E3泛素连接酶RNF39在抗病毒固有免疫反应中的调控作用及其分子机制

Innate immunity is the first line against viral infection, and its optimal activation is crucial for our body to eradicate invading viruses quickly and maintain immune homeostasis. Thus, investigating the regulatory mechanisms of innate immune responses is important for the elucidation of the interaction between virus and host and the mechanisms of viral immune escape. RNF39, an E3 ubiquitin ligase is encoded within the major histocompatibility complex class I (MHC class I) region. We found that RNF39 deficiency and knockdown both significantly enhanced RNA virus infection induced IFN-β expression. An interaction between RNF39 and DDX3 (an important adaptor in antiviral innate immune signal pathway) was detected by co-immunoprecipitation (Co-IP). In addition, RNF39 promoted DDX3 ubiquitination. These data indicated that RNF39 inhibited RIG-I-induced signaling activation, and DDX3 may be its target. However, the exact mechanisms of RNF39 in regulating innate immune responses and the potential roles of RNF39 in viral infection related diseases remain unknown. In the present proposal, we plan to use RNF39 deficient mice, several model virus (such as VSV, SeV and HSV-1) and varieties of molecular and immunology techniques, to explore the functions of RNF39 in the regulation of antiviral innate immunity, reveal the possible molecular mechanisms (whether RNF39 regulates PRR-induced signaling by promoting DDX3 protein degradation via ubiquitin-proteasome pathway), clarify the roles of RNF39 in RNA viral infection and DNA viral infection and supply ideal target for vaccine development and drug design.

固有免疫是机体抗病毒的第一道防线，其适度活化对于及时清除入侵病毒和维持免疫稳态至关重要；因此，揭示其调控机制对阐明病毒与宿主间相互作用及病毒免疫逃逸机制具有重要意义。E3泛素连接酶RNF39基因位于MHC-I类分子基因编码区；我们发现RNF39缺陷及干扰均明显促进RNA病毒感染诱导的IFN-β表达；免疫共沉淀发现RNF39特异性结合抗病毒固有免疫通路中的关键分子DDX3，促进其泛素化；以上提示RNF39抑制RIG-I信号通路活化，且靶点可能是DDX3；但其具体调控机制及在病毒感染中的作用尚不清楚。本研究利用RNF39基因敲除小鼠、病毒模型(RNA病毒VSV和SeV，DNA病毒HSV-1)，探讨RNF39在抗病毒固有免疫调控中的作用及分子机制（即调节作用是否通过介导DDX3的泛素化降解来实现）；揭示RNF39在RNA病毒和DNA病毒感染中的作用，为防治相关疾病的药物、疫苗研发提供理论依据。

病毒性疾病，如新型冠状病毒肺炎(COVID-19)、流感等，严重危害人类健康。揭示病毒和宿主的相互作用、阐明抗病毒固有免疫信号通路激活及调控机制，对于发掘潜在的抗病毒药物靶点，进而有效防控病毒性疾病，具有重要意义。在本项目资助下，我们以巨噬细胞为研究模型，对机体抗病毒免疫应答的调控进行研究，并取得了阶段性成果。负责人作为通讯作者在Sci.Adv、Nat.Immunol、Nat.Commun、J.Immunol等杂志发表7篇，5年平均影响因子10以上论文4篇；2021年获得国家自然科学基金委杰出青年基金资助。.我们发现E3泛素连接酶RNF39缺陷及干扰均明显促进RNA病毒感染诱导的IFN-β表达；免疫共沉淀发现RNF39可特异性结合抗病毒固有免疫通路中的关键分子DDX3X，并可通过其E3泛素连接酶活性介导DDX3X K48位偶联泛素化，促进DDX3X的蛋白酶体途径降解，从而抑制RLR介导的信号通路。同时，RNF39缺失增强了机体抗RNA病毒先天免疫反应并抑制病毒复制。这一系列研究成果进一步阐明了病毒感染过程中固有免疫激活和调控的分子机制，揭示和丰富了抗病毒固有免疫信号通路调节网络，可为抗病毒药物设计和疫苗研制提供新的靶点。