Cellular determinants of apoptosis in virus-infected cells

描述（由申请人提供）：该项目的目标是研究一种新发现的抗病毒途径是如何被激活和调节的，以及它在决定病毒发病机制和持久性方面的作用。I型干扰素（IFN）系统在先天性抗病毒应答中起主要作用。病毒感染激活转录因子IRF-3，其负责诱导IFN和其他抗病毒蛋白。我们最近的调查显示， IRF-3还激活IFN-非依赖性促凋亡途径，称为RIPA（RIG-I激活的IRF-3介导的凋亡途径）。为了触发RIPA，激活的IRF-3结合Bax并将其易位至线粒体以引起细胞凋亡; IRF-3的两种功能在遗传上是可分离的。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PI 3激酶的作用进行时间调节，XIAP和PI 3激酶也在病毒感染时被激活。在没有RIPA的情况下，病毒建立持续感染。在这里，我们建议进一步研究RIPA。在目的1中，我们将研究IRF-3是如何在RIPA中被激活的，作为特定丝氨酸残基磷酸化和特定赖氨酸残基泛素化的结果。遗传和生物化学分析将用于这一目的。在目标2中，我们将研究病毒如何暂时逃避RIPA，通过激活表皮生长因子受体，进而激活PI 3激酶，触发RIPA的负调控。在目标3中，我们将研究RIPA在决定病毒感染结果方面的生理相关性。在IRF-3的一种而非另一种作用中有缺陷的突变细胞将用于测量RIPA对病毒复制效率的作用 建立病毒持久性。最后，将使用转基因小鼠（包括本项目产生的新IRF-3突变体基因敲入小鼠）评估RIPA在控制病毒发病机制中的体内作用。

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.