与猪流行性腹泻病毒M蛋白互作的宿主蛋白的鉴定
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摘要
【背景】猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)膜蛋白(M)在病毒粒子的组装、膜融合和病毒复制等方面具有重要的作用,但M蛋白与宿主细胞的互作机制尚不清楚。【目的】利用免疫沉淀技术和液质联用技术筛选细胞内与PEDVM蛋白相互作用的蛋白,为揭示M蛋白在病毒增殖过程中发挥的功能提供研究基础。【方法】将MOI=0.1的PEDV DR13疫苗株接种于长成单层的Vero细胞,感染36 h后,收集细胞并进行裂解。利用抗M的单克隆抗体沉淀与M相互作用蛋白复合物,通过液相色谱串联质谱(LC-MS/MS)进行鉴定并利用细胞功能富集分析(Gene ontology,GO)对感染组鉴定到的细胞蛋白进行分析,确定两个细胞内源性蛋白为候选蛋白,进行免疫共沉淀(Co-IP)验证和共定位分析。【结果】基于鉴定蛋白的肽段数的方法分析显示,感染组与对照组相比,鉴定了218个与M蛋白相互作用的细胞内源性蛋白,分别与蛋白质合成、代谢、细胞信号通路转导等密切相关,选择细胞分裂周期蛋白42 (Cell division cycle 42,CDC42)、真核翻译起始因子3亚基L蛋白(eIF3L)为候选蛋白进行Co-IP(Co-immunoprecipitation)验证和共定位分析,结果证实CDC42、eIF3L蛋白分别与M蛋白在细胞内存在相互作用。【结论】鉴定出PEDV M蛋白能够与宿主细胞CDC42和eIF3L蛋白相互作用,并鉴定出其他可能与M蛋白发生相互作用的宿主蛋白60个,为开展PEDV与宿主细胞蛋白相互作用研究提供了重要理论依据。
[Background] The membrane protein(M) of porcine epidemic diarrhea virus(PEDV) plays an important role in the viral assembly process, membrane fusion and viral replication, but the mechanism of interaction between M protein and host cells is still unclear. [Objective] Co-immunoprecipitation technique coupled with LC-MS/MS were used to screen cellular proteins interacting with PEDV M protein, which can provide a foundation for revealing the function of M in viral multiplication. [Methods]PEDV DR13 vaccine strain was inoculated into monolayer of Vero cells at a MOI of 0.1. After 36 hours of infection, the cells were collected and lysed. Host cellular proteins that interact with the M protein of PEDV were immunoprecipitated using the M monoclonal antibody, then identified by LC-MS/MS, and analyzed by gene ontology(GO) annotation. Among them, two interested cellular proteins were further confirmed with Co-IP and cellular colocalization. [Results] Based on the analysis of the number of peptide segments, 218 cellular proteins interacting with M protein were identified. These host cellular proteins are closely related to protein synthesis, metabolism and cell signaling pathway transduction. Cell division cycle 42(CDC42) and eukaryotic translation initiation factor 3 subunit L(eIF3 L) were chosen for reverse Co-IP reconfirmation and colocalization analysis. The results showed that both CDC42 and eIF3 L protein interact with M protein. [Conclusion] This study identified that PEDV M protein could interact with CDC42 and eIF3 L proteins in host cells, and identified 60 other host proteins that might interact with M protein. The study is to provide an important theoretical basis for the study of the interaction between PEDV and host cell proteins.
[Background] The membrane protein(M) of porcine epidemic diarrhea virus(PEDV) plays an important role in the viral assembly process, membrane fusion and viral replication, but the mechanism of interaction between M protein and host cells is still unclear. [Objective] Co-immunoprecipitation technique coupled with LC-MS/MS were used to screen cellular proteins interacting with PEDV M protein, which can provide a foundation for revealing the function of M in viral multiplication. [Methods]PEDV DR13 vaccine strain was inoculated into monolayer of Vero cells at a MOI of 0.1. After 36 hours of infection, the cells were collected and lysed. Host cellular proteins that interact with the M protein of PEDV were immunoprecipitated using the M monoclonal antibody, then identified by LC-MS/MS, and analyzed by gene ontology(GO) annotation. Among them, two interested cellular proteins were further confirmed with Co-IP and cellular colocalization. [Results] Based on the analysis of the number of peptide segments, 218 cellular proteins interacting with M protein were identified. These host cellular proteins are closely related to protein synthesis, metabolism and cell signaling pathway transduction. Cell division cycle 42(CDC42) and eukaryotic translation initiation factor 3 subunit L(eIF3 L) were chosen for reverse Co-IP reconfirmation and colocalization analysis. The results showed that both CDC42 and eIF3 L protein interact with M protein. [Conclusion] This study identified that PEDV M protein could interact with CDC42 and eIF3 L proteins in host cells, and identified 60 other host proteins that might interact with M protein. The study is to provide an important theoretical basis for the study of the interaction between PEDV and host cell proteins.
引文
[1]Lee C.Porcine epidemic diarrhea virus:an emerging and re-emerging epizootic swine virus[J].Virology Journal,2015,12:193
[2]de Haan CAM,Vennema H,Rottier PJM.Assembly of the coronavirus envelope:homotypic interactions between the Mproteins[J].Journal of Virology,2000,74(11):4967-4978
[3]Ariel LA,Blake JL,Brenda GH.A conserved domain in the coronavirus membrane protein tail is important for virus assembly[J].Journal of Virology,2010,84(21):11418-11428
[4]Klumperman J,Locker JK,Meijer A,et al.Coronavirus Mproteins accumulate in the Golgi complex beyond the Site of virion budding[J].Journal of Virology,1994,68(10):6523-6534
[5]de Haan CAM,Kuo LL,Masters PS,et al.Coronavirus particle assembly:primary structure requirements of the membrane protein[J].Journal of Virology,1998,72(8):6838-6850
[6]Xu XG,Zhang HL,Zhang Q,et al.Porcine epidemic diarrhea virus M protein blocks cell cycle progression at S-phase and its subcellular localization in the porcine intestinal epithelial cells[J].Acta Virology,2015,59(3):265-275
[7]Song D,Park B.Porcine epidemic diarrhoea virus:a comprehensive review of molecular epidemiology,diagnosis,and vaccines[J].Virus Genes,2012,44(2):167-175
[8]Zhang QZ,Shi KC,Yoo D.Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1[J].Virology,2016,489:252-268
[9]Wang Q,Li YW,Dong H,et al.Identification of host cellular proteins that interact with the M protein of a highly pathogenic porcine reproductive and respiratory syndrome virus vaccine strain[J].Virology Journal,2017,14(1):39
[10]Qadir MI,Parveen A,Ali M.CDC42:role in cancer management[J].Chemical Biology&Drug Design,2015,86(4):432-439
[11]Johnson DI,Pringle JR.Molecular characterization of CDC42,a Saccharomyces cerevisiae gene involved in the development of cell polarity[J].Journal of Cell Biology,1990,111(1):143-152
[12]Cai Q,Todorovic A,Andaya A,et al.Distinct regions of human eIF3 are sufficient for binding to the HCV IRES and the 40Sribosomal subunit[J].Journal of Molecular Biology,2010,403(2):185-196
[13]San-Juan-Vergara H,Sampayo-Escobar V,Reyes N,et al.Cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells[J].Journal of Virology,2012,86(3):1832-1843
[14]Díaz-Salinas MA,Silva-Ayala D,López S,et al.Rotaviruses reach late endosomes and require the cation-dependent mannose-6-phosphate receptor and the activity of cathepsin proteases to enter the cell[J].Journal of Virology,2014,88(8):4389-4402
[15]Swaine T,Dittmar MT.CDC42 use in viral cell entry processes by RNA viruses[J].Viruses,2015,7(12):6526-6536
[16]Quinn K,Brindley MA,Weller ML,et al.Rho GTPases modulate entry of Ebola virus and vesicular stomatitis virus pseudotyped vectors[J].Journal of Virology,2009,83(19):10176-10186
[17]Fraser CS,Lee JY,Mayeur GL,et al.The j-subunit of human translation initiation factor eIF3 is required for the stable binding of e IF3 and its subcomplexes to 40S ribosomal subunits in vitro[J].Journal of Biological Chemistry,2004,279(10):8946-8956
[18]Morris-Desbois C,Réty S,Ferro M,et al.The human protein HSPC021 interacts with Int-6 and is associated with eukaryotic translation initiation factor 3[J].Journal of Biological Chemistry,2001,276(49):45988-45995
[19]Gardner CL,Ryman KD.Yellow fever:a reemerging threat[J].Clinics in Laboratory Medicine,2010,30(1):237-260
[20]Easton LE,Locker N,Lukavsky PJ.Conserved functional domains and a novel tertiary interaction near the pseudoknot drive translational activity of hepatitis C virus and hepatitis C virus-like internal ribosome entry sites[J].Nucleic Acids Research,2009,37(16):5537-5549
[21]Valente ST,Gilmartin GM,Mott C,et al.Inhibition of HIV-1replication by eIF3F[J].Proceedings of the National Academy of Sciences of the United States of America,2009,106(11):4071-4078
[22]Schoggins JW,Wilson SJ,Panis M,et al.A diverse range of gene products are effectors of the type I interferon antiviral response[J].Nature,2011,472(7344):481-485
[23]Morais ATS,Terzian ACB,Duarte DVB,et al.The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication[J].Virology Journal,2013,10:205
[2]de Haan CAM,Vennema H,Rottier PJM.Assembly of the coronavirus envelope:homotypic interactions between the Mproteins[J].Journal of Virology,2000,74(11):4967-4978
[3]Ariel LA,Blake JL,Brenda GH.A conserved domain in the coronavirus membrane protein tail is important for virus assembly[J].Journal of Virology,2010,84(21):11418-11428
[4]Klumperman J,Locker JK,Meijer A,et al.Coronavirus Mproteins accumulate in the Golgi complex beyond the Site of virion budding[J].Journal of Virology,1994,68(10):6523-6534
[5]de Haan CAM,Kuo LL,Masters PS,et al.Coronavirus particle assembly:primary structure requirements of the membrane protein[J].Journal of Virology,1998,72(8):6838-6850
[6]Xu XG,Zhang HL,Zhang Q,et al.Porcine epidemic diarrhea virus M protein blocks cell cycle progression at S-phase and its subcellular localization in the porcine intestinal epithelial cells[J].Acta Virology,2015,59(3):265-275
[7]Song D,Park B.Porcine epidemic diarrhoea virus:a comprehensive review of molecular epidemiology,diagnosis,and vaccines[J].Virus Genes,2012,44(2):167-175
[8]Zhang QZ,Shi KC,Yoo D.Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1[J].Virology,2016,489:252-268
[9]Wang Q,Li YW,Dong H,et al.Identification of host cellular proteins that interact with the M protein of a highly pathogenic porcine reproductive and respiratory syndrome virus vaccine strain[J].Virology Journal,2017,14(1):39
[10]Qadir MI,Parveen A,Ali M.CDC42:role in cancer management[J].Chemical Biology&Drug Design,2015,86(4):432-439
[11]Johnson DI,Pringle JR.Molecular characterization of CDC42,a Saccharomyces cerevisiae gene involved in the development of cell polarity[J].Journal of Cell Biology,1990,111(1):143-152
[12]Cai Q,Todorovic A,Andaya A,et al.Distinct regions of human eIF3 are sufficient for binding to the HCV IRES and the 40Sribosomal subunit[J].Journal of Molecular Biology,2010,403(2):185-196
[13]San-Juan-Vergara H,Sampayo-Escobar V,Reyes N,et al.Cholesterol-rich microdomains as docking platforms for respiratory syncytial virus in normal human bronchial epithelial cells[J].Journal of Virology,2012,86(3):1832-1843
[14]Díaz-Salinas MA,Silva-Ayala D,López S,et al.Rotaviruses reach late endosomes and require the cation-dependent mannose-6-phosphate receptor and the activity of cathepsin proteases to enter the cell[J].Journal of Virology,2014,88(8):4389-4402
[15]Swaine T,Dittmar MT.CDC42 use in viral cell entry processes by RNA viruses[J].Viruses,2015,7(12):6526-6536
[16]Quinn K,Brindley MA,Weller ML,et al.Rho GTPases modulate entry of Ebola virus and vesicular stomatitis virus pseudotyped vectors[J].Journal of Virology,2009,83(19):10176-10186
[17]Fraser CS,Lee JY,Mayeur GL,et al.The j-subunit of human translation initiation factor eIF3 is required for the stable binding of e IF3 and its subcomplexes to 40S ribosomal subunits in vitro[J].Journal of Biological Chemistry,2004,279(10):8946-8956
[18]Morris-Desbois C,Réty S,Ferro M,et al.The human protein HSPC021 interacts with Int-6 and is associated with eukaryotic translation initiation factor 3[J].Journal of Biological Chemistry,2001,276(49):45988-45995
[19]Gardner CL,Ryman KD.Yellow fever:a reemerging threat[J].Clinics in Laboratory Medicine,2010,30(1):237-260
[20]Easton LE,Locker N,Lukavsky PJ.Conserved functional domains and a novel tertiary interaction near the pseudoknot drive translational activity of hepatitis C virus and hepatitis C virus-like internal ribosome entry sites[J].Nucleic Acids Research,2009,37(16):5537-5549
[21]Valente ST,Gilmartin GM,Mott C,et al.Inhibition of HIV-1replication by eIF3F[J].Proceedings of the National Academy of Sciences of the United States of America,2009,106(11):4071-4078
[22]Schoggins JW,Wilson SJ,Panis M,et al.A diverse range of gene products are effectors of the type I interferon antiviral response[J].Nature,2011,472(7344):481-485
[23]Morais ATS,Terzian ACB,Duarte DVB,et al.The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication[J].Virology Journal,2013,10:205