猪瘟病毒NS4B蛋白对RLRs介导的天然免疫应答的影响及机制

猪瘟病毒（CSFV）NS4B蛋白在病毒的复制过程中发挥重要作用，前期研究发现CSFV NS4B能抑制猪肺泡巨噬细胞中RIG-I和MDA5介导的天然免疫效应分子（IFN-β和IL-6）的表达，但其调控RLRs介导的天然免疫应答的机制还不清楚。本项目拟在此基础上，分析NS4B对RLRs信号通路蛋白介导的天然免疫应答的影响，进一步通过酵母双杂交、激光共聚焦、Co-IP、GST pull-down等技术筛选与NS4B相互作用的“靶点蛋白”，并确定NS4B与“靶点蛋白”相互作用的关键区域和位点。利用CRISPR/Cas9敲除技术分析“靶点蛋白”对RLRs介导天然免疫应答的影响，结合NS4B蛋白、NS4B关键区域和NS4B突变体对“靶点蛋白”的表达及其功能的影响，明确NS4B抑制RLRs介导的天然免疫应答的机制，为揭示CSFV NS4B在CSFV致病机理中的作用提供理论依据。

Classical swine fever virus (CSFV) non-structural protein 4B (NS4B) plays an important role in virus replication. Our studies have found that CSFV NS4B inhibited the expression of IFN-β and IL-6 mediated by retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) in porcine alveolar macrophages (PAM), but the mechanism remains unclear. Based on the inhibition of RLRs-mediated innate immune response by CSFV NS4B, this study will determine the mechanism of that NS4B regulated RLRs-mediated innate immune response. Firstly, the effect of NS4B on RLRs signaling protein-mediated innate immune response will be investigated. In addition, this project will select the “target proteins” interacting with CSFV NS4B by yeast two-hybrid screening, laser scanning confocal microscope, Co-IP and GST pull-down. Furthermore, the key domain and amino acid of NS4B that interacts with “target proteins” will be confirmed. The effect of “target proteins” on RLRs-mediated innate immune response will be analyzed by CRISPR/Cas9 knockout technologies, and the effect of NS4B, NS4B key domain and NS4B mutants on target proteins expression and function will be performed to clear the mechanism of RLRs-mediated innate immune response regulated by CSFV NS4B. The results will provide theoretical basis for revealing the role of NS4B in CSFV pathogenesis.