塞内卡病毒A结构蛋白VP1诱导PK-15细胞凋亡
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摘要
塞内卡病毒A(SVA)作为一种新发病病原,致病机制仍不清楚。通过显微镜观察发现,SVA感染PK-15细胞后能使细胞产生明显的细胞病变(CPE),同时伴随着严重的细胞凋亡。为了深入研究SVA诱导凋亡的情况,在验证SVA各蛋白真核质粒正常表达后,通过Annexin V-FITC/PI双染流式方法检测SVA各蛋白诱导凋亡的情况,Annexin V-FITC/PI和Hoechst染色后显微镜观察磷脂酰丝氨酸和核凝聚程度,通过Western blotting和RT-PCR技术分析SVA-VP1对凋亡通路中主要调控分子Caspase3、Caspase8和Bax蛋白质和mRNA水平的影响,发现SVA-VP1能够显著诱导细胞发生早期和晚期凋亡,并且能够促进凋亡蛋白Caspase3、Caspase8及Bax蛋白和mRNA水平的上调。本研究结果为深入研究SVA调控宿主细胞凋亡的分子机制和致病机制奠定了理论基础。
As a new pathogen, the pathogenesis of Senecavirus A(SVA) remains unclear. Through microscopic observation, it was found that SVA can infect PK-15 cells and causes significant cytopathic(CPE). Concomitant with CPE, apoptosis is commonly observed in infected cells. In this study, the function of each individual SVA protein in the induction of apoptosis was studied. Firstly, using Western blotting confirmed the successful expression of SVA proteins. Subsequently, the SVA proteins were screened for their apoptotic function using flow cytometry with Annexin V-FITC/PI double staining. Then, the extent of externalized phospholipid phosphatidylserine and nuclear condensation induced by VP1 was analyzed by fluorescence microscope using Annexin V-FITC/PI and Hoechst staining. Finally, Western blotting and RT-PCR technique were used to analyze the effects of VP1 on the protein and mRNA levels of the major apoptosis regulatory molecules Caspase3, Caspase8 and Bax. SVA-VP1 was found to significantly induce early and late apoptosis in cells. And it also could promote the up-regulation of pro-apoptotic protein Caspase3, Caspase8 and Bax protein and mRNA levels. This study provides a theoretical basis for further research in molecular mechanism and the pathogenesis of SVA-regulated apoptosis.
As a new pathogen, the pathogenesis of Senecavirus A(SVA) remains unclear. Through microscopic observation, it was found that SVA can infect PK-15 cells and causes significant cytopathic(CPE). Concomitant with CPE, apoptosis is commonly observed in infected cells. In this study, the function of each individual SVA protein in the induction of apoptosis was studied. Firstly, using Western blotting confirmed the successful expression of SVA proteins. Subsequently, the SVA proteins were screened for their apoptotic function using flow cytometry with Annexin V-FITC/PI double staining. Then, the extent of externalized phospholipid phosphatidylserine and nuclear condensation induced by VP1 was analyzed by fluorescence microscope using Annexin V-FITC/PI and Hoechst staining. Finally, Western blotting and RT-PCR technique were used to analyze the effects of VP1 on the protein and mRNA levels of the major apoptosis regulatory molecules Caspase3, Caspase8 and Bax. SVA-VP1 was found to significantly induce early and late apoptosis in cells. And it also could promote the up-regulation of pro-apoptotic protein Caspase3, Caspase8 and Bax protein and mRNA levels. This study provides a theoretical basis for further research in molecular mechanism and the pathogenesis of SVA-regulated apoptosis.
引文
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[26] ELMORE S.Apoptosis:a review of programmed cell death[J].Toxicol Pathol,2007,35(4):495-516.
[27] ZHOU X C,JIANG W B,LIU Z S,et al.Virus infection and death receptor-mediated apoptosis[J].Viruses,2017,9(11):316.
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[29] MILES L A,BURGA L N,GARDNER E E,et al.Anthrax toxin receptor 1 is the cellular receptor for Seneca Valley virus[J].J Clin Invest,2017,127(8):2957-2967.
[30] QIAN S H,FAN W C,LIU T T,et al.Seneca valley virus suppresses host type I interferon production by targeting adaptor proteins MAVS,TRIF,and TANK for cleavage[J].J Virol,2017,91(16):e00823-17.
[31] XUE Q,LIU H S,ZHU Z X,et al.Seneca valley virus 3Cpro abrogates the IRF3- and IRF7-mediated innate immune response by degrading IRF3 and IRF7[J].Virology,2018,518:1-7.
[32] XUE Q,LIU H S,ZHU Z X,et al.Seneca valley virus 3C protease negatively regulates the type I interferon pathway by acting as a viral deubiquitinase[J].Antiviral Res,2018,160:183-189.
[33] LI P F,ZHANG X L,CAO W J,et al.RIG-I is responsible for activation of type I interferon pathway in Seneca Valley virus-infected porcine cells to suppress viral replication[J].Virol J,2018,15(1):162.
[34] STOTT E J,KILLINGTON R A.Rhinoviruses[J].Annu Rev Microbiol,1972,26:503-524.
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[37] PALMENBERG A C,GERN J E.Classification and evolution of human rhinoviruses[J].Methods Mol Biol,2015,1221:1-10.
[38] PALMENBERG A C,SPIRO D,KUZMICKAS R,et al.Sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution[J].Science,2009,324(5923):55-59.
[39] HULL H F,WARD N A,HULL B P,et al.Paralytic poliomyelitis:Seasoned strategies,disappearing disease[J].Lancet,1994,343(8909):1331-1337.
[40] CROFT S N,WALKER E J,GHILDYAL R.Picornaviruses and apoptosis:subversion of cell death[J].MBIO,2017,8(5):E01009-17.
[41] BELOV G A,ROMANOVA L I,TOLSKAYA E A,et al.The major apoptotic pathway activated and suppressed by poliovirus[J].J Virol,2003,77(1):45-56.
[42] IRURZUN A,ARROYO J,ALVAREZ A,et al.Enhanced intracellular calcium concentration during poliovirus infection[J].J Virol,1995,69(8):5142-5146.
[43] BRISAC C,TéOULé F,AUTRET A,et al.Calcium flux between the endoplasmic reticulum and mitochondrion contributes to poliovirus-induced apoptosis[J].J Virol,2010,84(23):12226-12235.
[44] WANG J C,WANG Y Q,LIU J,et al.A critical role of n-myc and stat interactor (nmi) in foot-and-mouth disease virus (FMDV) 2C-induced apoptosis[J].Virus Res,2012,170(1-2):59-65.
[45] PENG J M,LIANG S M,LIANG C M.VP1 of foot-and-mouth disease virus induces apoptosis via the akt signaling pathway[J].J Biol Chem,2004,279(50):52168-52174.
[2] HALES L M,KNOWLES N J,REDDY P S,et al.Complete genome sequence analysis of Seneca Valley virus-001,a novel oncolytic picornavirus[J].J Gen Virol,2008,89(5):1265-1275.
[3] CHEN Z H,YUAN F F,LI Y H,et al.Construction and characterization of a full-length cDNA infectious clone of emerging porcine Senecavirus A[J].Virology,2016,497:111-124.
[4] FALLAUX F J,BOUT A,VAN DER VELDE I,et al.New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses[J].Hum Gene Ther,1998,9(13):1909-1917.
[5] REDDY P S,BURROUGHS K D,HALES L M,et al.Seneca valley virus,a systemically deliverable oncolytic picornavirus,and the treatment of neuroendocrine cancers[J].JNCI,2007,99(21):1623-1633.
[6] SINGH K,CORNER S,CLARK S G,et al.Seneca valley virus and vesicular lesions in a pig with idiopathic vesicular disease[J].J Vet Sci Technol,2012,3(6):123.
[7] PASMA T,DAVIDSON S,SHAW S L.Idiopathic vesicular disease in swine in Manitoba[J].Can Vet J,2008,49(1):84-85.
[8] VANNUCCI F A,LINHARES D C L,BARCELLOS D E S N,et al.Identification and complete genome of seneca valley virus in vesicular fluid and sera of pigs affected with idiopathic vesicular disease,Brazil[J].Transbound Emerg Dis,2015,62(6):589-593.
[9] LEME R A,ZOTTI E,ALC?NTARA B K,et al.Senecavirus A:an emerging vesicular infection in brazilian pig herds[J].Transbound Emerg Dis,2015,62(6):603-611.
[10] LEME R A,OLIVEIRA T E,ALCANTARA B K,et al.Clinical manifestations of senecavirus a infection in neonatal pigs,Brazil,2015[J].Emerg Infect Dis,2016,22(7):1238-1241.
[11] LEME R A,ALFIERI A F,ALFIERI A A.Update on senecavirus infection in pigs[J].Viruses,2017,9(7):170.
[12] WU Q,ZHAO X,BAI Y,et al.The first identification and complete genome of Senecavirus A affecting pig with idiopathic vesicular disease in China[J].Transbound Emerg Dis,2017,64(5):1633-1640.
[13] ZHANG X,XIAO J,BA L,et al.Identification and genomic characterization of the emerging Senecavirus A in southeast China,2017[J].Transbound Emerg Dis,2018,65(2):297-302.
[14] ZHU Z,YANG F,CHEN P,et al.Emergence of novel Seneca Valley virus strains in China,2017[J].Transbound Emerg Dis,2017,64(4):1024-1029.
[15] JOSHI L R,FERNANDES M H,CLEMENT T,et al.Pathogenesis of Senecavirus A infection in finishing pigs[J].J Gen Virol,2016,97(12):3267-3279.
[16] SAENG-CHUTO K,RODTIAN P,TEMEEYASEN G,et al.The first detection of Senecavirus A in pigs in Thailand,2016[J].Transbound Emerg Dis,2018,65(1):285-288.
[17] SUN D,VANNUCCI F,KNUTSON T P,et al.Emergence and whole-genome sequence of Senecavirus A in Colombia[J].Transbound Emerg Dis,2017,64(5):1346-1349.
[18] WANG H W,LI C,ZHAO B,et al.Complete genome sequence and phylogenetic analysis of Senecavirus A isolated in Northeast China in 2016[J].Arch Virol,2017,162(10):3173-3176.
[19] LIU J,REN X,LI Z,et al.Genetic and phylogenetic analysis of reemerged novel Seneca Valley virus strains in Guangdong province,2017[J].Transbound Emerg Dis,2018,65(3):614-617.
[20] ARENDS M J,MORRIS R G,WYLLIE A H.Apoptosis.The role of the endonuclease[J].Am J Pathol,1990,136(3):593-608.
[21] DANTHI P.Viruses and the diversity of cell death[J].Annu Rev Virol,2016,3:533-553.
[22] TEODORO J G,BRANTON P E.Regulation of apoptosis by viral gene products[J].J Virol,1997,71(3):1739-1746.
[23] HUBER H J,DUESSMANN H,WENUS J,et al.Mathematical modelling of the mitochondrial apoptosis pathway[J].Biochim Biophys Acta,2011,1813(4):608-615.
[24] ICHIM G,TAIT S W G.A fate worse than death:apoptosis as an oncogenic process[J].Nat Rev Cancer,2016,16(8):539-548.
[25] GALLUZZI L,BRENNER C,MORSELLI E,et al.Viral control of mitochondrial apoptosis[J].PLoS Pathog,2008,4(5):e1000018.
[26] ELMORE S.Apoptosis:a review of programmed cell death[J].Toxicol Pathol,2007,35(4):495-516.
[27] ZHOU X C,JIANG W B,LIU Z S,et al.Virus infection and death receptor-mediated apoptosis[J].Viruses,2017,9(11):316.
[28] EVERETT H,MCFADDEN G.Apoptosis:an innate immune response to virus infection[J].Trends Microbiol,1999,7(4):160-165.
[29] MILES L A,BURGA L N,GARDNER E E,et al.Anthrax toxin receptor 1 is the cellular receptor for Seneca Valley virus[J].J Clin Invest,2017,127(8):2957-2967.
[30] QIAN S H,FAN W C,LIU T T,et al.Seneca valley virus suppresses host type I interferon production by targeting adaptor proteins MAVS,TRIF,and TANK for cleavage[J].J Virol,2017,91(16):e00823-17.
[31] XUE Q,LIU H S,ZHU Z X,et al.Seneca valley virus 3Cpro abrogates the IRF3- and IRF7-mediated innate immune response by degrading IRF3 and IRF7[J].Virology,2018,518:1-7.
[32] XUE Q,LIU H S,ZHU Z X,et al.Seneca valley virus 3C protease negatively regulates the type I interferon pathway by acting as a viral deubiquitinase[J].Antiviral Res,2018,160:183-189.
[33] LI P F,ZHANG X L,CAO W J,et al.RIG-I is responsible for activation of type I interferon pathway in Seneca Valley virus-infected porcine cells to suppress viral replication[J].Virol J,2018,15(1):162.
[34] STOTT E J,KILLINGTON R A.Rhinoviruses[J].Annu Rev Microbiol,1972,26:503-524.
[35] LEWIS-ROGERS N,CRANDALL K A.Evolution of picornaviridae:an examination of phylogenetic relationships and cophylogeny[J].Mol Phylogenet Evol,2010,54(3):995-1005.
[36] RACANIELLO R.Picornaviridae:the viruses and their replication[M]//Fields Virology.KNIPE D M,HOWLEY P M.4th ed.Philadelphia:Lippincott Raven press,2013.
[37] PALMENBERG A C,GERN J E.Classification and evolution of human rhinoviruses[J].Methods Mol Biol,2015,1221:1-10.
[38] PALMENBERG A C,SPIRO D,KUZMICKAS R,et al.Sequencing and analyses of all known human rhinovirus genomes reveal structure and evolution[J].Science,2009,324(5923):55-59.
[39] HULL H F,WARD N A,HULL B P,et al.Paralytic poliomyelitis:Seasoned strategies,disappearing disease[J].Lancet,1994,343(8909):1331-1337.
[40] CROFT S N,WALKER E J,GHILDYAL R.Picornaviruses and apoptosis:subversion of cell death[J].MBIO,2017,8(5):E01009-17.
[41] BELOV G A,ROMANOVA L I,TOLSKAYA E A,et al.The major apoptotic pathway activated and suppressed by poliovirus[J].J Virol,2003,77(1):45-56.
[42] IRURZUN A,ARROYO J,ALVAREZ A,et al.Enhanced intracellular calcium concentration during poliovirus infection[J].J Virol,1995,69(8):5142-5146.
[43] BRISAC C,TéOULé F,AUTRET A,et al.Calcium flux between the endoplasmic reticulum and mitochondrion contributes to poliovirus-induced apoptosis[J].J Virol,2010,84(23):12226-12235.
[44] WANG J C,WANG Y Q,LIU J,et al.A critical role of n-myc and stat interactor (nmi) in foot-and-mouth disease virus (FMDV) 2C-induced apoptosis[J].Virus Res,2012,170(1-2):59-65.
[45] PENG J M,LIANG S M,LIANG C M.VP1 of foot-and-mouth disease virus induces apoptosis via the akt signaling pathway[J].J Biol Chem,2004,279(50):52168-52174.