Zhou Jing, Chen Jing, Zhang Xiao-Min, Gao Zhi-Can, Liu Chun-Chun, Zhang Yun-Na, Hou Jin-Xiu, Li Zhao-Yao, Kan Lin, Li Wen-Liang, Zhou Bin
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Rugao Animal Husbandry and Veterinary Station, Rugao, China.
J Virol. 2018 Mar 14;92(7). doi: 10.1128/JVI.02147-17. Print 2018 Apr 1.
Mx proteins are interferon (IFN)-induced GTPases that have broad antiviral activity against a wide range of RNA and DNA viruses; they belong to the dynamin superfamily of large GTPases. In this study, we confirmed the anti-classical swine fever virus (CSFV) activity of porcine Mx1 and showed that porcine Mx2 (poMx2), human MxA (huMxA), and mouse Mx1 (mmMx1) also have anti-CSFV activity Small interfering RNA (siRNA) experiments revealed that depletion of endogenous poMx1 or poMx2 enhanced CSFV replication, suggesting that porcine Mx proteins are responsible for the antiviral activity of interferon alpha (IFN-α) against CSFV infection. Confocal microscopy, immunoprecipitation, glutathione -transferase (GST) pulldown, and bimolecular fluorescence complementation (BiFC) demonstrated that poMx1 associated with NS5B, the RNA-dependent RNA polymerase (RdRp) of CSFV. We used mutations in the poMx1 protein to elucidate the mechanism of their anti-CSFV activity and found that mutants that disrupted the association with NS5B lost all anti-CSV activity. Moreover, an RdRp activity assay further revealed that poMx1 undermined the RdRp activities of NS5B. Together, these results indicate that porcine Mx proteins exert their antiviral activity against CSFV by interacting with NS5B. Our previous studies have shown that porcine Mx1 (poMx1) inhibits classical swine fever virus (CSFV) replication and , but the molecular mechanism of action remains largely unknown. In this study, we dissect the molecular mechanism of porcine Mx1 and Mx2 against CSFV Our results show that poMx1 associates with NS5B, the RNA-dependent RNA polymerase of CSFV, resulting in the reduction of CSFV replication. Moreover, the mutants of poMx1 further elucidate the mechanism of their anti-CSFV activities.
Mx蛋白是干扰素（IFN）诱导的GTP酶，对多种RNA和DNA病毒具有广泛的抗病毒活性；它们属于大型GTP酶的发动蛋白超家族。在本研究中，我们证实了猪Mx1的抗经典猪瘟病毒（CSFV）活性，并表明猪Mx2（poMx2）、人MxA（huMxA）和小鼠Mx1（mmMx1）也具有抗CSFV活性。小干扰RNA（siRNA）实验表明，内源性poMx1或poMx2的缺失会增强CSFV复制，这表明猪Mx蛋白负责干扰素α（IFN-α）对CSFV感染的抗病毒活性。共聚焦显微镜、免疫沉淀、谷胱甘肽-S-转移酶（GST）下拉和双分子荧光互补（BiFC）实验表明，poMx1与CSFV的RNA依赖性RNA聚合酶（RdRp）NS5B相互作用。我们利用poMx1蛋白中的突变来阐明其抗CSFV活性的机制，发现破坏与NS5B相互作用的突变体失去了所有抗CSFV活性。此外，RdRp活性测定进一步表明，poMx1会破坏NS5B的RdRp活性。这些结果共同表明，猪Mx蛋白通过与NS5B相互作用发挥其对CSFV的抗病毒活性。我们之前的研究表明，猪Mx1（poMx1）可抑制经典猪瘟病毒（CSFV）复制，但其分子作用机制在很大程度上仍不清楚。在本研究中，我们剖析了猪Mx1和Mx2针对CSFV的分子机制。我们的结果表明，poMx1与CSFV的RNA依赖性RNA聚合酶NS5B相互作用，导致CSFV复制减少。此外，poMx1的突变体进一步阐明了其抗CSFV活性的机制。
