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

水痘带状疱疹病毒：细胞融合依赖性发病机制的分子控制

• 批准号: 8401103
• 负责人: Ann Arvin
• 金额: $ 39.27万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8401103
• 关键词: Acetylation; Address; Affect; Antiviral Agents; Arginine; Biological; Biological Assay; Cell fusion; Cell membrane; Cells; Characteristics; Chickenpox; Complex; Confocal Microscopy; Cytoplasmic Tail; Disease; Drug Design; Event; Evolution; Exhibits; Ganglia; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic Transcription; Giant Cells; Glycoproteins; Goals; Herpes zoster disease; Herpesviridae; Herpesvirus Type 3; Human; Immunocompromised Host; In Vitro; Infection; Kinetics; Life; Luciferases; Lysine; Mass Spectrum Analysis; Measures; Mediating; Modification; Molecular; Mutagenesis; Mutation; Neurons; Ontology; Pain; Pathogenesis; Pathway interactions; Pharmaceutical Preparations; Post-Translational Protein Processing; Precipitation; Process; Production; Protein Tyrosine Kinase; Proteins; RNA; RNA Sequences; Recruitment Activity; Reporter; Research; Role; SCID Mice; Sensory Ganglia; Signal Pathway; Signal Transduction; Skin; Spinal Ganglia; Surface; System; T-Lymphocyte; Technology; Testing; Time; Tissues; Transfection; Tropism; Tyrosine; Tyrosine Phosphorylation; Ubiquitination; Vaccines; Viral Proteins; Virion; Virulence; Virus; Virus Diseases; Virus Replication; Vulnerable Populations; Xenograft procedure; attenuation; base; cell growth regulation; genetic regulatory protein; human tissue; in vivo; interest; knock-down; mouse model; mutant; novel; prevent; research study; satellite cell; small hairpin RNA; spatiotemporal; varicella-zoster virus glycoprotein B; virus pathogenesis

DESCRIPTION (provided by applicant): Varicella-zoster virus (VZV) is a medically important human ¿-herpesvirus that causes varicella (chickenpox) and leads to zoster (shingles) upon reactivation from latently infected sensory ganglia. Varicella can be serious and is life-threatening in immunocompromised patients. VZV exhibits tropism for T cells, skin and neurons during infection of the human host and overcomes the usual constraint against fusion between fully differentiated host cells to form multinucleated polykaryocytes, a hallmark of VZV pathogenesis. Glycoprotein B (gB) along with the gH/gL heterodimer is known to be critical for fusion of the virion envelope with the target cell membrane during herpesvirus entry. Our novel concept is that VZV mediates cell-cell fusion through a gB-dependent intracellular signaling function. This is based on our new evidence that preventing tyrosine phosphorylation of the gB cytoplasmic domain (gBcyt) leads to anomalies in cell-cell fusion and syncytia formation in vitro. Our application will investigate how the gBcyt modulates cell- cell fusion mechanisms via intracellular signaling pathways to produce the characteristic syncytia in vitro and fusion of epidermal cells and neuron-satellite cells caused by VZV infection of skin and ganglia in vivo. In Aim 1 we will determine how VZV modifies cellular regulation to favor transcription of genes that facilitate cell-cell fusion and syncytia formation by applying the high-throughput whole-transcriptome sequencing technology, RNA-seq, to our new fusion assay and our virus mutants, which carry mutations in the gBcyt residues that affect cell-cell fusion. To quantify the effects o tyrosine phosphorylation, the spatiotemporal evolution of syncytia formation will be measured in real-time for VZV and the gBcyt mutants. To determine the role of genes in cell fusion, as identified by RNA-seq, we will perform gene perturbation experiments to assess their biological significance in the context of VZV replication. Aim 2 will determine how the gBcyt regulates intracellular signaling events in cell fusion via post-translational modifications of tyrosine and/r lysine residues by cellular or viral proteins. Mass spectrometry will be used to identify cellular and viral proteins that interact with the gBcyt domain in its tyrosine-phosphorylated and non-phosphorylated forms. Lysine mutagenesis studies will be performed to assess the effects of acetylation and ubiquitination posttranslational modifications on VZV fusion and virulence. Finally, Aim 3 will establish whether the gBcyt modulates polykaryocyte formation to optimize VZV infection of skin and DRG. Our mutant viruses will be compared to wild type VZV for replication competencies in human skin and neuronal tissue using novel reporter viruses. We will establish the role of newly identified genes required for cell-cell fusion using a novel shRNA carrying virus. Given the significance of polykaryocyte formation for pathogenesis, deciphering how VZV regulates this process has the potential to yield new strategies for vaccine virus attenuation and antiviral drug design to ease the burden on vulnerable populations. PUBLIC HEALTH RELEVANCE: Varicella-zoster virus (VZV) is a medically important human herpesvirus that causes chicken pox and shingles. These diseases can be serious, painful and life-threatening in immunocompromised patients. Drug treatments to prevent pain are ineffective and current vaccines are not recommended for the immunocompromised. A hall mark of VZV disease is the fusion of cells in human skin and neuronal tissue. We have strong evidence that molecular events inside cells infected with VZV are controlled by a protein, glycoprotein B, found on the surface of the virus. The goal of our research will be to provide a fundamental understanding of how VZV glycoprotein B triggers cells in human tissues to fuse and cause disease. These studies have great potential to provide common themes with other human herpesvirus viruses that will be directly relevant to developing safer vaccines and more effective drug treatments.

描述（通过应用提供）：水痘带状疱疹病毒（VZV）是一种重要的人类„ - hepesvirus，导致水痘（水痘），并在受到潜在感染的感觉神经节的重新激活后导致带状疱疹（带状疱疹）。在免疫功能低下的患者中，水痘可能是严重的，并且正在威胁生命。 VZV在人类宿主感染过程中表现出对T细胞，皮肤和神经元的向流，并克服了完全分化的宿主细胞融合的通常约束，形成了多核核细胞，这是VZV发病机理的标志。已知糖蛋白B（GB）以及GH/GL异二聚体对于在疱疹病毒进入期间与靶细胞膜融合至关重要。我们的新概念是，VZV通过依赖GB的细胞内信号传导函数介导细胞细胞融合。这是基于我们的新证据，即防止GB细胞质结构域（GBCYT）的酪氨酸磷酸化导致细胞细胞融合和合成症的体外形成异常。我们的应用将研究GBCYT如何通过细胞内信号传导途径调节细胞细胞融合机制，以在体外产生特征性的突触以及表皮细胞和神经元 - 卫星细胞的融合，并由体内的皮肤和神经神经节感染引起。在AIM 1中，我们将通过将高通量的全移交组测序技术RNA-Seq应用于我们的新融合分析和我们的病毒突变体中，从而促进细胞调节的基因转录，从而促进促进细胞融合和突触形成的基因转录，并​​在GBCYT中携带突变，从而影响了Cell-Cell-Cell-Cell-Cell-Cell-Cell-Cell-Cell fiusion。为了量化O酪氨酸磷酸化的影响，将为VZV和GBCYT突变体实时测量突触形成的空间时间演变。为了确定基因在RNA-Seq确定的细胞融合中的作用，我们将执行基因扰动实验，以评估其在VZV复制背景下的生物学意义。 AIM 2将确定GBCYT如何通过细胞或病毒蛋白通过酪氨酸和/或赖氨酸残留的翻译后修饰来调节细胞融合中的细胞内信号传导事件。质谱法将用于鉴定与酪氨酸磷酸化和非磷酸化形式中与GBCYT结构域相互作用的细胞和病毒蛋白。将进行赖氨酸诱变研究，以评估翻译后修饰对VZV融合和病毒的乙酰化和泛素化的影响。最后，AIM 3将确定GBCYT是否调节多氯辛形成以优化皮肤和DRG的VZV感染。我们的突变病毒将与野生型VZV进行比较，以使用新的记者病毒进行人体皮肤和神经元组织的复制能力。我们将使用新的shRNA确定细胞融合所需的新鉴定的基因的作用 携带病毒。鉴于多氯藻形成对发病机理的重要性，解密的VZV如何调节此过程有可能产生疫苗病毒衰减和抗病毒药物设计的新策略，从而缓解易受伤害人群的伯嫩。 公共卫生相关性：水痘带状疱疹病毒（VZV）是一种医学上重要的人类疱疹病毒，会导致鸡肉痘和带状疱疹。这些疾病在免疫功能低下的患者中可能严重，痛苦和威胁生命。防止疼痛的药物治疗无效，不建议进行免疫功能低下的疫苗。 VZV疾病的大厅标记是人皮肤和神经元组织中细胞的融合。我们有强有力的证据表明，感染VZV的细胞内部事件由在病毒表面发现的蛋白质，糖蛋白B控制。我们研究的目的是提供对VZV糖蛋白B如何触发人体组织中的细胞融合和引起疾病的基本了解。这些研究具有与其他人类疱疹病毒病毒一起提供常见主题的巨大潜力，这些病毒将与开发安全的疫苗和更有效的药物治疗直接相关。