The Role of TRIM23 in Autophagy Mediated Antiviral Defenses

项目总结 自噬，细胞处理细胞内容物的过程，已经被 越来越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。 几个已知在干扰素介导的免疫中起关键作用的分子也是相互关联的 自噬的重要调节者。在这些分子中，具有自噬和 干扰素介导的免疫是几个TRIM(三方基序)蛋白家族成员。然而， 鉴于TRIM蛋白作为抗病毒限制因子或 调节干扰素反应的特征已经很好了，我们对TRIM的作用有更多的了解 对病毒感染作出反应的自噬中的蛋白质仍然是最基本的。 这项拟议的研究建立在加克实验室最近的一项发现基础上，该发现确定了 TRIM23作为自噬介导的宿主防御广泛范围的关键调节因子 病毒。TRIM23与天然免疫分子TBK1相互作用并激活，促进 TBK1介导的选择性自噬受体p62的磷酸化，最终 引发病毒清除和宿主抵抗。从机制上讲，N-末端环E3连接酶 TRIM23诱导非典型的非降解性K27连锁的C端ARF自身泛素化 GTPase结构域，这是TRIM23所独有的。ARF泛素化导致酶活性增强 TRIM23可以降解GTP，激活TBK1，并介导病毒抗性。TRIM23耗尽或 不同细胞类型的基因靶向研究表明，TRIM23介导的自噬具有抗病毒作用 对包括HSV-1在内的几种病毒的活性。 使用分子、生化、细胞生物学和结构方法相结合 病毒感染研究，我们将精确详细地定义TRIM23如何在 病毒感染。这项研究将深入了解TRIM23的激活机制 病毒感染期间的上游调节器(目标1)。我们将进一步确定分子 TRIM23激活TBK1的机制及TRIM23-E3的作用 连接酶和GTP酶在TBK1激活中的作用。最后，我们将确定生理学的 利用体外细胞研究TRIM23在抗病毒宿主抵抗和病毒致病机制中的作用 TRIM23基因敲除小鼠的培养系统和感染研究(目标2)。我们的研究将提供 自噬介导的宿主防御中关键途径的分子理解 可指导新型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.