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| Gene Synthesis | pcDNA3.0-poMx1-HA and pEGFP- poMx1 have been described previously(Zhou et al., 2018). Mouse Mx1 (GenBank accession number: NP_034976) and Mx2 (GenBank accession number: NP_038634), which were kindly provided by Dr. Song Gao (Sun Yat-sen University Cancer Center, Guangzhou, China), were cloned into pcDNA3.0 and pEGFP-C1, resulting in pcDNA3.0-mmMx1-HA, pcDNA3.0-mmMx2-HA, pEGFP-mmMx1 and pEGFP-mmMx2. The mmMx1 mutants K49A, G83R, A222V, A516V, G540E and ΔL4 (deletion of residues 499–541) were constructed by directed mutagenesis (GenScript, Biotech, Corp, China) of pcDNA3.0-mmMx1-HA. The wild-type and mutant mmMx1 were also cloned into pGEX-4T-1 for GST pull down assays and VC for BiFC assays. CSFV Core, NS2, NS3, NS4B, NS5A and NS5B were cloned into the p3×FLAG-CMV™-7.1 for confocal and Co-IP assays. Core and NS5B were also cloned into VN for BiFC assays. The mutant mmMx1 (R614E), along with CSFV Core, was cloned into pET-28a and NS5B was cloned into pColdI for RdRp assays. All plasmids constructs were confirmed by DNA sequencing. All amplification primers used in this study are listed in Table 2. HEK293T or PK-15 cells were transfected with plasmids (2.5 μg each) using Lipofectamine 3000 (Invitrogen) in 6-well plates according to the manufacturer’s instructions. At 6 h post transfection (hpt), the transfection mixture was replaced with fresh DMEM containing 2% FBS and cells were incubated for an additional 48 h. | Get A Quote |
Mx proteins are interferon-induced GTPases that have broad antiviral activity against a wide range of RNA and DNA viruses. We previously demonstrated that porcine Mx1 protein (poMx1) inhibited the replication of classical swine fever virus (CSFV), an economically important Pestivirus, and that mouse Mx1 did so as well. It is unknown why the nucleus-localizing mouse Mx1 inhibits CSFV replication which occurs in the cytoplasm. To the end, we assessed the anti-CSFV actions of wild type mouse Mx1 and seven previously reported mutants (K49A, G83R, A222V, A516V, G540E, R614E and ΔL4) and identified the molecular mechanism of R614E action against CSFV replication. A series of experiments revealed that mmMx1 (R614E) ... More
