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

描述（由申请方提供）：水痘-带状疱疹病毒（VZV）是一种医学上重要的人类疱疹病毒，可引起水痘（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）。水痘可能是严重的，是危及生命的免疫功能低下的患者。VZV在感染人类宿主期间表现出对T细胞、皮肤和神经元的嗜性，并且克服了对完全分化的宿主细胞之间融合以形成多核多核细胞（VZV发病机制的标志）的通常限制。已知糖蛋白B（gB）沿着gH/gL异二聚体对于疱疹病毒进入期间病毒体包膜与靶细胞膜的融合是关键的。我们的新概念是，VZV介导的细胞-细胞融合，通过一个gB依赖的细胞内信号传导功能。这是基于我们的新证据，即阻止gB胞质结构域（gBcyt）的酪氨酸磷酸化导致细胞-细胞融合和体外合胞体形成异常。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合胞体，以及在体内由皮肤和神经节的VZV感染引起的表皮细胞和神经元-卫星细胞的融合。在目标1中，我们将确定VZV如何修改细胞调节，以有利于基因的转录，促进细胞-细胞融合和合胞体形成，通过应用高通量全转录组测序技术，RNA-seq，我们的新的融合检测和我们的病毒突变体，其中携带突变的gBcyt残基，影响细胞-细胞融合。为了量化酪氨酸磷酸化的影响，将实时测量VZV和gBcyt突变体的合胞体形成的时空演变。为了确定基因在细胞融合中的作用，如通过RNA-seq鉴定的，我们将进行基因扰动实验以评估它们在VZV复制背景下的生物学意义。目的2将确定gBcyt如何通过细胞或病毒蛋白对酪氨酸和/或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。质谱法将用于鉴定与酪氨酸磷酸化和非磷酸化形式的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，以评估乙酰化和泛素化翻译后修饰对VZV融合和毒力的影响。最后，目的3将确定gBcyt是否调节多核细胞形成以优化皮肤和DRG的VZV感染。我们的突变病毒将比较野生型VZV在人类皮肤和神经元组织中使用新的报告病毒的复制能力。我们将使用一种新的shRNA来确定新发现的细胞-细胞融合所需基因的作用 携带病毒鉴于多核细胞形成对发病机制的重要性，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。

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.