Cellular Determinants of Apoptosis in Virus-Infected Cells

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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.