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

描述（由申请人提供）：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）。对于免疫功能低下的患者，水痘可能是严重的，并危及生命。在感染人类宿主期间，VZV表现出对T细胞、皮肤和神经元的趋向性，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。糖蛋白B （gB）和gH/gL异源二聚体在疱疹病毒进入过程中对病毒粒子包膜与靶细胞膜融合至关重要。我们的新概念是，VZV通过依赖gb的细胞内信号功能介导细胞-细胞融合。这是基于我们的新证据，即阻止酪氨酸磷酸化的gB细胞质结构域（gBcyt）导致细胞-细胞融合和合胞体形成的异常。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，在体外产生特征性合胞体，并在体内由VZV感染皮肤和神经节引起表皮细胞和神经元卫星细胞的融合。在Aim 1中，我们将通过将高通量全转录组测序技术RNA-seq应用于我们的新融合实验和我们的病毒突变体，确定VZV如何修改细胞调控，以促进促进细胞融合和合胞体形成的基因的转录。为了量化酪氨酸磷酸化的影响，将实时测量VZV和gBcyt突变体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，正如RNA-seq所鉴定的那样，我们将进行基因扰动实验来评估它们在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/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，Aim 3将确定gBcyt是否调节多核细胞的形成以优化VZV感染皮肤和DRG。我们的突变病毒将与野生型VZV进行比较，用新的报告病毒在人类皮肤和神经组织中的复制能力。我们将利用一种新的shRNA来确定细胞-细胞融合所需的新鉴定基因的作用

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.