Cellular Determinants of Apoptosis in Virus-Infected Cells

描述(申请人提供)：由于病毒性疾病是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。被病毒感染的细胞既可以死亡，也可以存活；如果它活着，它可能会永久感染病毒。这项提议是为了研究决定上述选择的分子机制，即感染病毒的细胞是死亡还是存活并产生更多病毒。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。致病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感染细胞的先天反应，感染细胞可能发生凋亡或存活，并能够建立持续感染，这两种结果对宿主机体具有相反的影响。我们已经做了新的观察，在不表达细胞转录因子IRF-3的细胞中，副粘病毒不是裂解的，因为IRF-3在感染的细胞中启动了凋亡反应。此外，通过抑制PI3激酶的活性，这种反应大大加快。这些观察使我们形成了这样的假设，即IRF-3决定了溶血性感染和非溶血性感染之间的选择，而PI3激酶决定了溶血性感染的时机。将追求三个具体目标来检验这一假设。在第一个目标中，我们将确定IRF-3的哪些已知特性是必需的，以及哪些已知的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度表达或通过siRNA来消融它们。最后，将通过插入突变或适当的shRNA文库和细胞存活作为终点分析来对整个人类基因组进行无偏见的遗传筛选。在第二个目标中，我们将确定病毒凋亡中使用的是内在途径还是外在途径。我们还将确定PI3激酶的特定同工酶及其下游靶点，它们是阻断快速细胞凋亡所必需的。在第三个目标中，我们将比较持续感染细胞和急性感染细胞的特性。为此，我们将研究病毒生命周期的不同阶段。此外，还将评估持续感染细胞中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.