动脉炎病毒PRRSV和EAV的Nsp4蛋白对mTANK蛋白的降解  

Arteritis virus PRRSV and EAV Nsp4 protein degrades mTANK

作　　者：吴磊 袁旭 洪锦璇 陈叶 李训良 宋中宝 WU Lei;YUAN Xu;HONG Jinxuan;CHEN Ye;LI Xunliang;SONG Zhongbao(College of Animal Sciences(College of Bee Science),Fujian Agriculture and Forestry University;Key Laboratory of Fujian-Taiwan Animal Pathogen Biology,Fuzhou,Fujian 350002,China)

机构地区：[1]福建农林大学动物科学学院(蜂学学院) [2]闽台动物病原生物学福建省高校重点实验室,福建福州350002

出　　处：《福建农林大学学报（自然科学版）》2022年第5期675-681,共7页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：福建省自然科学基金项目(2020J05026);福建农林大学科技创新项目(CXZX2020060A).

摘　　要：为研究猪繁殖与呼吸综合征病毒(PRRSV)和马动脉炎病毒(EAV)等动脉炎病毒的Nsp4蛋白在病毒免疫逃逸中的作用,通过免疫沉淀和质谱技术筛选到与Nsp4互作的宿主蛋白mTANK.将pCAGGS-PRRSV-Nsp4-Flag、pCAGGS-EAV-Nsp4-Flag质粒分别与pCAGGS-mTANK-HA质粒共转染HEK293T细胞,发现Nsp4可特异性降解mTANK.为了进一步揭示其中的机制,利用蛋白酶体抑制剂MG-132、凋亡途径抑制剂Z-VAD-FMK、溶酶体抑制剂NHCl和自噬抑制剂3-MA分别处理共转染的细胞,发现这些抑制剂处理并不影响Nsp4对mTANK的降解作用,随后构建了PRRSV Nsp4(E39、E64、E118)和EAV Nsp4(E39、E65、E120)的酶活性突变体质粒.将突变体质粒分别与pCAGGS-mTANK-HA质粒共转染后发现,Nsp4蛋白酶活性缺失后无法降解mTANK.进一步研究发现,mTANK降解产生的片段分别位于30和25~30 ku附近.综上,PRRSV和EAV的Nsp4蛋白可通过其3C样蛋白酶活性特异性切割宿主蛋白mTANK.In order to study the role of Nsp4 protein of arteritis virus, porcine reproductive and respiratory syndrome virus(PRRSV) and equine infectious arteritis virus(EAV), in virus immune escape, the interaction between host protein mTANK and Nsp4 was screened through immunoprecipitation and mass spectrometry. After co-transfecting the pCAGGS-PRRSV-Nsp4-Flag and pCAGGS-EAV-Nsp4-Flag with pCAGGS-mTANK-HA plasmid into 293 T cells, respectively, the Nsp4 protein was shown to specifically degrade mTANK. In order to reveal the mechanism behind the specific degradation, the co-transfected cells were further treated with proteasome inhibitor MG-132, apoptosis pathway inhibitor Z-VAD-FMK, lysosomal inhibitor NHCl, and autophagy inhibitor 3-MA, respectively. It was found that these treatments did not affect the degradation of mTANK protein by Nsp4 protein. Subsequently, the enzymatic activity mutant plasmids of PRRSV Nsp4(E39, E64, E118) and EAV Nsp4(E39, E65, E120) proteins were constructed. After co-transfecting the mutant plasmids with the mTANK plasmid, the PRRSV and EAV Nsp4 mutants were unable to degrade the mTANK protein. Further experiment showed that the sizes of the fragments produced by the degradation were around 30 and 25-30 ku, respectively. In summary, the arteritis virus Nsp4 protein can specifically cleave the host protein mTANK through its 3 C-like serine protease activity.

关 键 词：动脉炎病毒 猪繁殖与呼吸综合征病毒 马动脉炎病毒 NSP4 mTANK 降解 

分 类 号：S855.3[农业科学—临床兽医学]