Cellular Determinants of Apoptosis in Virus-Infected Cells

描述（由申请人提供）：由于病毒性疾病是对社会的主要健康风险，了解它们是如何引起的以及我们的自然防御系统如何保护我们是很重要的。被病毒感染的细胞要么死亡，要么存活；如果它活了下来，它可能会永久感染这种病毒。这一建议是为了研究决定上述选择的分子机制，即，病毒感染的细胞是死亡还是存活并产生更多的病毒。许多主要的病毒性疾病都是由副粘病毒科引起的，如呼吸道合胞病毒、副流感病毒、麻疹病毒和腮腺炎病毒。发病机制的性质和严重程度是由宿主反应决定的，宿主反应的一个主要组成部分是受感染细胞的先天反应，这些细胞可能发生凋亡，也可能存活并能够建立持续感染，这两种结果对宿主机体的影响是相反的。我们观察到，在不表达细胞转录因子IRF-3的细胞中，副粘病毒不会裂解，因为IRF-3在感染细胞中启动了凋亡反应。此外，这种反应通过抑制PI3激酶的活性而大大加速。这些观察结果使我们提出假设，IRF-3决定了裂解和非裂解感染之间的选择，PI3激酶决定了裂解的时间。为了验证这一假设，将追求三个具体目标。在第一个目标中，我们将确定哪些已知的IRF-3特性是必需的，哪些已知的IRF-3调节基因可能介导细胞凋亡。这些实验将使用单个蛋白质的过表达或siRNA消融。最后，通过插入突变或适当的shRNA文库和细胞存活作为终点试验，对整个人类基因组进行无偏倚的遗传筛选。在第二个目标中，我们将确定是内在途径还是外在途径在病毒凋亡中使用。我们还将确定PI3激酶的特异性同工酶及其下游靶点，这些靶点是阻断快速凋亡所需的。在第三个目标中，我们将比较持续感染细胞与急性感染细胞的特性。为此目的，将研究病毒生命周期的不同步骤。此外，将评估持续感染细胞的先天免疫反应状态。

DESCRIPTION (provided by applicant): Because viral diseases are major health risks to the society, it is important to understand how they are caused and how our natural defense systems can protect us. A virus-infected cell can either die or it can live; if it lives it may be permanently infected with the virus. This proposal is to investigate the molecular mechanism that dictates the above choice, namely, whether a virus-infected cell dies or lives and produces more viruses. Many major viral diseases are caused by Paramyxoviridae, such as respiratory syncytial virus, parainfluzenza virus, measles virus and mumps virus. The nature and the severity of pathogenesis is determined by the host response, a major component of which is the innate response of the infected cell which may undergo apoptosis or live and be capable to establish persistent infection, the two outcomes having opposite effects on the host organism. We have made the novel observation that in cells not expressing the cellular transcription factor IRF-3, paramyxoviruses are not lytic because IRF-3 initiates an apoptotic response in infected cells. Moreover, this response is greatly accelerated by inhibiting the activity of PI3 kinases. These observations led us to formulate the hypothesis that IRF-3 determines the choice between lytic and non-lytic infection and PI3 kinases determine the timing of the lysis. Three specific aims will be pursued to test the hypothesis. In the first aim, we will determine which known properties of IRF-3 are needed and which known IRF-3 regulated genes may be mediating apoptosis. These experiments will use over- expression of individual proteins or their ablation by siRNA. Finally, unbiased genetic screens of the whole human genome will be conducted by using insertional mutagenesis or an appropriate shRNA library and cell survival as the end point assay. In the second aim, we will determine whether the intrinsic or the extrinsic pathway is used in viral apoptosis. We will also identify the specific isozyme of PI3 kinase and its downstream targets that are required for blocking the rapid apoptosis. In the third aim, we will compare the properties of the persistently infected cell with those of the acutely infected cells. For this purpose, the different steps of the viral life cycle will be examined. Moreover, the status of the innate immune response in the persistently infected cells will be evaluated.