ICP0-induced Cellular Factors Promote HSV-1 Replication

描述（由申请人提供）：长期以来，人们已经知道应激刺激可以诱导体内和体外潜伏期的HSV-1再激活，但负责激活病毒基因表达的细胞因子的身份尚不清楚。在相关的静脉中，单纯疱疹病毒-1的即时早期调控蛋白ICP0是病毒和细胞基因表达的广泛而强烈的转录激活因子。在离体小鼠模型中，ICP0是有效的病毒复制所必需的，特别是在低感染的多重性下，以及从神经元潜伏期中有效的再激活。我们之前已经证明，应激诱导的细胞因子可以在ICP0零突变体感染的细胞中补偿ICP0的活性，这表明ICP0的一个功能可能是“开启”应激诱导的细胞因子。这些细胞因子包括，但可能不限于，周期蛋白依赖性激酶。在本项目的Aim 1中，将使用微阵列和Western blot分析来鉴定同时受应激和ICP0影响的细胞因子。在Aim 2中，将测试受应激和ICP0影响的特定细胞因子是否能够通过使用siRNA和可用的敲除细胞系抑制ICP0的活性来替代ICP0的功能。ICP0激活所有类别HSV-1基因的表达。在Aim 3中，将在转染了启动子特异性报告质粒并受到应激的细胞中鉴定由应激诱导的细胞icp0互补活性激活的病毒基因类别。这些实验旨在确定应激诱导和icp诱导的促进HSV-1复制的细胞因子，并可能在未来的实验中帮助确定负责HSV-1神经元潜伏期再激活的细胞因子。

DESCRIPTION (provided by applicant): Stressful stimuli have long been known to induce reactivation of HSV-1 from latency in vivo and in vitro, yet the identity of the cellular factors responsible for the activation of viral gene expression is not known. In a related vein, the HSV-1 immediate-early regulatory protein, ICP0, is a broad and strong transcriptional activator of viral and cellular gene expression. ICP0 is required for efficient viral replication, especially at low multiplicities of infection, and for efficient reactivation from neuronal latency in an ex vivo mouse model. We have shown previously that stress-induced cellular factors can compensate for the activities of ICP0 in ICP0 null mutant-infected cells, suggesting that one function of ICP0 may be to "turn on" cellular factors induced by stress. These cellular factors include, but are likely not limited to, cyclin-dependent kinases. In Aim 1 of this project, microarray and Western blot analyses will be used to identify the cellular factors whose expression is affected by both stress and ICP0. In Aim 2, specific cellular factors affected by both stress and ICP0 will be tested for their ability to substitute for the functions of ICP0 by inhibiting their activities using siRNA and available knockout cell lines. ICP0 activates expression of all classes of HSV-1 genes. In Aim 3, the classes of viral genes activated by the stress-induced cellular, ICP0-complementing activities will be identified in cells transfected with promoter-specific reporter plasmids and subjected to stress. These experiments are designed to identify the stress-induced and ICP0-induced cellular factors that promote HSV-1 replication and may, in future experiments, assist in identifying the cellular factors responsible for reactivation of HSV-1 for neuronal latency.