Cellular determinants of apoptosis in virus-infected cells

描述(由申请人提供)：该项目的目标是调查新发现的抗病毒途径是如何被激活和调节的，以及它在确定病毒致病机制和持久性方面所起的作用。I型干扰素系统在先天抗病毒反应中发挥重要作用。病毒感染激活了转录因子IRF-3，该因子负责诱导干扰素和其他抗病毒蛋白的产生。我们最近的调查显示， IRF-3还激活一条非干扰素依赖的促凋亡通路，称为RIG-I激活的IRF-3介导的凋亡通路(RIPA)。为了触发RIPA，激活的IRF-3与Bax结合并将其移位到线粒体以引起细胞凋亡；IRF-3的这两个功能在基因上是可以分离的。RipA被许多RNA和DNA病毒激活，并抑制病毒复制和致病。RipA通过XIAP和PI3激酶的作用进行时间调节，PI3激酶也在病毒感染时被激活。在没有RIPA的情况下，病毒会建立持续感染。在这里，我们建议对RIPA进行进一步调查。在目标1中，我们将研究特定丝氨酸残基的磷酸化和特定赖氨酸残基的泛素化是如何在RIPA中激活IRF-3的。遗传和生化分析将用于这一目的。在目标2中，我们将研究病毒如何通过激活表皮生长因子受体来触发对RIPA的负调控，进而激活PI3激酶，从而暂时逃避RIPA。在目标3中，我们将研究RIPA在决定病毒感染结局中的生理学相关性。在IRF-3的一个作用上有缺陷，但在另一个作用上没有缺陷的突变细胞将被用来测量RIPA在病毒复制效率上的作用 以及建立病毒持久性。最后，转基因小鼠，包括为该项目培育的新的IRF-3突变敲入小鼠，将被用于评估RIPA在体内对控制病毒致病的贡献。

DESCRIPTION (provided by applicant): The goal of the project is to investigate how a newly discovered antiviral pathway is activated and regulated and what its role is in determining viral pathogenesis and persistence. The type I interferon (IFN) system plays a major role in innate antiviral response. Virus infection activates the transcription factor IRF-3, which is responsible for the induction of IFN and other antiviral proteins. Recent investigation by us has revealed that IRF-3 also activates an IFN-independent pro-apoptotic pathway, named RIPA (RIG-I-activated IRF-3-mediated Pathway of Apoptosis). To trigger RIPA, activated IRF-3 binds Bax and translocates it to mitochondria to cause apoptosis; the two functions of IRF-3 are genetically separable. RIPA is activated by many RNA and DNA viruses and inhibits both viral replication and pathogenesis. RIPA is temporally regulated through the action of XIAP and PI3 kinase, which is also activated upon virus infection. In the absence of RIPA, viruses establish persistent infection. Here we propose to investigate RIPA further. In Aim 1, we will investigate how IRF-3 is activated in RIPA as a consequence of phosphorylation of specific serine residues and ubiquitynation of specific lysine residues. Genetic and biochemical analyses will be used for this purpose. In Aim 2, we will investigate how viruses evade RIPA temporarily, by triggering negative regulation of RIPA though activation of the epidermal growth factor receptor, which in turn activates PI3 kinase. In Aim 3, we will investigate the physiological relevance of RIPA in determining the outcome of virus infection. Mutant cells defective in one, but not the other, action of IRF-3, will be used for measuring the role of RIPA on the efficiency of virus replication and establishing viral persistence. Finally, genetically modified mice, including new IRF-3 mutant knock-in mice which have been generated for this project, will be used to assess the in vivo contribution of RIPA in controlling viral pathogenesis.