology. This possibility requires careful consideration at this critical point in the pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we review observations relevant to the risks of ADE of disease, and their potential implications for SARS-CoV-2 infection. At present, there are no known clinical findings, immunological assays or biomarkers that can differentiate any severe viral infection from immune-enhanced disease, whether by measuring antibodies, T cells or intrinsic host responses. In vitro systems and animal models do not predict the risk of ADE of disease, in part because protective and potentially detrimental antibody-mediated mechanisms are the same and designing animal models depends on understanding how antiviral host responses may become harmful in humans. The implications of our lack of knowledge are twofold. First, comprehensive studies are urgently needed to define clinical correlates of protective immunity against SARS-CoV-2. Second, because ADE of disease cannot be reliably predicted after either vaccination or treatment with antibodies-regardless of what virus is the causative agent-it will be essential to depend on careful analysis of safety in humans as immune interventions for COVID-19 move forward.
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http://dx.doi.org/10.1038/s41586-020-2538-8DOI Listing
August 2020

Deletion of Herpes Simplex Virus 1 MicroRNAs miR-H1 and miR-H6 Impairs Reactivation.

J Virol 2020 07 16;94(15). Epub 2020 Jul 16.

Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, Florida, USA

During all stages of infection, herpes simplex virus 1 (HSV-1) expresses viral microRNAs (miRNAs). There are at least 20 confirmed HSV-1 miRNAs, yet the roles of individual miRNAs in the context of viral infection remain largely uncharacterized. We constructed a recombinant virus lacking the sequences for miR-H1-5p and miR-H6-3p (17dmiR-H1/H6). The seed sequences for these miRNAs are antisense to each other and are transcribed from divergent noncoding RNAs in the latency-associated transcript (LAT) promoter region. Comparing phenotypes exhibited by the recombinant virus lacking these miRNAs to the wild type (17+), we found that during acute infection in cell culture, 17dmiR-H1/H6 exhibited a modest increase in viral yields. Analysis of pathogenesis in the mouse following footpad infection revealed a slight increase in virulence for 17dmiR-H1/H6 but no significant difference in the establishment or maintenance of latency. Strikingly, explant of latently infected dorsal root ganglia revealed a decreased and delayed reactivation phenotype. Further, 17dmiR-H1/H6 was severely impaired in epinephrine-induced reactivation in the rabbit ocular model. Finally, we demonstrated that deletion of miR-H1/H6 increased the accumulation of the LAT as well as several of the LAT region miRNAs. These results suggest that miR-H1/H6 plays an important role in facilitating efficient reactivation from latency. While HSV antivirals reduce the severity and duration of clinical disease in some individuals, there is no vaccine or cure. Therefore, understanding the mechanisms regulating latency and reactivation as a potential to elucidate targets for better therapeutics is important. There are at least 20 confirmed HSV-1 miRNAs, yet the roles of individual miRNAs in the context of viral infection remain largely uncharacterized. The present study focuses on two of the miRNAs (miR-H1/H6) that are encoded within the latency-associated transcript (LAT) region, a portion of the genome that has been associated with efficient reactivation. Here, we demonstrate that the deletion of the seed sequences of these miRNAs results in a severe reduction in reactivation of HSV-1 in the mouse and rabbit models. These results suggest a linkage between these miRNAs and reactivation.
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http://dx.doi.org/10.1128/JVI.00639-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375377PMC
July 2020

Will Measles Virus or Humanity Win the International "Fitness" Challenge?

Authors:
Ann M Arvin

Annu Rev Virol 2019 09;6(1):iii-vii

Division of Infectious Diseases, Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94304, USA; email:

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http://dx.doi.org/10.1146/annurev-vi-06-072619-100011DOI Listing
September 2019

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

PLoS One 2018 25;13(10):e0206365. Epub 2018 Oct 25.

Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0206365PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6201953PMC
April 2019

Distinctive Roles for Type I and Type II Interferons and Interferon Regulatory Factors in the Host Cell Defense against Varicella-Zoster Virus.

J Virol 2018 11 12;92(21). Epub 2018 Oct 12.

Department of Pediatrics, Stanford University, Stanford, California, USA

Both type I and type II interferons (IFNs) have been implicated in the host defense against varicella-zoster virus (VZV), a common human herpesvirus that causes varicella and zoster. The purpose of this study was to compare their contributions to the control of VZV replication, to identify the signaling pathways that are critical for mediating their antiviral activity, and to define the mechanisms by which the virus counteracts their effects. Gamma interferon (IFN-γ) was much more potent than IFN-α in blocking VZV infection, which was associated with a differential induction of the interferon regulatory factor (IRF) proteins IRF1 and IRF9, respectively. These observations account for the clinical experience that while the formation of VZV skin lesions is initially controlled by local immunity, adaptive virus-specific T cell responses are required to prevent life-threatening VZV infections. While both type I and type II IFNs are involved in the control of herpesvirus infections in the human host, to our knowledge, their relative contributions to the restriction of viral replication and spread have not been assessed. We report that IFN-γ has more potent activity than IFN-α against VZV. Findings from this comparative analysis show that the IFN-α-IRF9 axis functions as a first line of defense to delay the onset of viral replication and spread, whereas the IFN-γ-IRF1 axis has the capacity to block the infectious process. Our findings underscore the importance of IRFs in IFN regulation of herpesvirus infection and account for the clinical experience of the initial control of VZV skin infection attributable to IFN-α production, together with the requirement for induction of adaptive IFN-γ-producing VZV-specific T cells to resolve the infection.
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http://dx.doi.org/10.1128/JVI.01151-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189497PMC
November 2018

Mass Cytometric Analysis of HIV Entry, Replication, and Remodeling in Tissue CD4+ T Cells.

Cell Rep 2017 07;20(4):984-998

Gladstone Institute of Virology and Immunology, San Francisco, CA 94158, USA; Department of Urology, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

To characterize susceptibility to HIV infection, we phenotyped infected tonsillar T cells by single-cell mass cytometry and created comprehensive maps to identify which subsets of CD4+ T cells support HIV fusion and productive infection. By comparing HIV-fused and HIV-infected cells through dimensionality reduction, clustering, and statistical approaches to account for viral perturbations, we identified a subset of memory CD4+ T cells that support HIV entry but not viral gene expression. These cells express high levels of CD127, the IL-7 receptor, and are believed to be long-lived lymphocytes. In HIV-infected patients, CD127-expressing cells preferentially localize to extrafollicular lymphoid regions with limited viral replication. Thus, CyTOF-based phenotyping, combined with analytical approaches to distinguish between selective infection and receptor modulation by viruses, can be used as a discovery tool.
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http://dx.doi.org/10.1016/j.celrep.2017.06.087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5560086PMC
July 2017

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

Curr Clin Microbiol Rep 2016 Dec 9;3(4):204-215. Epub 2016 Sep 9.

Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, California, 94305-5208.

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.
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http://dx.doi.org/10.1007/s40588-016-0044-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373811PMC
December 2016

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

J Virol 2017 Jan 16;91(1). Epub 2016 Dec 16.

Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, California, USA.

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 log), 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-log 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.
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http://dx.doi.org/10.1128/JVI.01707-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5165221PMC
January 2017

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

J Virol 2017 Jan 16;91(1). Epub 2016 Dec 16.

Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.

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.
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http://dx.doi.org/10.1128/JVI.01613-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5165202PMC
January 2017

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

J Virol 2016 08 27;90(16):7567-78. Epub 2016 Jul 27.

Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, California, USAOregon Health & Science University.

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.
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http://dx.doi.org/10.1128/JVI.00792-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4984616PMC
August 2016

Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication.

Virology 2016 May 23;492:82-91. Epub 2016 Feb 23.

Departments of Pediatrics and Microbiology & Immunology, Stan ford University School of Medicine, Stanford, CA, United States.

VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This study mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication.
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http://dx.doi.org/10.1016/j.virol.2016.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4826839PMC
May 2016

Longitudinal Kinetics of Cytomegalovirus-Specific T-Cell Immunity and Viral Replication in Infants With Congenital Cytomegalovirus Infection.

J Pediatric Infect Dis Soc 2016 Mar 11;5(1):14-20. Epub 2014 Sep 11.

Department of Pediatrics.

Background: Congenital cytomegalovirus (CMV) is reported to affect up to 1% of all live births in the United States. T-cell immunity may be important for controlling CMV replication in congenital CMV-infected infants. We describe the natural history of CMV-specific T-cell evolution and CMV replication in infants with congenital CMV infection.

Methods: Cytomegalovirus viral load, CMV urine culture, and CMV-specific CD4 and CD8 T-cell responses were assessed in a prospective longitudinal cohort of 51 infants with congenital CMV infection who were observed from birth to 3 years of age.

Results: We found a kinetic pattern of decreasing urinary CMV replication and increasing CMV-specific CD4 and CD8 T-cell responses during the first 3 years of life. We also found higher CMV-specific CD8 T-cell responses were associated with subsequent reduction of urine CMV viral load.

Conclusion: For infants with congenital CMV infection, our data suggest an age-related maturation of both CMV-specific CD4 and CD8 T-cell immunity that is associated with an age-related decline in urinary CMV replication.
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http://dx.doi.org/10.1093/jpids/piu089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4765489PMC
March 2016

Dissecting the Molecular Mechanisms of the Tropism of Varicella-Zoster Virus for Human T Cells.

J Virol 2016 Jan 20;90(7):3284-7. Epub 2016 Jan 20.

Departments of Pediatrics and Microbiology & Immunology, Stanford University, Stanford, California, USA

Studies of varicella-zoster virus (VZV) tropism for T cells support their role in viral transport to the skin during primary infection. Multiparametric single-cell mass cytometry demonstrates that, instead of preferentially infecting skin-homing T cells, VZV alters cell signaling and remodels surface proteins to enhance T cell skin trafficking. Viral proteins dispensable in skin, such as that encoded by open reading frame 66, are necessary in T cells. Interference with VZV T cell tropism may offer novel strategies for drug and vaccine design.
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http://dx.doi.org/10.1128/JVI.03375-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4794656PMC
January 2016

Single cell mass cytometry reveals remodeling of human T cell phenotypes by varicella zoster virus.

Methods 2015 Nov 23;90:85-94. Epub 2015 Jul 23.

Departments of Pediatrics, Stanford University, Stanford, CA 94025, USA; Departments of Microbiology & Immunology, Stanford University, Stanford, CA 94025, USA. Electronic address:

The recent application of mass cytometry (CyTOF) to biology provides a 'systems' approach to monitor concurrent changes in multiple host cell factors at the single cell level. We used CyTOF to evaluate T cells infected with varicella zoster virus (VZV) infection, documenting virus-mediated phenotypic and functional changes caused by this T cell tropic human herpesvirus. Here we summarize our findings using two complementary panels of antibodies against surface and intracellular signaling proteins to elucidate the consequences of VZV-mediated perturbations on the surface and in signaling networks of infected T cells. CyTOF data was analyzed by several statistical, analytical and visualization tools including hierarchical clustering, orthogonal scaling, SPADE, viSNE, and SLIDE. Data from the mass cytometry studies demonstrated that VZV infection led to 'remodeling' of the surface architecture of T cells, promoting skin trafficking phenotypes and associated with concomitant activation of T-cell receptor and PI3-kinase pathways. This method offers a novel approach for understanding viral interactions with differentiated host cells important for pathogenesis.
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http://dx.doi.org/10.1016/j.ymeth.2015.07.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655147PMC
November 2015

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

Proc Natl Acad Sci U S A 2015 May 27;112(19):6056-61. Epub 2015 Apr 27.

GlaxoSmithKline (GSK) Vaccines, Cambridge, MA 02139;

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.
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http://dx.doi.org/10.1073/pnas.1501176112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4434712PMC
May 2015

Varicella-zoster virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription.

Virology 2015 Jul 17;481:179-86. Epub 2015 Mar 17.

Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, CA, United States.

The VZV genome has two origins of DNA replication (oriS), each of which consists of an AT-rich sequence and three origin binding protein (OBP) sites called Box A, C and B. In these experiments, the mutation in the core sequence CGC of the Box A and C not only inhibited DNA replication but also inhibited both ORF62 and ORF63 expression in reporter gene assays. In contrast the Box B mutation did not influence DNA replication or flanking gene transcription. These results suggest that efficient DNA replication enhances ORF62 and ORF63 transcription. Recombinant viruses carrying these mutations in both sites and one with a deletion of the whole oriS were constructed. Surprisingly, the recombinant virus lacking both copies of oriS retained the capacity to replicate in melanoma and HELF cells suggesting that VZV has another origin of DNA replication.
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http://dx.doi.org/10.1016/j.virol.2015.02.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4437856PMC
July 2015

Autophagic flux without a block differentiates varicella-zoster virus infection from herpes simplex virus infection.

Proc Natl Acad Sci U S A 2015 Jan 22;112(1):256-61. Epub 2014 Dec 22.

Virology Laboratory, Department of Pediatrics, University of Iowa Children's Hospital, Iowa City, IA 52242; and

Autophagy is a process by which misfolded and damaged proteins are sequestered into autophagosomes, before degradation in and recycling from lysosomes. We have extensively studied the role of autophagy in varicella-zoster virus (VZV) infection, and have observed that vesicular cells are filled with >100 autophagosomes that are easily detectable after immunolabeling for the LC3 protein. To confirm our hypothesis that increased autophagosome formation was not secondary to a block, we examined all conditions of VZV infection as well as carrying out two assessments of autophagic flux. We first investigated autophagy in human skin xenografts in the severe combined immunodeficiency (SCID) mouse model of VZV pathogenesis, and observed that autophagosomes were abundant in infected human skin tissues. We next investigated autophagy following infection with sonically prepared cell-free virus in cultured cells. Under these conditions, autophagy was detected in a majority of infected cells, but was much less than that seen after an infected-cell inoculum. In other words, inoculation with lower-titered cell-free virus did not reflect the level of stress to the VZV-infected cell that was seen after inoculation of human skin in the SCID mouse model or monolayers with higher-titered infected cells. Finally, we investigated VZV-induced autophagic flux by two different methods (radiolabeling proteins and a dual-colored LC3 plasmid); both showed no evidence of a block in autophagy. Overall, therefore, autophagy within a VZV-infected cell was remarkably different from autophagy within an HSV-infected cell, whose genome contains two modifiers of autophagy, ICP34.5 and US11, not present in VZV.
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http://dx.doi.org/10.1073/pnas.1417878112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4291665PMC
January 2015

Varicella-zoster virus infections in patients treated with fingolimod: risk assessment and consensus recommendations for management.

JAMA Neurol 2015 Jan;72(1):31-9

Novartis Pharma AG, Basel, Switzerland.

Importance: Varicella-zoster virus (VZV) infections increasingly are reported in patients with multiple sclerosis (MS) and constitute an area of significant concern, especially with the advent of more disease-modifying treatments in MS that affect T-cell-mediated immunity.

Objective: To assess the incidence, risk factors, and clinical characteristics of VZV infections in fingolimod-treated patients and provide recommendations for prevention and management.

Design, Setting, And Participants: Rates of VZV infections in fingolimod clinical trials are based on pooled data from the completed controlled phases 2 and 3 studies (3916 participants) and ongoing uncontrolled extension phases (3553 participants). Male and female patients aged 18 through 55 years (18-60 years for the phase 2 studies) and diagnosed as having relapsing-remitting MS were eligible to participate in these studies. In the postmarketing setting, reporting rates since 2010 were evaluated.

Interventions: In clinical trials, patients received fingolimod at a dosage of 0.5 or 1.25 mg/d, interferon beta-1a, or placebo. In the postmarketing setting, all patients received fingolimod, 0.5 mg/d (total exposure of 54,000 patient-years at the time of analysis).

Main Outcomes And Measures: Calculation of the incidence rate of VZV infection per 1000 patient-years was based on the reporting of adverse events in the trials and the postmarketing setting.

Results: Overall, in clinical trials, VZV rates of infection were low but higher with fingolimod compared with placebo (11 vs 6 per 1000 patient-years). A similar rate was confirmed in the ongoing extension studies. Rates reported in the postmarketing settings were comparable (7 per 1000 patient-years) and remained stable over time. Disproportionality in reporting herpes zoster infection was higher for patients receiving fingolimod compared with those receiving other disease-modifying treatments (empirical Bayes geometric mean, 2.57 [90% CI, 2.26-2.91]); the proportion of serious herpes zoster infections was not higher than the proportion for other treatments (empirical Bayes geometric mean, 1.88 [90% CI, 0.87-3.70]). Corticosteroid treatment for relapses might be a risk factor for VZV reactivation.

Conclusions And Relevance: Rates of VZV infections in clinical trials were low with fingolimod, 0.5 mg/d, but higher than in placebo recipients. Rates reported in the postmarketing setting are comparable. We found no sign of risk accumulation with longer exposure. Serious or complicated cases of herpes zoster were uncommon. We recommend establishing the patient's VZV immune status before initiating fingolimod therapy and immunization for patients susceptible to primary VZV infection. Routine antiviral prophylaxis is not needed, but using concomitant pulsed corticosteroid therapy beyond 3 to 5 days requires an individual risk-benefit assessment. Vigilance to identify early VZV symptoms is important to allow timely antiviral treatment.
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http://dx.doi.org/10.1001/jamaneurol.2014.3065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391035PMC
January 2015

Single-cell mass cytometry analysis of human tonsil T cell remodeling by varicella zoster virus.

Cell Rep 2014 Jul 17;8(2):633-45. Epub 2014 Jul 17.

Department of Pediatrics, Stanford University, Stanford, CA 94025, USA; Department of Microbiology and Immunology, Stanford University, Stanford, CA 94025, USA. Electronic address:

Although pathogens must infect differentiated host cells that exhibit substantial diversity, documenting the consequences of infection against this heterogeneity is challenging. Single-cell mass cytometry permits deep profiling based on combinatorial expression of surface and intracellular proteins. We used this method to investigate varicella-zoster virus (VZV) infection of tonsil T cells, which mediate viral transport to skin. Our results indicate that VZV induces a continuum of changes regardless of basal phenotypic and functional T cell characteristics. Contrary to the premise that VZV selectively infects T cells with skin trafficking profiles, VZV infection altered T cell surface proteins to enhance or induce these properties. Zap70 and Akt signaling pathways that trigger such surface changes were activated in VZV-infected naive and memory cells by a T cell receptor (TCR)-independent process. Single-cell mass cytometry is likely to be broadly relevant for demonstrating how intracellular pathogens modulate differentiated cells to support pathogenesis in the natural host.
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http://dx.doi.org/10.1016/j.celrep.2014.06.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4127309PMC
July 2014

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

PLoS Pathog 2014 May 29;10(5):e1004173. Epub 2014 May 29.

Departments of Pediatrics and Microbiology & Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

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.
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http://dx.doi.org/10.1371/journal.ppat.1004173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038623PMC
May 2014

Molecular mechanisms of varicella zoster virus pathogenesis.

Nat Rev Microbiol 2014 Mar 10;12(3):197-210. Epub 2014 Feb 10.

Departments of Pediatrics and of Microbiology & Immunology, Stanford University School of Medicine, Stanford, California 94305, USA.

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.
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http://dx.doi.org/10.1038/nrmicro3215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4066823PMC
March 2014

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

J Virol 2013 May 20;87(9):5106-17. Epub 2013 Feb 20.

Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA.

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.
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http://dx.doi.org/10.1128/JVI.00102-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3624291PMC
May 2013