USP18分子通过Ⅰ型干扰素通路影响猪瘟病毒复制的研究
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摘要
猪瘟病毒(CSFV)感染无特定病原(SPF)猪后,分离猪外周血单个核细胞进行转录组分析,数据显示病毒感染后泛素特异性蛋白酶18(USP18)基因的转录水平明显上升。为研究USP18分子对CSFV复制的影响及其作用机制,本研究通过构建过表达USP18的细胞系及应用特异性小干扰RNA下调表达USP18的方法,采用荧光定量PCR验证USP18对CSFV增殖的影响,结果显示USP18分子能够促进CSFV的复制;通过检测IFN-β、NF-κB及ISRE的启动子活性及I型干扰素(IFN-I)信号通路的下游分子m RNA的转录水平,采用荧光定量PCR和双荧光酶报答试验分析USP18对IFN-I信号通路的影响,结果显示USP18分子抑制IFN-I信号通路。以上结果表明CSFV通过诱导USP18分子的上调表达,抑制了IFN-I途径,从而逃避天然免疫防御,进而促进自身的复制。本研究为明确CSFV与宿主之间的相互作用及CSFV致病机制提供参考依据和奠定基础。
Peripheral blood mononuclear cells(PBMCs) isolated from classical swine fever virus(CSFV)-infected specific-pathogen-free(SPF) pigs were examined by transcriptome analysis, and the data indicated that the m RNA transcription level of ubiquitin-specific protease 18(USP18) was significantly increased during CSFV infection. To investigate the effect of USP18 on the CSFV infection and its molecular mchanisms, PK-15(porcine kindey) cells were transduced by the lentivirus carrying USP18 gene and infected with CSFV shimen strain. The results demonatrated that overexpression of USP18 in PK-15 cells significantly enhanced the replication of CSFV, whereas knockdown of USP18 expression by specific small interfering RNAs significantly inhibited CSFV growth. Furthermore, overexpression of USP18 inhibited promoter activities of interferon beta(IFN-β), nuclear factor-kappa B(NF-κB) or interferon-stimulated response element(ISRE) in a dose-dependent manner, which resulted in the reduction of the ISGs m RNA transcription including Mx1 and GBP1. Taken together, our findings reveal that CSFV promotes up-regulation of USP18 to block the type I interferon signaling pathway and facilitates the virus replication.
Peripheral blood mononuclear cells(PBMCs) isolated from classical swine fever virus(CSFV)-infected specific-pathogen-free(SPF) pigs were examined by transcriptome analysis, and the data indicated that the m RNA transcription level of ubiquitin-specific protease 18(USP18) was significantly increased during CSFV infection. To investigate the effect of USP18 on the CSFV infection and its molecular mchanisms, PK-15(porcine kindey) cells were transduced by the lentivirus carrying USP18 gene and infected with CSFV shimen strain. The results demonatrated that overexpression of USP18 in PK-15 cells significantly enhanced the replication of CSFV, whereas knockdown of USP18 expression by specific small interfering RNAs significantly inhibited CSFV growth. Furthermore, overexpression of USP18 inhibited promoter activities of interferon beta(IFN-β), nuclear factor-kappa B(NF-κB) or interferon-stimulated response element(ISRE) in a dose-dependent manner, which resulted in the reduction of the ISGs m RNA transcription including Mx1 and GBP1. Taken together, our findings reveal that CSFV promotes up-regulation of USP18 to block the type I interferon signaling pathway and facilitates the virus replication.
引文
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[6] Fan Wei-guo, Xie Shi-qi, Zhao Xin-hao, et al. IFN-lambda4 desensitizes the response to IFN-alpha treatment in chronic hepatitis C through long-term induction of USP18[J]. J Gen Virol,2016, 97(9):2210-2220.
[7] Huang Meng-hao, Jiang Jian-dong, Peng Zong-gen. Recent advances in the anti-HCV mechanisms of interferon[J]. Acta Pharm Sin B, 2014, 4(4):241-247.
[8] Randall G, Chen Li-min, Panis M, et al. Silencing of USP18 potentiates the antiviral activity of interferon against hepatitis C virus infection[J]. Gastroenterology, 2006, 131(5):1584-1591.
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[10] Li Lin, Lei Qing-song, Zhang Shu-Jun, et al. Suppression of USP18 Potentiates the Anti-HBV Activity of Interferon Alpha in HepG2.2.15 Cells via JAK/STAT Signaling[J]. PLoS One,2016, 11(5):e0156496.
[11] Zhang Man, Zhang Meng-xing, Zhang Qiang, et al. USP18 recruits USP20 to promote innate antiviral response through deubiquitinating STING/MITA[J]. Cell Res, 2016, 26(12):1302-1319.
[12] Tanikawa T, Uchida Y, Saito T. Replication of a low-pathogenic avian influenza virus is enhanced by chicken ubiquitin-specific protease 18[J]. J Gen Virol, 2017, 98(9):2235-2247.
[13] Xu De-quan, Lillico SG, Barnett MW, et al. USP18 restricts PRRSV growth through alteration of nuclear translocation of NF-kappaB p65 and p50 in MARC-145 cells[J]. Virus Res,2012, 169(1):264-267.
[14] Shen Liang, Li Yong-feng, Chen Jia-ning, et al. Generation of a recombinant classical swine fever virus stably expressing the firefly luciferase gene for quantitative antiviral assay[J]. Antiviral Res, 2014, 109:15-21.
[15] Wang Jing-han, Sun Yuan, Meng Xing-yu, et al. Comprehensive evaluation of the host responses to infection with differentially virulent classical swine fever virus strains in pigs[J]. Virus Res,2018, 255:68-76.
[16] Li Lian-feng, Yu Jia-hui, Li Yong-feng, et al. Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GT-Pase Activity[J]. J Virol, 2016, 90(9):4412-4426.
[17] Zhao Jian-jun, Cheng Dan, Li Na, et al. Evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type viruses and C-strain vaccine of Classical swine fever virus[J]. Vet Microbiol, 2008, 126(1-3):1-10.
[18] Li Dan, Dong Hong, Li So, 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.
[19] Reed L M H. A simple method of estimating 50 percent endpoints[J]. Am J Hyg, 1938, 27:493-497.
[20] Takeuchi O, Akira S. Pattern recognition receptors and inflammation[J]. Cell, 2010, 140(6):805-820.
[21] Bauhofer O, Summerfield A, Sakoda Y, et al. C.lassical swine fever virus Npro interacts with interferon regulatory factor 3 and induces its proteasomal degradation[J]. J Virol, 2007, 81(7):3087-3096.
[22] Li Su, Wang Jing-han, Yang Qian, et al. Complex Virus-Host Interactions Involved in the Regulation of Classical Swine Fever Virus Replication:A Minireview[J]. Viruses, 2017, 9(7). pii:E171.
[2] Kellam P. Attacking pathogens through their hosts[J]. Genome Biol, 2006, 7(1):201.
[3] Metz P, Reuter A, Bender S, et al. Interferon-stimulated genes and their role in controlling hepatitis C virus[J]. J Hepatol,2013, 59(6):1331-1341.
[4] Ritchie K J, Hahn C S, Kim K I, et al. Role of ISG15 protease UBP43(USP18)in innate immunity to viral infection[J]. Nat Med, 10(12):1374-1378.
[5] Honke N, Shaabani N, Zhang Dong-Er, et al. Multiple functions of USP18[J]. Cell Death Dis, 2016, 7(11):e2444.
[6] Fan Wei-guo, Xie Shi-qi, Zhao Xin-hao, et al. IFN-lambda4 desensitizes the response to IFN-alpha treatment in chronic hepatitis C through long-term induction of USP18[J]. J Gen Virol,2016, 97(9):2210-2220.
[7] Huang Meng-hao, Jiang Jian-dong, Peng Zong-gen. Recent advances in the anti-HCV mechanisms of interferon[J]. Acta Pharm Sin B, 2014, 4(4):241-247.
[8] Randall G, Chen Li-min, Panis M, et al. Silencing of USP18 potentiates the antiviral activity of interferon against hepatitis C virus infection[J]. Gastroenterology, 2006, 131(5):1584-1591.
[9] Cai Jing, Liu Tian-de, Jiang Xiao-liu, et al. Downregulation of USP18 inhibits growth and induces apoptosis in hepatitis B virus-related hepatocellular carcinoma cells by suppressing BCL2L1[J]. Exp Cell Res, 2017, 358(2):315-322.
[10] Li Lin, Lei Qing-song, Zhang Shu-Jun, et al. Suppression of USP18 Potentiates the Anti-HBV Activity of Interferon Alpha in HepG2.2.15 Cells via JAK/STAT Signaling[J]. PLoS One,2016, 11(5):e0156496.
[11] Zhang Man, Zhang Meng-xing, Zhang Qiang, et al. USP18 recruits USP20 to promote innate antiviral response through deubiquitinating STING/MITA[J]. Cell Res, 2016, 26(12):1302-1319.
[12] Tanikawa T, Uchida Y, Saito T. Replication of a low-pathogenic avian influenza virus is enhanced by chicken ubiquitin-specific protease 18[J]. J Gen Virol, 2017, 98(9):2235-2247.
[13] Xu De-quan, Lillico SG, Barnett MW, et al. USP18 restricts PRRSV growth through alteration of nuclear translocation of NF-kappaB p65 and p50 in MARC-145 cells[J]. Virus Res,2012, 169(1):264-267.
[14] Shen Liang, Li Yong-feng, Chen Jia-ning, et al. Generation of a recombinant classical swine fever virus stably expressing the firefly luciferase gene for quantitative antiviral assay[J]. Antiviral Res, 2014, 109:15-21.
[15] Wang Jing-han, Sun Yuan, Meng Xing-yu, et al. Comprehensive evaluation of the host responses to infection with differentially virulent classical swine fever virus strains in pigs[J]. Virus Res,2018, 255:68-76.
[16] Li Lian-feng, Yu Jia-hui, Li Yong-feng, et al. Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GT-Pase Activity[J]. J Virol, 2016, 90(9):4412-4426.
[17] Zhao Jian-jun, Cheng Dan, Li Na, et al. Evaluation of a multiplex real-time RT-PCR for quantitative and differential detection of wild-type viruses and C-strain vaccine of Classical swine fever virus[J]. Vet Microbiol, 2008, 126(1-3):1-10.
[18] Li Dan, Dong Hong, Li So, 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.
[19] Reed L M H. A simple method of estimating 50 percent endpoints[J]. Am J Hyg, 1938, 27:493-497.
[20] Takeuchi O, Akira S. Pattern recognition receptors and inflammation[J]. Cell, 2010, 140(6):805-820.
[21] Bauhofer O, Summerfield A, Sakoda Y, et al. C.lassical swine fever virus Npro interacts with interferon regulatory factor 3 and induces its proteasomal degradation[J]. J Virol, 2007, 81(7):3087-3096.
[22] Li Su, Wang Jing-han, Yang Qian, et al. Complex Virus-Host Interactions Involved in the Regulation of Classical Swine Fever Virus Replication:A Minireview[J]. Viruses, 2017, 9(7). pii:E171.