GST pull-down结合质谱筛选猪圆环病毒2型ORF4潜在互作蛋白
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摘要
猪圆环病毒2型编码的ORF4蛋白是近年来发现的新蛋白。迄今为止,人们对ORF4所参与的细胞生物学过程知之甚少。本研究首先构建了带双标签的真核表达载体pCMV-N-Flag-GST,再将ORF4基因插入该载体中,形成pCMV-N-Flag-GST-ORF4。将质粒转染293T细胞表达ORF4后,通过GSTPull-down试验捕获细胞内潜在与ORF4互作的蛋白库。经SDS-PAGE分离及银染后,对所得的特异性条带进行质谱鉴定,筛选出5个与ORF4潜在互作的蛋白,包括丝氨酸/苏氨酸蛋白磷酸酶6催化亚基、α心肌蛋白、β肌动蛋白、SEC-14样蛋白5和肌球蛋白myosin 9。上述研究结果为深入揭示ORF4在病毒感染细胞过程中发挥的作用提供新的思路与方向。
A novel protein encoded by the open reading frame 4(ORF4) was recently discovered in porcine circovirus type 2(PCV2).However,little is known about the interaction proteins of ORF4 which hindered better understanding the biological functions of ORF4 in the life cycle of PCV2.In the present study,the ORF4 was inserted into the multiple cloning site of pCMV-N-Flag-GST,yielding recombinant plasmid pCMV-N-Flag-GST-ORF4.The recombinant plasmid was transfected into 293 T cells and the intracellular interaction complex of ORF4 were enriched and separated by GST pull-down and SDS-PAGE,sequentially.The potential interacting proteins of PCV2 ORF4 were stained with silver and identified by mass spectrometry(MS).Finally,five candidate ORF4-interacting proteins,including Serine/threonine-protein phosphatase 6 catalytic subunit,alpha cardiac muscle 1,actin,SEC14-like protein 5 and myosin 9 were identified.These results would benefit a better understanding of the biological function of ORF4 in PCV2 infected cells.
A novel protein encoded by the open reading frame 4(ORF4) was recently discovered in porcine circovirus type 2(PCV2).However,little is known about the interaction proteins of ORF4 which hindered better understanding the biological functions of ORF4 in the life cycle of PCV2.In the present study,the ORF4 was inserted into the multiple cloning site of pCMV-N-Flag-GST,yielding recombinant plasmid pCMV-N-Flag-GST-ORF4.The recombinant plasmid was transfected into 293 T cells and the intracellular interaction complex of ORF4 were enriched and separated by GST pull-down and SDS-PAGE,sequentially.The potential interacting proteins of PCV2 ORF4 were stained with silver and identified by mass spectrometry(MS).Finally,five candidate ORF4-interacting proteins,including Serine/threonine-protein phosphatase 6 catalytic subunit,alpha cardiac muscle 1,actin,SEC14-like protein 5 and myosin 9 were identified.These results would benefit a better understanding of the biological function of ORF4 in PCV2 infected cells.
引文
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[27]Sasaki H,Nakamura M,Ohno T,et al.Myosin-actin interaction plays an important role in human immunodeficiency virus type 1 release from host cells.Proc Natl Acad Sci USA,1995,92(6):2026-2030.
[28]Arii J,Goto H,Suenaga T,et al.Non-muscle myosin IIAis a functional entry receptor for herpes simplex virus-1.Nature,2010,467(7317):859-862.
[29]Moseley JB,Goode BL.The yeast actin cytoskeleton:from cellular function to biochemical mechanism.Microbiol Mol Biol Rev,2006,70(3):605-645.
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[31]Franklin-Tong VE,Gourlay CW.A role for actin in regulating apoptosis/programmed cell death:evidence spanning yeast,plants and animals.Biochem J,2008,413(3):389-404.
[2]Opriessnig T,Meng XJ,Halbur PG.Porcine circovirus type 2-associated disease:update on current terminology,clinical manifestations,pathogenesis,diagnosis,and intervention strategies.J Vet Diagn Invest,2007,19(6):591-615.
[3]Breitbart M,Delwart E,Rosario K,et al.ICTV virus taxonomy profile:Circoviridae.J Gen Virol,2017,98(8):1997-1998.
[4]Zhou JY,Chen QX,Ye JX,et al.Serological investigation and genomic characterization of PCV2isolates from different geographic regions of Zhejiang province in China.Vet Res Commun,2006,30(2):205-220.
[5]Mankertz A,Mankertz J,Wolf K,et al.Identification of a protein essential for replication of porcine circovirus.JGen Virol,1998,79(2):381-384.
[6]Nawagitgul P,Morozov I,Bolin SR,et al.Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein.J Gen Virol,2000,81(9):2281-2287.
[7]Blanchard P,MahéD,Cariolet R,et al.Protection of swine against post-weaning multisystemic wasting syndrome(PMWS)by porcine circovirus type 2(PCV2)proteins.Vaccine,2003,21(31):4565-4575.
[8]Liu J,Chen I,Kwang J.Characterization of a previously unidentified viral protein in porcine circovirus type2-infected cells and its role in virus-induced apoptosis.JVirol,2005,79(13):8262-8274.
[9]He JL,Cao JJ,Zhou N,et al.Identification and functional analysis of the novel ORF4 protein encoded by porcine circovirus Type 2.J Virol,2013,87(3):1420-1429.
[10]Lv QZ,Guo KK,Xu H,et al.Identification of putative ORF5 protein of porcine circovirus type 2 and functional analysis of GFP-fused ORF5 protein.PLoSONE,2015,10(6):e0127859.
[11]Gao ZZ,Dong QF,Jiang YH,et al.ORF4-protein deficient PCV2 mutants enhance virus-induced apoptosis and show differential expression of mRNAs in vitro.Virus Res,2014,183:56-62.
[12]Lv QZ,Guo KK,Wang T,et al.Porcine circovirus type2 ORF4 protein binds heavy chain ferritin.J Biosci,2015,40(3):477-485.
[13]Lv QZ,Guo KK,Zhang GF,et al.The ORF4 protein of porcine circovirus type 2 antagonizes apoptosis by stabilizing the concentration of ferritin heavy chain through physical interaction.J Gen Virol,2016,97(7):1636-1646.
[14]Lin C,Gu JY,Wang HJ,et al.Caspase-dependent apoptosis induction via viral protein ORF4 of porcine circovirus 2 binding to mitochondrial adenine nucleotide translocase 3.J Virol,2018,92(10):e00238-18.
[15]Coligan JE.Short Protocols in Protein Science.Beijing:Science Press,2007:294-297(in Chinese).科林根JE.精编蛋白质科学实验指南.北京:科学出版社,2007:294-297.
[16]Zamarin D,Garcia-Sastre A,Xiao XY,et al.Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1.PLoS Pathog,2005,1(1):e4.
[17]Pollard TD,Cooper JA.Actin,a Central player in cell shape and movement.Science,2009,326(5957):1208-1212.
[18]Sellers JR,Goodson HV.Motor proteins 2:myosin.Protein Profile,1995,2(12):1323-1423.
[19]Yam PT,Wilson CA,Ji L,et al.Actin-myosin network reorganization breaks symmetry at the cell rear to spontaneously initiate polarized cell motility.J Cell Biol,2007,178(7):1207-1221.
[20]Sodeik B.Mechanisms of viral transport in the cytoplasm.Trends Microbiol,2000,8(10):465-472.
[21]Smith AE,Helenius A.How viruses enter animal cells.Science,2004,304(5668):237-242.
[22]Charlton CA,Volkman LE.Penetration of Autographa californica nuclear polyhedrosis virus nucleocapsids into IPLB Sf 21 cells induces actin cable formation.Virology,1993,197(1):245-254.
[23]Cao JJ,Lin C,Wang HJ,et al.Circovirus transport proceeds via direct interaction of the cytoplasmic dynein IC1 subunit with the viral capsid protein.J Virol,2015,89(5):2777-2791.
[24]He JL.Identification of putative ORF4 within porcine circovirus type 2 and gene expression profile of host immune organ after PCV2 infection[D].Hangzhou:Zhejiang University,2012(in Chinese).何嘉玲.猪圆环病毒2型编码蛋白4的鉴定及其感染后的宿主免疫器官基因表达谱分析[D].杭州:浙江大学,2012.
[25]Welch MD,Mullins RD.Cellular control of actin nucleation.Ann Rev Cell Dev Biol,2002,18:247-288.
[26]Lehmann MJ,Sherer NM,Marks CB,et al.Actin-and myosin-driven movement of viruses along filopodia precedes their entry into cells.J Cell Biol,2005,170(2):317-325.
[27]Sasaki H,Nakamura M,Ohno T,et al.Myosin-actin interaction plays an important role in human immunodeficiency virus type 1 release from host cells.Proc Natl Acad Sci USA,1995,92(6):2026-2030.
[28]Arii J,Goto H,Suenaga T,et al.Non-muscle myosin IIAis a functional entry receptor for herpes simplex virus-1.Nature,2010,467(7317):859-862.
[29]Moseley JB,Goode BL.The yeast actin cytoskeleton:from cellular function to biochemical mechanism.Microbiol Mol Biol Rev,2006,70(3):605-645.
[30]Heeren G,Jarolim S,Laun P,et al.The role of respiration,reactive oxygen species and oxidative stress in mother cell-specific ageing of yeast strains defective in the RAS signalling pathway.FEMS Yeast Res,2004,5(2):157-167.
[31]Franklin-Tong VE,Gourlay CW.A role for actin in regulating apoptosis/programmed cell death:evidence spanning yeast,plants and animals.Biochem J,2008,413(3):389-404.