宿主TRIM56蛋白与猪瘟病毒N~(pro)蛋白的相互作用及其对猪瘟病毒复制的影响
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摘要
目的分析宿主TRIM56蛋白与猪瘟病毒(classical swine fever virus,CSFV)N~(pro)蛋白之间的相互作用,并检测TRIM56对CSFV增殖的影响。方法 siRNA干扰法下调细胞中TRIM56表达后接种CSFV,利用Western blot及荧光定量RT-PCR法检测TRIM56表达下调对CSFV增殖的影响;利用免疫共沉淀技术(Co-IP)和Western blot法分析TRIM56与N~(pro)之间的相互作用关系;利用激光共聚焦试验检测TRIM56与N~(pro)共定位于细胞内的具体位置。结果下调TRIM56表达后接种CSFV,细胞中CSFV基因组拷贝数和滴度均明显升高。TRIM56与N~(pro)能特异性相互作用,并共定位于HEK 293T细胞的胞桨内。结论宿主TRIM56蛋白与CSFV的N~(pro)蛋白存在相互作用,并且能够负调控CSFV的增殖。
Objective To analyze the interaction between TRIM56 and N~(pro) protein of classical swine fever virus(CSFV)and investigate the effect of host TRIM56 on replication of the virus. Methods PK-15 cells in which the expression of TRIM56 was down-regulated by using si RNA were inoculated with CSFV. The effect of the down-regulation of TRIM56 expression on proliferation of CSFV was evaluated by Western blot and fluorescent quantitative RT-PCR. The interaction between TRIM56 and N~(pro)was confirmed by co-immunoprecipitation(Co-IP) assay and Western blot. The co-location of TRIM56 and N~(pro)was determined by laser confocal microscopy. Results Both the genomic copy number and titer of CSFV of down-regulation of TRIM56 expression increased significantly. TRIM56 and N~(pro)were interacted specifically and colocalized in the cytoplasm of HEK 293 T cells. Conclusion Host TRIM56 showed interaction with CSFV N~(pro)protein and negatively regulated the replication of CSFV.
Objective To analyze the interaction between TRIM56 and N~(pro) protein of classical swine fever virus(CSFV)and investigate the effect of host TRIM56 on replication of the virus. Methods PK-15 cells in which the expression of TRIM56 was down-regulated by using si RNA were inoculated with CSFV. The effect of the down-regulation of TRIM56 expression on proliferation of CSFV was evaluated by Western blot and fluorescent quantitative RT-PCR. The interaction between TRIM56 and N~(pro)was confirmed by co-immunoprecipitation(Co-IP) assay and Western blot. The co-location of TRIM56 and N~(pro)was determined by laser confocal microscopy. Results Both the genomic copy number and titer of CSFV of down-regulation of TRIM56 expression increased significantly. TRIM56 and N~(pro)were interacted specifically and colocalized in the cytoplasm of HEK 293 T cells. Conclusion Host TRIM56 showed interaction with CSFV N~(pro)protein and negatively regulated the replication of CSFV.
引文
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[9]RAJSBAUM R,GARCIA-SASTRE A,VERSTEEG G A.TRIM-munity:the roles of the TRIM E3-ubiquitin ligase family in innate antiviral immunity[J].J Mol Biol,2014,426(6):1265-1284.
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[12]GOTTIPATI K,RUGGLI N,GERBER M,et al.The structure of classical swine fever virus Npro:a novel cysteine autoprotease and zinc-binding protein involved in subversion of type I interferon induction[J].PLoS Pathog,2013,9(10):e1003704.doi:10.1371/journal.ppat.1003704.
[13]ZHANG S Q,TAN B,DING Y L,et al.Complete genome sequence and pathogenesis of bovine viral diarrhea virus JL-1isolate from cattle in China[J].Virol J,2014,11(1):67.doi:10.1186/1743-422X-11-67.
[14]王翠辉,那雷,王晓钧.TRIM蛋白参与天然免疫信号转导的研究进展[J].中国预防兽医学报,2016,38(2):166-170.
[15]VERSTEEG G A,RAJSBAUM R,SANCHEZ-APARICIO MT,et al.The E3-ligase TRIM family of proteins regulates signaling pathways triggered by immune pattern-recognition receptors[J].Immunity,2013,38(2):384-398.
[16]LIU B M,LI N L,WANG J,et al.Overlapping and distinct molecular determinants dictating the antiviral activities of TRIM56 against Flaviviruses and Coronavirus[J].J Virol,2014,88(23):13821-13835.
[17]SHEN Y,LI N L,WANG J,et al.TRIM56 is an essential component of the TLR3 antiviral signaling pathway[J].J Biol Chem,2012,287(43):36404-36413.
[18]LIU B M,LI N L,SHEN Y,et al.The C-terminal tail of TRIM56dictates antiviral restriction of influenza A and B viruses by impeding viral RNA synthesis[J].J Virol,2016,90(9):4369-4382.
[2]RAWLING N D,WALLER M,BARRETT A,et al.MEROPS:the database of proteolytic enzymes,their substrates and inhibitors[J].Nucleic Acids Res,2014,42(1):D503-509.
[3]TARRADAS J,DE LA TORRE M E,ROSELL R,et al.The impact of CSFV on the immune response to control infection[J].Virus Res,2014,185(7):82-91.
[4]FIEBACH A R,GUZYLACK-PIRIOU L,PYTHON S,et al.Classical swine fever virus N(pro)limits type I interferon induction in plasmacytoid dendritic cells by interacting with interferon regulatory factor 7[J].J Virol,2011,85(16):8002-8011.
[5]LI D,DONG H,LI S,et al.Hemoglobin subunit beta interacts with the capsid protein and antagonizes the growth of classical swine fever virus[J].J Virol,2013,87(10):5707-5717.
[6]METZ P,REUTER A,BENDER S,et al.Interferon-stimulated genes and their role in controlling hepatitis C virus[J].JHepatol,2013,59(6):1331-1341.
[7]LESTER S N,LI K.Toll-like receptors in antiviral innate immunity[J].J Mol Biol,2014,426(6):1246-1264.
[8]BAUMANN J G.Intracellular restriction factors in mammalian cells-An ancient defense system finds a modern foe[J].Curr HIV Res,2006,4(2):141-168.
[9]RAJSBAUM R,GARCIA-SASTRE A,VERSTEEG G A.TRIM-munity:the roles of the TRIM E3-ubiquitin ligase family in innate antiviral immunity[J].J Mol Biol,2014,426(6):1265-1284.
[10]KANE M,ZANG T M,RIHN S J,et al.Identification of Interferon-stimulated genes with antiretroviral activity[J].Cell Host Microbe,2016,20(3):392-405.
[11]WANG J,LIU B M,WANG N,et al.TRIM56 is a virus-and interferon-inducible E3 ubiquitin ligase that restricts pestivirus infection[J].J Virol,2011,85(8):3733-3745.
[12]GOTTIPATI K,RUGGLI N,GERBER M,et al.The structure of classical swine fever virus Npro:a novel cysteine autoprotease and zinc-binding protein involved in subversion of type I interferon induction[J].PLoS Pathog,2013,9(10):e1003704.doi:10.1371/journal.ppat.1003704.
[13]ZHANG S Q,TAN B,DING Y L,et al.Complete genome sequence and pathogenesis of bovine viral diarrhea virus JL-1isolate from cattle in China[J].Virol J,2014,11(1):67.doi:10.1186/1743-422X-11-67.
[14]王翠辉,那雷,王晓钧.TRIM蛋白参与天然免疫信号转导的研究进展[J].中国预防兽医学报,2016,38(2):166-170.
[15]VERSTEEG G A,RAJSBAUM R,SANCHEZ-APARICIO MT,et al.The E3-ligase TRIM family of proteins regulates signaling pathways triggered by immune pattern-recognition receptors[J].Immunity,2013,38(2):384-398.
[16]LIU B M,LI N L,WANG J,et al.Overlapping and distinct molecular determinants dictating the antiviral activities of TRIM56 against Flaviviruses and Coronavirus[J].J Virol,2014,88(23):13821-13835.
[17]SHEN Y,LI N L,WANG J,et al.TRIM56 is an essential component of the TLR3 antiviral signaling pathway[J].J Biol Chem,2012,287(43):36404-36413.
[18]LIU B M,LI N L,SHEN Y,et al.The C-terminal tail of TRIM56dictates antiviral restriction of influenza A and B viruses by impeding viral RNA synthesis[J].J Virol,2016,90(9):4369-4382.