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

基本信息

批准号：
8472440
负责人：
Ann Arvin
金额：
$ 36.92万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
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/8472440
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 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 transcriptome sequencing varicella-zoster virus glycoprotein B virus pathogenesis

项目摘要

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.

描述（由申请人提供）：水痘带状疱疹病毒（VZV）是一种具有重要医学意义的人类 ¿ - 引起水痘（水痘）并在潜伏感染的感觉神经节重新激活后导致带状疱疹（带状疱疹）的情况可能很严重，并且在免疫功能低下的患者中会危及生命。人类宿主并克服了完全分化的宿主细胞之间融合形成多核多核细胞的通常限制，这是 VZV 发病机制的一个标志。已知糖蛋白 B (gB) 与 gH/gL 异二聚体对于疱疹病毒进入过程中病毒体包膜与靶细胞膜的融合至关重要。我们的新概念是 VZV 通过 gB 依赖性细胞内信号传导介导细胞与细胞的融合。这是基于我们的新证据，即阻止 gB 细胞质结构域 (gBcyt) 的酪氨酸磷酸化会导致细胞-细胞融合和合胞体异常。我们的应用将研究 gBcyt 如何通过细胞内信号通路调节细胞-细胞融合机制，以在体外产生特征性合胞体，以及在体内由 VZV 感染皮肤和神经节引起的表皮细胞和神经元卫星细胞的融合。 1 我们将通过应用高通量全转录组测序技术来确定 VZV 如何修改细胞调控以促进促进细胞间融合和合胞体形成的基因转录， RNA-seq，针对我们新的融合测定和我们的病毒突变体，它们在影响细胞-细胞融合的 gBcyt 残基中携带突变。为了量化酪氨酸磷酸化的影响，将实时测量合胞体形成的时空演化。为了确定 RNA-seq 确定的基因在细胞融合中的作用，我们将进行基因扰动实验来评估它们在背景下的生物学意义。目标 2 将通过确定细胞或病毒蛋白，gBcyt 如何通过酪氨酸和/r 赖氨酸残基的翻译后修饰来调节细胞融合中的细胞内信号传导事件。将进行酪氨酸磷酸化和非磷酸化形式的 gBcyt 结构域的赖氨酸诱变研究，以评估乙酰化和非磷酸化的影响。最后，目标 3 将确定 gBcyt 是否调节多核细胞形成以优化 VZV 对皮肤和 DRG 的感染，并将使用我们的突变病毒与野生型 VZV 在人类皮肤和神经组织中的复制能力进行比较。我们将使用新型 shRNA 确定细胞与细胞融合所需的新鉴定基因的作用。鉴于多核细胞形成对于发病机制的重要性，破译 VZV 如何调节这一过程有可能产生疫苗病毒减毒和抗病毒药物设计的新策略，以减轻脆弱人群的负担。