Luciferase activity was measured 24 h post-transfection. a significant decrease in the expression of interferons and interferon-stimulating genes in cells infected with SeV. Similar results were obtained from experiments using I226R-overexpressed PK15 and 3D4/21 cells stimulated with vesicular stomatitis virus. We observed that I226R inhibited the activation of both nuclear factor-kappa B (NF-B) and interferon regulatory factor 3 (IRF3). Furthermore, it was shown that overexpression of I226R suppressed IRF3 activation and caused the degradation of NF-B essential modulator (NEMO) protein. The I226R-induced NEMO degradation could be prevented by treatment with MG132, a proteasome inhibitor. Together, 6-Maleimido-1-hexanol these results reveal that the ASFV I226R protein impairs antiviral responses, likely through multiple mechanisms including the suppression of NF-B and IRF3 activation, to counteract innate immune responses during the viral infection. gene. The presence of the gene was first reported in 1996 by transcriptional analysis [31]. Thereafter, a subsequent report revealed that the I226R protein could be expressed in the early and late stages of viral infection [32]. Recently, it was reported that I226R knockout attenuated ASFV in pigs. Moreover, pigs infected with I226R-deleted virus were resistant to infection by its virulent parental virus, indicating that I226R is a promising candidate to design recombinant vaccines for genotype II ASF [33]. However, the biological function of the I226R protein is largely unknown. In 6-Maleimido-1-hexanol FACD this study, we reveal that the ASFV I226R protein can antagonize innate immunity by inhibiting the activation of NF-B and the IRF3 signaling pathway. Our findings 6-Maleimido-1-hexanol uncover a new mechanism of how ASFV evades innate immunity, and provide a new theoretical basis in designing a recombinant vaccine. 2. Materials and Methods 2.1. Cells, Virus and Plasmids For this study, 6-Maleimido-1-hexanol 293T, MDCK, PK15, 3D4/21 cells were purchased from American Type Culture Collection (ATCC). The 293T, MDCK, and PK15 cells were maintained in Dulbeccos Modified Eagles Medium (DMEM, Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), penicillin (100 U/mL), and streptomycin (100 g/mL). The 3D4/21 cells were maintained in Roswell Park Memorial 6-Maleimido-1-hexanol Institute 1640 Medium (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA), containing 10% FBS, 1% nonessential amino acids (Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 g/mL streptomycin. All cells were cultured at 37 C under 5% CO2 atmosphere, as previously described [34]. Sendai virus (SeV) was generated and propagated in specific-pathogen-free chicken embryos, as previously described [35]. The virus titer was determined by a hemagglutination test, as described previously [36]. Vesicular stomatitis virus (VSV) was preserved in our laboratory and propagated in PK15 cells. The A137R, K145R, A151R, KP177R, K205R, I215L, I226R, I243L, E248R, or C257L coding sequence of the ASFV isolate China/2018/AnhuiXCGQ (Gen-108 Bank accession number: “type”:”entrez-nucleotide”,”attrs”:”text”:”MK128995.1″,”term_id”:”1517383423″,”term_text”:”MK128995.1″MK128995.1), fused with the FLAG tag, was optimized and synthesized by Genewiz Biotechnology Co., Ltd. (Suzhou, China) and cloned into a pcDNA3.1(?) vector. Recombinant plasmids were confirmed by restriction digestion and sequencing. Other human genes (RIG-I, MAVS, RIP1, TRAF2, TRAF6, TAK1, IRF3), fused with specific peptide tags, were also cloned into indicated vectors, as described previously [37]. The plasmids IKK-Flag, p65-Flag, and NEMO-Flag were purchased from Wuhan Miaoling Biotechnology Co., Ltd. (Wuhan, China). The luciferase reporter plasmids used in this study included an IFN- promoter reporter plasmid (IFN–Luc), carrying promoter sequences of IFN- used to examine the activation of the IFN- promoter; an NF-B-Luc plasmid, harboring NF-B-binding elements used to detect the activation of NF-B; a (pRDIII-I)4-Luc plasmid, carrying IRF3-binding elements used to examine the activity of IRF3; and a control reporter plasmid, pRL-TK. All these reporter plasmids were gifts from Dr. Chunfu Zheng (Fujian Medical University, Fuzhou, China) [38,39]. An ISRE luciferase reporter plasmid (ISRE-Luc), harboring interferon (IFN)-stimulated response elements used to detect IFN production and secretion, was purchased from YEASEN Biotechnology Co.,.