Identifying functional targets of HSV-1 ICP0-directed degradation

单纯疱疹病毒（HSV-1）在人类中引起疾病，可导致口腔、生殖器或眼部溃疡，或在极少数情况下导致危及生命的脑炎。1型单纯疱疹病毒的生命周期有两个不同的阶段：裂解期和潜伏期。一种HSV-1立即早期蛋白，即感染细胞蛋白0 (ICP0)，是决定感染是溶解性还是潜伏性的关键因素。ICP0是一种110 kda的蛋白，通过其E3泛素（Ub）连接酶活性有效地激活病毒基因表达。通常，E3 Ub连接酶是连接和聚合Ub（一种76个氨基酸的蛋白质）到靶蛋白的途径的组成部分，标记它们被蛋白酶体降解。现有证据表明，ICP0介导了其反激活活性所需的几种细胞蛋白的降解。由于检测所需的特定蛋白质的纯度、溶解度和数量的限制，通过蛋白质组学方法确定icp定向降解的特定靶标一直具有挑战性。虽然已经发现了ICP0在病毒感染过程中的一些功能，但被ICP0标记为降解的细胞蛋白的身份在很大程度上是未知的。在这些新的靶标被确定之前，尚不清楚ICP0的E3 Ub连接酶活性在其反激活活性中所起的确切作用。我们研究的长期目标是在分子水平上了解病毒-宿主相互作用如何调节HSV-1的生命周期。本应用程序的目的是使用一种独特的筛选程序来鉴定由ICP0降解的细胞蛋白，确定这些ICP0靶点在HSV-1基因表达和裂解复制中所起的作用。我们的中心假设是ICP0介导了新的细胞蛋白的降解；我们认为这些蛋白的缺失促进了病毒基因的高效表达和复制。我们的方法是使用尖端屏幕识别icp0介导的降解的功能目标。我们开展这些研究的基本原理是，通过了解ICP0如何通过泛素化促进生产性感染，ICP0降解的靶标可用于开发新的治疗方法来破坏HSV复制及其相关疾病。为此，提出了以下具体目标：目标#1：确定HSV-1 E3 Ub连接酶ICP0的靶点；目的2：确定ICP0靶点在HSV- 1基因表达和复制中的作用。从这个提议中，我们期望用一种创新的方法来鉴定参与ICP0生物学的细胞蛋白，这是一个重要的贡献，可以用来分离和鉴定在蛋白质水解和病毒发病过程中起重要作用的基因和途径。

Herpes simplex virus (HSV-1) causes diseases in humans, which can result in oral, genital, or ocular sores, or in rare occasions, life-threatening encephalitis. HSV-1 has two distinct phases of its viral life cycle: lytic and latent. An HSV-1 immediate-early protein, infected cell protein 0 (ICP0), is a key determinant as to whether an infection will be lytic or latent. ICP0 is a 110-KDa protein that potently transactivates viral gene expression via its E3 ubiquitin (Ub) ligase activity. Typically, E3 Ub ligases are components of pathways that attach and polymerize Ub (a 76 amino acid protein) to target proteins, marking them for degradation by the proteasome. Available evidence suggests that ICP0 mediates the degradation of several cellular proteins required for its transactivating activity. Efforts to identify specific targets of ICP0-directed degradation by proteomic approaches have been challenging because of limitations in the purity, solubility, and amount of a given protein required for its detection. While several functions of ICP0 during viral infection have been discovered, the identities of cellular proteins marked for degradation by ICP0 are largely unknown. Until these novel targets have been identified, it is unclear the exact role ICP0’s E3 Ub ligase activity plays in its transactivating activity. The long-term goal of our studies is to understand at the molecular level how virus-host interactions regulate the HSV-1 life cycle. The objective of this application is to a use a unique screening procedure to identify cellular proteins whose degradation is directed ICP0, determining the role these ICP0 targets play in HSV-1 gene expression and lytic replication. Our central hypothesis is that ICP0 mediates the degradation of novel cellular proteins; we propose that the loss of these proteins promotes efficient viral gene expression and replication. Our approach is to identify functional targets of ICP0-mediated degradation using a cutting edge screen. Our rationale for carrying out these studies is that by understanding how ICP0 facilitates productive infection via ubiquitination, targets of ICP0 degradation could be used to develop new therapies to impair HSV replication and its associated diseases. For this purpose, the following specific aims are proposed: Aim #1: Identify targets of the HSV-1 E3 Ub ligase, ICP0; Aim #2: Determine the contribution ICP0 targets play in HSV- 1 gene expression and replication. From this proposal we expect to identify cellular proteins involved in the biology of ICP0 using an innovative approach, which is a significant contribution that can used to isolate and identify genes and pathways that play important roles involved proteolysis and viral pathogenesis.