在已结题的项目研究中发现猪瘟病毒 NS5A蛋白与NF-κB必需调节蛋白NEMO和信号转导与转录激活因子3（STAT3）相互作用。NEMO是IκB 激酶（IKK）复合物的调节亚单位，调控IKK和NF-κB激活。预研结果显示，NS5A 126-250aa片段介导NS5A与NEMO相互作用及抑制NF-κB信号。荧光素酶报告基因分析表明NS5A抑制STAT3转录活性。这些结果显示NS5A通过阻断NF-κB和STAT3信号通路抑制宿主抗病毒天然免疫反应。本项目拟用免疫沉淀等方法，分析NS5A对 NEMO泛素化、与其它信号分子结合等与IKK激活有关事件的影响，分析NS5A对STAT3磷酸化、二聚体化等环节的抑制作用，阐明NS5A蛋白阻断NF-κB和STAT3信号通路进而抑制宿主抗病毒天然免疫的分子机制。研究成果将为深入揭示猪瘟病毒免疫抑制的分子机制和创新防控策略提供科学依据。

Result of our finished project showed that CSFV NS5A interacted with cellular nuclear factor- kappaB (NF-κB) essentioal modulator (NEMO or IKKγ) and signal transducer and activator of transcription 3 (STAT3). NEMO is a regulatory subunit of IκB kinase (IKK) complex, regulating IKK and NF-κB activation induced by diverse stimulations. The IKK complex contains two catalytic subunits of IKKα, IKKβ and a regulatory subunit of NEMO. Upon IKK activation, IKKβ phosphorylates IκBα proteins on two N-terminal serine residues and then results in proteasomal degradation of IκBα, which allows NF-κB nuclear translocation and activation of canonical NF-κB pathway. STAT3 is a cytoplasmic transcription factor. In response to stimulus signals, STAT3 is phosphorylated at its conserved 705 tyrosine residue and which in turn results in STAT3 homo- and hetero-dimerization with STAT1and translocation into nucleus and regulates the target genes transcription. NF-κB and STAT3 are involved in regulation of inflammation, innate and adaptive immunity, apoptosis, and tumorigenesis. Preliminary study showed that the 126-250aa fragment is responsible for the interaction of NS5A with NEMO and the inhibition of NF-κB signaling induced by TNFα. Luciferase reporter analysis showed that NS5A inhibited STAT3 transcriptional activity. Therefore, we speculates that NS5A inhibits host antiviral innate immune by blocking NF-κB and STAT3signal pathway. In this project, we will analyse the inhibition of NEMO ubiquitination, phosphorylation and its binding to other signal moleculars, and the degaradation of NEMO mediated by NS5A, analyse the inhibition of STAT3 phosphorylation, dimerization, nuclear translocation and the degaradation of STAT3 mediated by NS5A. Based on these analysis, the mechanism of the inhibition of NF-κB and STAT3 signaling pathway and further inhibition of host antiviral innate immune response by NS5A will be elucidated. The results will provide sciencific evidences for revealing the immunosuppressive mechanism of CSFV and innovating the prevention and control strategies.