RNA解螺旋酶DDX5在抗病毒天然免疫反应中的调控作用及分子机制研究

Viral infection is a serious threat to human public health. Antiviral innate immunity is the first line of defense against viral infection. DEAD-box helicase 5 (DDX5) is a multifunctional protein, which is involved in a variety of biological processes. In recent years, many DDX family members have been reported to be involved in regulating antiviral innate immune response. However, the regulatory role and molecular mechanism of DDX5 in antiviral innate immune response are still not known. Our previous study found that the expression level of DDX5 protein was significantly upregulated in virus-infected mouse macrophages. Furthermore, DDX5 knockdown mediated by specific siRNA significantly inhibited virus infection-induced the increase of IFN-α/β expression and the phosphorylation of IRF3. Moreover, we found that DDX5 specifically interacted with protein phosphatase 2A catalytic subunit of beta (PP2A-Cβ), measured by mass spectrometry and co-immunoprecipitation assays. The above results indicated that DDX5 may facilitate the activation of IRF3 by interacting with PP2A-Cβ, thereby positively regulating the antiviral innate immune response. Based on that, this study aims to further explore the regulatory mechanism of DDX5 in the antiviral innate immune response in vitro and vivo, in order to provide new ideas for clinical treatment of viral infectious diseases, and to provide new potential targets for the development of antiviral drugs.

病毒感染严重威胁着人类公共卫生安全。抗病毒天然免疫是机体抵抗病毒感染的第一道防线。RNA解螺旋酶DDX5 (DEAD-box helicase 5) 是一种多功能生物活性蛋白，参与调控多种生物学过程。近年来，多个DDX家族成员被报道参与调控抗病毒天然免疫反应，然而DDX5在该过程中的调控作用及分子机制尚不清楚。我们前期研究发现DDX5蛋白在病毒感染的小鼠巨噬细胞中显著上调表达；siRNA敲低DDX5表达后能显著抑制病毒感染诱导的IFN-α/β表达以及IRF3磷酸化；质谱鉴定及免疫共沉淀实验发现DDX5与蛋白磷酸酶2A催化亚基beta（PP2A-Cβ）特异性互作，提示DDX5可能通过PP2A-Cβ影响IRF3活化，从而调控抗病毒天然免疫反应。本项目拟在体内外水平进一步探究DDX5在抗病毒天然免疫反应中的调控作用及其分子机制，为临床治疗病毒感染疾病提供新思路，为研发抗病毒药物提供新的潜在靶点。

病毒感染严重威胁着人类公共卫生安全。天然免疫是机体抵抗病毒感染的第一道防线。I-IFN 是机体抗病毒天然免疫反应中最重要的细胞因子之一，对机体抵抗病毒入侵起着至关重要的作用。RNA解螺旋酶DDX5是一种多功能生物活性蛋白，参与调控多种生物学过程。近年来，多个DDX家族成员被报道参与调控抗病毒天然免疫反应，然而DDX5在该过程中的调控作用及分子机制尚不清楚。本课题通过使用小鼠腹腔原代巨噬细胞，体外实验发现VSV或HSV感染促进DDX5上调表达；敲低DDX5显著促进VSV或HSV诱导的I-IFN及炎症因子的表达并抑制病毒复制；进一步发现敲低DDX5显著促进VSV诱导的IRF3和P65磷酸化蛋白水平上调；通过蛋白质免疫共沉淀实验及质谱分析发现蛋白磷酸酶 2A 催化亚基 beta（PP2A-Cβ）与DDX5发生互作，且VSV刺激增强蛋白间互作；敲低PP2A-Cβ显著增强VSV诱导的IRF3磷酸化蛋白水平上调及IFN-β表达；体内动物实验进一步发现敲低DDX5增强IFN-β表达，抑制VSV复制。这些结果揭示了RNA解螺旋酶DDX5通过与PP2A-Cβ蛋白互作，抑制IRF3磷酸化和激活，从而负调控抗病毒天然免疫反应的机制。本项目的实施丰富了对于机体抗病毒天然免疫反应的调控机理的认识，并且为精准研发抗病毒药物提供科学依据。