泛素连接酶TRIM调控抗病毒天然免疫反应的机制研究

Innate immunity is the first line against virus infection. After viral infection, the viral molecules such as RNA and DNA can be recognized by innate immune receptors including TLR, RLR and DNA sensors, leading to the production of proinflammatory cytokines and interferons, which are essential for the elimination of viral infection. Except for the expression of proinflammatory cytokines and type I interferons, the innate immune system also expresses varieties of feedback molecules after virus infection. These molecules are thought to regulate the secretion of proinflammatory cytokines and type I interferons and to prevent excessive activation of the innate immune responses. We have previously demonstrated that TLR3/4 activation by LPS or poly(I:C) induced the expression of tripartite motif protein 26 (TRIM26) in macrophages. TRIM26 expression was also induced by Sendai virus (SeV) infection. Importantly, TRIM26 protein was distributed to nucleus after SeV and vesicular stomatitis virus (VSV) infection or IFN-β stimulation. These data indicate that TRIM26 may play an important role in the regulation of antiviral innate immune responses. Indeed, we found that TRIM26 overexpression attenuated TLR3-, TLR4-, RLR- and dsDNA sensor-induced IFN-β promoter activation, while, VSV replication was increased upon TRIM26 overexpression. In contrast, siRNA knockdown of TRIM26 expression increased IFN-β production and decreased VSV replication. Decreased IFN-β production and increased VSV replication was also found in primary macrophages from TRIM26 transgenic mice, compared to the macrophages from WT mice. In this project, we propose to use TRIM26 transgenic mice and various of cells including macrophages and HEK293 cells to investigate the functions of TRIM26 in the producton of IFN-β and antiviral immune responses. We will also use varieties of molecular techniques such as Dual-Luciferase Reporter Assay, co-immunoprecipitation, in vitro and in vivo ubiquitination to identify the targets that are regulated by TRIM26. Finally, we will figure out how the targets were regulated by TRIM26. TRIM26 is located in the MHC class I coding region, but the function of TRIM26 in the immune regulation is not known. Our study is the first to explore the functions of TRIM26 on the reguation of innate immune resonses and may provide a new threputic target for the antiviral drug design. Given that TRIM26 expression and nuclear translocation was induced by virus infection, our study may provide some new concept of virus-host interaction and new mechanisms for the virus invasion of the innate immune system.

天然免疫作为机体抗病毒感染的"第一道防线"，对于病毒的识别和清除起着至关重要的作用。前期工作发现，病毒感染可诱导TRIM26蛋白的表达以及核转位；TRIM26过表达可显著抑制IFN-β的活化，并促进VSV病毒的复制，而siRNA干扰可促进IFN-β的生成，抑制VSV病毒复制；TRIM26转基因鼠的原代巨噬细胞，活化后IFN-β的表达大大降低，而VSV的复制显著增高。这些结果提示，TRIM26可作为反馈调节因子调控抗病毒天然免疫反应。本课题拟利用巨噬细胞、HEK293 等细胞和TRIM26转基因鼠，结合各种分子技术手段深入探讨TRIM26在抗病毒天然免疫中的功能，并通过研究TRIM26与抗病毒信号转导途径中关键分子的相互作用，揭示其作用的分子机制。本研究首次探讨TRIM26在抗感染天然免疫中的功能及其作用机制，将为抗病毒天然免疫应答的调控机制提供新思路，以及抗感染药物的设计提供新的潜在靶点。

病毒感染机体后，天然免疫系统首先被激活，导致转录因子IRF3的活化以及I型干扰素（IFN-β）的合成，进而诱导大量抗病毒基因的表达，最终清除病毒感染。转录因子IRF3的磷酸化、二聚化和入核是其活化的重要标志，但是活化入核的IRF3在细胞核内如何终止，并不清楚。我们的研究发现，病毒感染可诱导泛素连接酶TRIM26的表达，特别重要的是，病毒感染后TRIM26可从细胞质转移到细胞核，在细胞核内与活化形式IRF3发生结合，促进IRF3进行K48位连接的泛素化修饰，并最终导致IRF3的蛋白酶体降解。进一步研究发现，TRIM26可以促进野生型IRF3（IRF3 WT）及组成型活化形式IRF3（IRF3 5D）的泛素化修饰和降解，而对于非活化形式的IRF3（IRF3 5A）没有作用；同时，IRF3的细胞核定位信号（NLS）缺失后，也不能被TRIM26进行泛素化修饰和降解；同样，TRIM26的细胞核定位信号（NLS）缺失后，病毒感染不能诱导TRIM26突变体入核，也丧失了对IRF3的降解作用。与TRIM26降解IRF3一致，TRIM26可以抑制TLR3/4, RLR及胞浆DNA介导的IFN-β的产生；利用TRIM26转基因小鼠发现，VSV病毒感染后，TRIM26转基因鼠产生的IFN-β较野生型小鼠降低，而病毒复制增强，转基因小鼠对病毒感染更敏感。本课题揭示了活化形式IRF3在细胞核内的终止机制，并且揭示了一条新的病毒逃逸机制，为I型干扰素表达调控机制提出新的认识，为抗病毒感染药物研发提供了新的靶点。