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 in vitro and showed that porcine Mx2 (poMx2), human MxA (huMxA), and mouse Mx1 (mmMx1) also have anti-CSFV activity in vitro. 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 S-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.

IMPORTANCE Our previous studies have shown that porcine Mx1 (poMx1) inhibits classical swine fever virus (CSFV) replication in vitro and in vivo, but the molecular mechanism of action remains largely unknown. In this study, we dissect the molecular mechanism of porcine Mx1 and Mx2 against CSFV in vitro. 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GTPases，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酶的动力超家族。在这项研究中，我们证实了猪Mx1的体外抗经典猪瘟病毒（CSFV）活性，并显示猪Mx2（poMx2），人MxA（huMxA）和小鼠Mx1（mmMx1）在体外也具有抗CSFV活性。体外。小型干扰RNA（siRNA）实验表明，内源性poMx1或poMx2的耗尽增强了CSFV复制，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）证明poMx1与NS5B有关，NS5B是CSFV的RNA依赖性RNA聚合酶（RdRp）。我们使用poMx1蛋白中的突变阐明了其抗CSFV活性的机制，发现破坏与NS5B关联的突变体失去了所有抗CSV活性。此外，RdRp活性测定进一步揭示了poMx1破坏了NS5B的RdRp活性。总之，这些结果表明，猪Mx蛋白通过与NS5B相互作用而发挥了针对CSFV的抗病毒活性。

重要性我们先前的研究已经表明，猪的Mx1（poMx1）抑制猪瘟病毒（CSFV）复制在体外和体内，但作用的分子机制仍知之甚少。在这项研究中，我们剖析了猪Mx1和Mx2体外抗CSFV的分子机制。我们的结果表明poMx1与NS5B（CSFV的RNA依赖性RNA聚合酶）缔合，导致CSFV复制的减少。此外，poMx1的突变体进一步阐明了其抗CSFV活性的机制。