The Role of TRIM23 in Autophagy Mediated Antiviral Defenses

项目摘要 自噬是细胞处理细胞内容物的过程， 越来越多地被认为是抗病毒宿主防御的重要途径。有趣的是，最近 研究表明，自噬和抗病毒I型干扰素反应是错综复杂的， 连接，并且已知在IFN介导的免疫中起关键作用的几种分子也是 重要的自噬调节因子。在这些在自噬中具有双重作用的分子中， IFN介导的免疫是几个TRIM（三重基序）蛋白家族成员。然而，在这方面， 而TRIM蛋白作为抗病毒限制因子或 调节IFN反应已经得到很好的表征，我们对TRIM作用的了解 自噬中的蛋白质对病毒感染的反应仍然是初步的。 这项拟议中的研究建立在Gack实验室最近的一项发现的基础上， TRIM 23作为自噬介导的宿主防御广泛的免疫缺陷病毒的关键调节因子， 病毒TRIM 23与先天免疫分子TBK 1相互作用并激活先天免疫分子TBK 1， TBK 1介导的选择性自噬受体p62的磷酸化， 触发病毒清除和宿主抗性。从机制上讲，N-末端RING E3连接酶 TRIM 23诱导C-末端ARF的非典型非降解性K27连接的自身泛素化 GTdR结构域，其是TRIM 23所特有的。ARF泛素化导致增强的 TRIM 23水解GTP，激活TBK 1，并介导病毒抗性。TRIM 23耗竭或 在各种细胞类型中的基因靶向显示TRIM 23介导的自噬赋予抗病毒活性， 对包括HSV-1在内的几种病毒的活性。 利用分子、生物化学、细胞生物学和结构方法，结合 在病毒感染研究中，我们将精确详细地定义TRIM 23如何介导自噬， 病毒感染这项研究将通过以下方式深入了解TRIM 23激活的机制： 病毒感染过程中的上游调节因子（Aim 1）。我们将进一步确定 TRIM 23如何激活TBK 1以及TRIM 23- E3的酶活性的作用的详细信息 连接酶和GTP酶在TBK 1活化中起作用。最后，我们将确定生理 TRIM 23与抗病毒宿主抗性和使用体外细胞病毒发病机制的相关性 培养系统和TRIM 23敲除小鼠中的感染研究（Aim 2）。我们的研究将提供 对自噬介导的宿主防御中的关键途径的分子理解， 可以指导新抗病毒药物的合理设计。

PROJECT SUMMARY Autophagy, the process by which cells dispose of cellular contents, has been increasingly appreciated as an important pathway in antiviral host defenses. Intriguingly, recent studies demonstrated that autophagy and the antiviral type I IFN response are intricately connected, and several molecules known to play key roles in IFN-mediated immunity are also important regulators of autophagy. Among these molecules with dual roles in autophagy and IFN-mediated immunity are several TRIM (tripartite motif) protein family members. However, whereas the molecular mechanisms by which TRIM proteins act as antiviral restriction factors or regulate IFN responses have been well characterized, our knowledge about the role of TRIM proteins in autophagy in response to viral infection is still rudimentary. The proposed study builds on a recent discovery by the Gack laboratory that identified TRIM23 as a critical regulator of autophagy-mediated host defense against a broad range of viruses. TRIM23 interacts with and activates the innate immune molecule TBK1, promoting TBK1-mediated phosphorylation of the selective autophagy receptor p62, which ultimately triggers viral clearance and host resistance. Mechanistically, the N-terminal RING E3 ligase of TRIM23 induces atypical non-degradative K27-linked auto-ubiquitination of the C-terminal ARF GTPase domain, which is unique to TRIM23. ARF ubiquitination results in enhanced activity of TRIM23 to hydrolyze GTP, activate TBK1, and mediate virus resistance. TRIM23 depletion or gene-targeting in various cell types showed that TRIM23-mediated autophagy confers antiviral activity against several viruses including HSV-1. Using molecular, biochemical, cell biological and structural approaches combined with virus infection studies, we will define in precise detail how TRIM23 mediates autophagy during viral infection. This study will yield insight into the mechanisms of TRIM23 activation by upstream regulators during viral infection (Aim 1). We will further determine the molecular details of how TRIM23 activates TBK1 and the role the enzymatic activities of TRIM23 – E3 ligase and GTPase – play in TBK1 activation. Finally, we will determine the physiological relevance of TRIM23 for antiviral host resistance and viral pathogenesis using in vitro cell culture systems and infection studies in TRIM23 knockout mice (Aim 2). Our studies will provide a molecular understanding of a key pathway in the autophagy-mediated host defense, which may guide the rational design of new antivirals.