ICP0-induced Cellular Factors Promote HSV-1 Replication

描述(由申请人提供)：长期以来，应激刺激在体内和体外都能从潜伏期诱导HSV-1重新激活，但负责激活病毒基因表达的细胞因素的身份尚不清楚。在相关的脉络中，HSV-1立即早期调节蛋白ICP0是病毒和细胞基因表达的广泛和强大的转录激活因子。ICP0对于病毒的有效复制是必需的，特别是在低感染复数的情况下，以及在体外小鼠模型中从神经元潜伏期有效地重新激活。我们以前已经证明，在ICP0缺失突变感染的细胞中，应激诱导的细胞因子可以补偿ICP0的活性，提示ICP0的一个功能可能是“激活”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。在该项目的目标1中，将使用微阵列和Western印迹分析来确定其表达受压力和ICP0影响的细胞因子。在目标2中，将测试受应激和ICP0影响的特定细胞因子通过使用siRNA和可用的基因敲除细胞系来抑制其活性来替代ICP0功能的能力。ICP0可激活所有类型HSV-1基因的表达。在目标3中，通过应激诱导的细胞ICP0互补活性激活的病毒基因将在转染启动子特异报告质粒并受到应激的细胞中得到鉴定。这些实验旨在确定压力诱导和ICP0诱导的促进HSV-1复制的细胞因子，并在未来的实验中可能有助于确定导致神经元潜伏期HSV-1重新激活的细胞因素。

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.