新型冠状病毒PLpro负调控DDX3诱导的β干扰素抗病毒免疫通路  

SARS-CoV-2 PLpro negatively regulates interferon-βimmune pathway induced by DDX3

作　　者：王明宇 陈晓娟 孟欢 邵丽婷 焦园园 李文倩 李萍 邢雅玲 WANG Mingyu;CHEN Xiaojuan;MENG Huan;SHAO Liting;JIAO Yuanyuan;LI Wenqian;LI Ping;XING Yaling(Bioinformatics Center,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Institute of Radiation Medicine,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China;Department of Clinical Laboratory,Beijing Ditan Hospital,Capital Medicine University,Beijing 100015,China)

机构地区：[1]军事科学院军事医学研究院生物信息中心,北京100850 [2]军事科学院军事医学研究院辐射医学研究所,北京100850 [3]首都医科大学附属地坛医院检验科,北京100015

出　　处：《军事医学》2024年第6期453-460,共8页Military Medical Sciences

基　　金：国家自然科学基金面上项目(82072285)。

摘　　要：目的发现严重急性呼吸综合征冠状病毒2(SARS-CoV-2)木瓜样蛋白酶(PLpro)的宿主互作分子,探索其生物学意义。方法邻近蛋白标记结合质谱分析筛选宿主互作分子DEAD-box解旋酶3(DDX3);免疫荧光法分析PLpro与DDX3的胞内共定位;免疫共沉淀法分析PLpro与DDX3的相互作用,以及PLpro对DDX3与IκB激酶ε(IKKε)和TANK结合激酶1(TBK1)、DDX3与线粒体抗病毒信号蛋白(MAVS)互作的影响;荧光素酶报告基因法检测PLpro对β干扰素(IFN-β)通路的影响和对底物的酶切效率。结果DDX3能够与PLpro发生细胞内共定位和相互作用;PLpro可能抑制DDX3-MAVS复合体形成,抑制DDX3-IKKε-TBK1之间的相互作用,负调控Ⅰ型干扰素通路;DDX3过表达情况下,PLpro/PLP-TM对底物位点的切割效率显著升高,而DDX3表达降低时,切割效率则显著降低。结论DDX3可能是PLpro的宿主相互作用分子之一;PLpro负调控DDX3活化的IFN-β表达,为病毒复制提供有利的免疫环境,提升蛋白酶切割活性,共同促进病毒复制。Objective To discover the host factor interacting with severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)papain-like protease(PLpro)and explore the potential mechanism.Methods The second-generation proximity-dependent biotin identification(BioID2)approach combined with mass spectrometry analysis was used to search for the potential host factors.Immunofluorescence and co-immunoprecipitation(Co-IP)assay were used to verify the interactions between DEAD-box helicase 3(DDX3)and PLpro.The influence of PLpro on DDX3-inhibitor of kappa B kinaseε(IKKε)-TANK-binding kinase 1(TBK1)and DDX3-mitochondrial antiviral signaling protein(MAVS)complexes was also investigated by Co-IP.The effect of PLpro on interferon-β(IFN-β)immune pathway and the protease activity on substrates were studied via luciferase activity assay.Results DDX3 could co-locate and interact with PLpro intracellularly.PLpro might possibly inhibit both the formation of DDX3-MAVS complex and the interactions between DDX3-IKK-ε-TBK1.PLpro could negatively regulate typeⅠinterferon pathway.Overexpression of DDX3 could lead to a significant increase in the cleavage activity of PLpro/PLP-TM that might be significantly decreased in case of inventions with DDX3 expressions.Conclusion DDX3 may be one of the host factors that interact with SARS-CoV-2 PLpro.PLpro negatively regulates IFN-βimmune pathway induced by DDX3,which may provide a favorable immune environment for virus replication.

关 键 词：严重急性呼吸综合征冠状病毒2 木瓜样蛋白酶 DEAD-box解旋酶3 抗病毒天然免疫 

分 类 号：Q27[生物学—细胞生物学] Q937[医药卫生—免疫学] R392.1[医药卫生—基础医学]