TRIM14 PRY/SPRY结构域介导RLRs和cGAS通路非依赖性抗病毒机制研究

TRIM14 is known to play an important role in host defense against viral infection by positively regulating the activation of retinoic acid-induced gene type I-like receptor (RLRs) and cyclic GMP-AMP synthase (cGAS) pathways. However, the direct antiviral mechanism of TRIM14 in innate immune response remains poorly understood. Based on our previous work on innate responses to Hepatitis C virus (HCV) and Influenza A virus (IAV), we have found that TRIM14 also has direct antiviral function which is independent of RLRs and cGAS pathways. TRIM14 PRY/SPRY domain can interact and form complex with HCV NS5A and IAV NP proteins, and subsequently promote their ubiquitination and degradation, but what other host factors are involved in this process has not been determined. In this project, we will utilize co-immunoprecipitation-protein mass spectrometry, CRISPR/Cas9 gene knockout techniques to systematically examine the protein-protein interactions of PRY/SPRY domain, and to identify the key E3 ubiquitin ligase involved in mediating the ubiquitination of the target viral proteins. Together, these studies will elucidate the mechanism by which TRIM14 PRY/SPRY domain promotes the ubiquitination and degradation of viral proteins through the formation of multi-molecular ubiquitin ligase complexes, and will provide potential molecular targets for the development of new antiviral drugs.

已知TRIM14蛋白可通过正向调控视黄酸诱导基因I样受体（RLRs）和环GMP-AMP合成酶（cGAS）信号通路激活发挥重要抗病毒效应。然而对于TRIM14直接抗病毒作用却认识甚少。在丙型肝炎病毒（HCV）和甲型流感病毒（IAV）的研究工作中，我们发现TRIM14具有不依赖于RLRs和cGAS信号通路的抗病毒功能。TRIM14通过PRY/SPRY结构域识别HCV NS5A和IAV NP蛋白，并介导其泛素化修饰与降解，但抑制病毒侵染复制的具体机制尚不清楚。本项目将对PRY/SPRY结构域蛋白-蛋白相互作用进行系统解析，并借助免疫共沉淀-蛋白质谱、CRISPR/Cas9基因敲除等手段寻找与其作用的E3泛素连接酶，阐明TRIM14 PRY/SPRY结构域如何通过多分子泛素连接酶复合物的形成调控病毒蛋白泛素化修饰与降解从而发挥抗病毒效应的分子机制，为开发新型抗病毒药物提供潜在的分子靶点。

已知TRIM14蛋白可通过正向调控视黄酸诱导基因I样受体（RLRs）和环GMP-AMP合成酶（cGAS）信号通路激活发挥重要抗病毒效应。然而对于TRIM14直接抗病毒作用却认识甚少。在丙型肝炎病毒（HCV）、甲型流感病毒（IAV）和严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的研究工作中，我们发现TRIM14具有不依赖于RLRs和cGAS信号通路的抗病毒功能。TRIM14通过PRY/SPRY结构域识别HCV NS5A、IAV NP和SARS-CoV-2 N蛋白，并介导其泛素化修饰与降解。对PRY/SPRY结构域蛋白-蛋白相互作用进行系统解析，并借助免疫共沉淀、CRISPR/Cas9基因敲除等手段筛选并鉴定出STUB1可作为关键E3泛素连接酶，参与调控了TRIM14 PRY/SPRY结构域调控病毒靶蛋白泛素化降解过程。本项目的开展阐明了TRIM14 PRY/SPRY结构域如何通过多分子泛素连接酶复合物的形成调控病毒蛋白泛素化修饰与降解从而发挥抗病毒效应的分子机制，为开发新型抗病毒药物提供潜在的分子靶点。