ICP0-induced Cellular Factors Promote HSV-1 Replication

描述（由申请人提供）：长期以来已知应激刺激可诱导HSV-1在体内和体外从潜伏期再活化，但负责病毒基因表达活化的细胞因子的身份尚不清楚。在相关静脉中，HSV-1立即早期调节蛋白ICP 0是病毒和细胞基因表达的广泛且强的转录激活剂。ICP 0是有效病毒复制所必需的，特别是在低感染复数下，并且是从离体小鼠模型中的神经元潜伏期有效再激活所必需的。我们以前已经表明，应激诱导的细胞因子可以补偿ICP 0空质粒感染的细胞中ICP 0的活性，这表明ICP 0的一个功能可能是“打开”应激诱导的细胞因子。这些细胞因子包括但可能不限于细胞周期蛋白依赖性激酶。在本项目的目标1中，将使用微阵列和蛋白质印迹分析来鉴定其表达受应激和ICP 0影响的细胞因子。在目标2中，将使用siRNA和可用的敲除细胞系通过抑制受应激和ICP 0影响的特定细胞因子的活性来测试它们替代ICP 0功能的能力。ICP 0激活所有类型的HSV-1基因的表达。在目标3中，将在用启动子特异性报告质粒转染并经受应激的细胞中鉴定由应激诱导的细胞ICP 0互补活性激活的病毒基因的类别。这些实验旨在鉴定促进HSV-1复制的应激诱导和ICP 0诱导的细胞因子，并且在未来的实验中，可能有助于鉴定负责HSV-1重新激活神经元潜伏期的细胞因子。

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.