家蚕IAP互作蛋白的筛选、鉴定及其对BmNPV增殖的影响

doi:10.3864/j.issn.0578-1752.2019.03.016

Abstract

Abstract:

【Objective】 As an important part of insect immune response, apoptosis plays an important role in the interaction between virus and host. The study of apoptosis-related genes is of great value in elucidating their roles in the process of virus infection. The objective of this study is to screen the interaction proteins of Bombyx mori inhibitor of apoptosis proteins (BmIAP), verify the regulatory relationships between them and Bmiap in the process of B. mori nucleopolyhedrovirus (BmNPV) infection and their roles in the proliferation of BmNPV, and to provide a theoretical basis for exploring the molecular mechanism of the interaction between host and BmNPV.【Method】 The proteins interacting with BmIAP in the process of BmNPV infection in B. mori cells were screened by immunoprecipitation, and the specific differential bands were analyzed by LC-MS/MS. The obtained proteins were identified according to the molecular weight of the protein and bioinformatics method. The candidate gene was obtained by molecular cloning technique, and the domain of the candidate gene was predicted by SMART online tools and the multiple sequence alignment analysis was performed with BioEdit. Co-localization of BmIAP and candidate protein was verified by immunofluorescence assay, and their interaction was further verified by immunoprecipitation. The candidate gene was overexpressed and knocked out by eukaryotic expression and CRISPR/Cas9 gene editing system, respectively. The expression of corresponding genes was detected by qRT-PCR to determine the regulatory relationship between Bmiap and Bmpp5 in the process of BmNPV infection. Similarly, the expression of baculovirus Vp39 was detected after overexpression and knockout of Bmpp5 to determine the effect of Bmpp5 on the proliferation of BmNPV.【Result】 Seven host proteins and one BmNPV protein which may interact with BmIAP were obtained by immunoprecipitation and further analysis identified a candidate gene Bmpp5 associated with apoptosis. The open reading frame (ORD) of Bmpp5 is 1 473 bp, encoding 490 amino acids. The predicted molecular weight of BmPP5 is about 56 kD. It contains three TRP domains and one PP2Ac domain. It is highly conserved among insects. Immunofluorescence assay showed that BmIAP and BmPP5 were co-localized in cytoplasm, and the results of immunoprecipitation showed that they could interact with each other. In the process of BmNPV infection, after overexpression of Bmiap, the expression of Bmpp5 was significantly up-regulated, while after knockout of Bmiap, the expression of Bmpp5 was significantly down-regulated, suggesting that Bmiap could promote the expression of Bmpp5. Bmiap was significantly up-regulated after overexpression of Bmpp5, while Bmiap was significantly down-regulated after knockout of Bmpp5, suggesting that Bmpp5 could also promote the expression of Bmiap. Overexpression of Bmpp5 could promote the proliferation of BmNPV, and knockdown of Bmpp5 could inhibit the proliferation of BmNPV, indicating that the expression of Bmpp5 was conducive to the proliferation of BmNPV. 【Conclusion】 A protein BmPP5, which interacts with BmIAP was identified and highly conserved in insects. In the process of BmNPV infection, Bmiap and Bmpp5 can promote each other. It is also proved that Bmpp5 can promote the replication and proliferation of BmNPV.

Key words: Bombyx mori, inhibitor of apoptosis protein (IAP), interaction protein, BmNPV, BmPP5

CHEN Peng,BAO XiYan,KANG TaoTao,DONG ZhanQi,ZHU Yan,PAN MinHui,LU Cheng. Screening and Identification of Proteins Interacting with Bombyx mori IAP and Their Effects on BmNPV Proliferation[J].Scientia Agricultura Sinica, 2019, 52(3): 558-567.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.chinaagrisci.com/EN/10.3864/j.issn.0578-1752.2019.03.016

https://www.chinaagrisci.com/EN/Y2019/V52/I3/558

Figures/Tables 7

Table 1

Table 2

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

References 38

[1]	VAN OERS M M, VLAK J M . Baculovirus genomics. Current Drug Targets, 2007,8(10):1051-1068.
[2]	NGUYEN Q, PALFREYMAN R W, CHAN L C, REID S, NIELSEN L K . Transcriptome sequencing and microarray development for a Helicoverpa zea cell line to investigate in vitro insect cell-baculovirus interactions. PLoS ONE, 2012,7(5):e36324. doi: 10.1371/journal.pone.0036324 pmid: 22629315
[3]	VANDERGAAST R, SCHULTZ K L, CERIO R J, FRIESEN P D . Active depletion of host cell inhibitor-of-apoptosis proteins triggers apoptosis upon baculovirus DNA replication. Journal of Virology, 2011,85(16):8348-8358. doi: 10.1128/JVI.00667-11 pmid: 3147993
[4]	MITCHELL J K, FRIESEN P D . Baculoviruses modulate a proapoptotic DNA damage response to promote virus multiplication. Journal of Virology, 2012,86(24):13542-13553. doi: 10.1128/JVI.02246-12 pmid: 23035220
[5]	SALEM T Z, ZHANG F, XIE Y, THIEM S M . Comprehensive analysis of host gene expression in Autographa californica nucleopolyhedrovirus-infected Spodoptera frugiperda cells. Virology, 2011,412(1):167-178.
[6]	BAO Y Y, LV Z Y, LIU Z B, XUE J, XU Y P, ZHANG C X . Comparative analysis of Bombyx mori nucleopolyhedrovirus responsive genes in fat body and haemocyte of B. mori resistant and susceptible strains. Insect Molecular Biology, 2010,19(3):347-358. doi: 10.1111/j.1365-2583.2010.00993.x pmid: 20201979
[7]	MEHRABADI M, HUSSAIN M, ASGARI S . MicroRNAome of Spodoptera frugiperda cells (Sf9) and its alteration following baculovirus infection. The Journal of General Virology, 2013,94(6):1385-1397.
[8]	YU X, ZHOU Q, LI S C, LUO Q, CAI Y, LIN W C, CHEN H, YANG Y, HU S, YU J . The silkworm (Bombyx mori) microRNAs and their expressions in multiple developmental stages. PLoS ONE, 2008,3(8):e2997. doi: 10.1371/journal.pone.0002997 pmid: 2500172
[9]	XUE J, QIAO N, ZHANG W, CHENG R L, ZHANG X Q, BAO Y Y, XU Y P, GU L Z, HAN J D, ZHANG C X . Dynamic interactions between Bombyx mori nucleopolyhedrovirus and its host cells revealed by transcriptome analysis. Journal of Virology, 2012,86(13):7345-7359. doi: 10.1128/JVI.07217-12 pmid: 22532689
[10]	蒋亚明, 董战旗, 陈婷婷, 胡楠, 董非凡, 黄亮, 唐良彤, 潘敏慧 . 杆状病毒LEF-11蛋白自身相互作用关键区域的鉴定. 中国农业科学, 2017,50(20):4028-4035.
	JIANG Y M, DONG Z Q, CHEN T T, HU N, DONG F F, HUANG L, TANG L T, PAN M H . Identification the key areas of Bombyx mori nucleopolyhedrovirus LEF-11 self-interaction. Scientia Agricultura Sinica, 2017,50(20):4028-4035. (in Chinese)
[11]	CLEM R J . Baculoviruses and apoptosis: a diversity of genes and responses. Current Drug Targets, 2007,8(10):1069-1074. doi: 10.2174/138945007782151405 pmid: 17979666
[12]	MONTEIRO F, CARINHAS N, CARRONDO M J, BERNAL V , ALVES P M . Toward system-level understanding of baculovirus-host cell interactions: from molecular fundamental studies to large-scale proteomics approaches. Frontiers in Microbiology, 2012, 3: Article 391. doi: 10.3389/fmicb.2012.00391 pmid: 3494084
[13]	GUY M P, FRIESEN P D . Reactive-site cleavage residues confer target specificity to baculovirus P49, a dimeric member of the P35 family of caspase inhibitors. Journal of Virology, 2008,82(15):7504-7514. doi: 10.1128/JVI.00231-08 pmid: 18508888
[14]	HUANG N, WU W, YANG K, PASSARELLI A L, ROHRMANN G F, CLEM R J . Baculovirus infection induces a DNA damage response that is required for efficient viral replication. Journal of Virology, 2011,85(23):12547-12556. doi: 10.1128/JVI.05766-11 pmid: 3209345
[15]	MITCHELL J K, BYERS N M, FRIESEN P D . Baculovirus F-box protein LEF-7 modifies the host DNA damage response to enhance virus multiplication. Journal of Virology, 2013,87(23):12592-12599. doi: 10.1128/JVI.02501-13 pmid: 24027328
[16]	SHIRATA N, IKEDA M, KOBAYASHI M . Identification of a Hyphantria cunea nucleopolyhedrovirus (NPV) gene that is involved in global protein synthesis shutdown and restricted Bombyx mori NPV multiplication in a B. mori cell line . Virology, 2010,398(2):149-157. doi: 10.1016/j.virol.2009.11.049 pmid: 20034650
[17]	KANNAN R P, HENSLEY L L, EVERS L E, LEMON S M, MCGIVERN D R . Hepatitis C virus infection causes cell cycle arrest at the level of initiation of mitosis. Journal of Virology, 2011,85(16):7989-8001. doi: 10.1128/JVI.00280-11 pmid: 3147967
[18]	DING L, HUANG Y, DAI M, ZHAO X, DU Q, DONG F, WANG L, HUO R, ZHANG W, XU X, TONG D . Transmissible gastroenteritis virus infection induces cell cycle arrest at S and G2/M phases via p53-dependent pathway. Virus Research, 2013,178(2):241-251. doi: 10.1016/j.virusres.2013.09.036 pmid: 24095767
[19]	EVERETT H, MCFADDEN G . Apoptosis: an innate immune response to virus infection. Trends in Microbiology, 1999,7(4):160-165. doi: 10.1016/S0966-842X(99)01487-0 pmid: 10217831
[20]	HUANG Y, LIU H, LI S, TANG Y, WEI B, YU H, WANG C . MAVS-MKK7-JNK2 defines a novel apoptotic signaling pathway during viral infection. PLoS Pathogens, 2014,10(3):e1004020. doi: 10.1371/journal.ppat.1004020 pmid: 3961361
[21]	ZMASEK C M, GODZIK A . Evolution of the animal apoptosis network//BAEHRECKE E H, GREEN D R, KORNBLUTH S, SALVESEN G S. Cold Spring Harbor Perspectives in Biology, 2013,5:a008649.
[22]	ZHANG J Y, PAN M H, SUN Z Y, HUANG S J, YU Z S, LIU D, ZHAO D H, LU C . The genomic underpinnings of apoptosis in the silkworm,Bombyx mori. BMC Genomics, 2010,11:611. doi: 10.1186/1471-2164-11-611 pmid: 3091752
[23]	HUANG Q, DEVERAUX Q L, MAEDA S, STENNICKE H R, HAMMOCK B D, REED J C . Cloning and characterization of an inhibitor of apoptosis protein (IAP) from Bombyx mori. Biochimica et Biophysica Acta, 2001,1499(3):191-198. doi: 10.1016/S0167-4889(00)00105-1 pmid: 11341966
[24]	HAMAJIMA R, IWAMOTO A, TOMIZAKI M, SUGANUMA I, KITAGUCHI K, KOBAYASHI M, YAMADA H, IKEDA M . Functional analysis of inhibitor of apoptosis 1 of the silkworm Bombyx mori. Insect Biochemistry and Molecular Biology, 2016,79:97-107. doi: 10.1016/j.ibmb.2016.10.012 pmid: 27989836
[25]	PAN M H, CAI X J, LIU M, LV J, TANG H, TAN J, LU C . Establishment and characterization of an ovarian cell line of the silkworm,Bombyx mori. Tissue and Cell, 2010,42(1):42-46.
[26]	ZHANG J, HE Q, ZHANG C D, CHEN X Y, CHEN X M, DONG Z Q, LI N, KUANG X X, CAO M Y, LU C, PAN M H . Inhibition of BmNPV replication in silkworm cells using inducible and regulated artificial microRNA precursors targeting the essential viral gene lef-11. Antiviral Research, 2014,104:143-152. doi: 10.1016/j.antiviral.2014.01.017 pmid: 24486953
[27]	张倩, 刘太行, 董小龙, 吴云飞, 杨基贵, 周亮, 潘彩霞, 潘敏慧 . 家蚕CDK11与RNPS1和9G8相互作用的鉴定. 中国农业科学, 2017,50(22):4398-4407.
	ZHANG Q, LIU T H, DONG X L, WU Y F, YANG J G, ZHOU L, PAN C X, PAN M H . Identification of the interactions of CDK11 with RNPS1 and 9G8 in the silkworm (Bombyx mori). Scientia Agricultura Sinica, 2017,50(22):4398-4407. (in Chinese)
[28]	DONG Z Q, CHEN T T, ZHANG J, HU N, CAO M Y, DONG F F, JIANG Y M, CHEN P, LU C, PAN M H . Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells. Antiviral Research, 2016,130:50-57. doi: 10.1016/j.antiviral.2016.03.009 pmid: 26979473
[29]	ZHOU B H, WANG H W, ZHAO Z S, LIU M, YAN W C, ZHAO J, ZHANG Z, XUE F Q . A novel serine/threonine protein phosphatase type 5 from second-generation merozoite of Eimeria tenella is associated with diclazuril-induced apoptosis. Parasitology Research, 2013,112(4):1771-1780. doi: 10.1007/s00436-013-3336-0 pmid: 23417098
[30]	WANG J, ZHU J, DONG M, YU H, DAI X, LI K . Inhibition of protein phosphatase 5 (PP5) suppresses survival and growth of colorectal cancer cells. Biotechnology and Applied Biochemistry, 2015,62(5):621-627. doi: 10.1002/bab.1308 pmid: 25322973
[31]	BERTHELET J, DUBREZ L . Regulation of apoptosis by inhibitors of apoptosis (IAPs). Cells, 2013,2(1):163-187. doi: 10.3390/cells2010163 pmid: 3972657
[32]	UREN A G, COULSON E J, VAUX D L . Conservation of baculovirus inhibitor of apoptosis repeat proteins (BIRPs) in viruses, nematodes, vertebrates and yeasts. Trends in Biochemical Sciences, 1998,23(5):159-162. doi: 10.1016/S0968-0004(98)01198-0 pmid: 9612077
[33]	SWINGLE M R, HONKANEN R E, CISZAK E M . Structural basis for the catalytic activity of human serine/threonine protein phosphatase-5. The Journal of Biological Chemistry, 2004,279(32):33992-33999. doi: 10.1074/jbc.M402855200 pmid: 15155720
[34]	YANG J, ROE S M, CLIFF M J, WILLIAMS M A, LADBURY J E, COHEN P T, BARFORD D . Molecular basis for TPR domain- mediated regulation of protein phosphatase 5. The EMBO Journal, 2005,24(1):1-10. doi: 10.1038/sj.emboj.7600496
[35]	MORITA K, SAITOH M, TOBIUME K, MATSUURA H, ENOMOTO S, NISHITOH H, ICHIJO H . Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress. The EMBO Journal, 2001,20(21):6028-6036. doi: 10.1093/emboj/20.21.6028 pmid: 11689443
[36]	WECHSLER T, CHEN B P, HARPER R, MOROTOMI-YANO K, HUANG B C, MEEK K, CLEAVER J E, CHEN D J, WABL M . DNA-PKcs function regulated specifically by protein phosphatase 5. Proceedings of the National Academy of Sciences of the United States of America, 2004,101(5):1247-1252. doi: 10.1073/pnas.0307765100 pmid: 14734805
[37]	YUAN M, HUANG Z, WEI D, HU Z, YANG K, PANG Y . Identification of Autographa californica nucleopolyhedrovirus ac93 as a core gene and its requirement for intranuclear microvesicle formation and nuclear egress of nucleocapsids. Journal of Virology, 2011,85(22):11664-11674. doi: 10.1128/JVI.05275-11 pmid: 21880748
[38]	ONO C, KAMAGATA T, TAKA H, SAHARA K, ASANO S, BANDO H . Phenotypic grouping of 141 BmNPVs lacking viral gene sequences. Virus Research, 2012,165(2):197-206. doi: 10.1016/j.virusres.2012.02.016 pmid: 22421381

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

引物名称 Primer name	引物序列 Primer sequence
pIZ/V5-Flag-Bmiap-F	5′-CGCGGATCCATGGACTACAAAGACGATGACGACAAGGAGTTGACGAAAGTTGCTA-3′
pIZ/V5-Flag-Bmiap-R	5′-CCGCTCGAGTCACTTGTCGTCATCGTCTTTGTAGTCCGAGAAGTAGAGCCGCACCGCA-3′
pIZ/V5-HA-Bmpp5-F	5′-CGCGGATCCATGTACCCATACGATGTTCCAGATTACGCTTCTAATCACGAAGAAATCACTG-3′
pIZ/V5-HA-Bmpp5-R	5′-CCGCTCGAGTTAAGCGTAATCTGGAACATCGTATGGGTATTGGCAGAGGAAATTGAAG-3′
Knockdown-Bmiap-F	5′-AAGTGCCCGACATGCGTCGTGAAG-3′
Knockdown-Bmiap-R	5′-AAACCTTCACGACGCATGTCGGGC-3′
Knockdown-Bmpp5-F	5′-AAGTGGTCGAGTGCTTGTAATGCA-3′
Knockdown-Bmpp5-R	5′-AAACTGCATTACAAGCACTCGACC-3′
Bmiap-qRT-PCR-F	5′-CCTTAGTGACTCCTGCTTTACGAA-3′
Bmiap-qRT-PCR-R	5′-TAGAAACTTGCAAATGGCTTGTG-3′
Bmpp5-qRT-PCR-F	5′-GGTTTCCGTGGGGAGGTG-3′
Bmpp5-qRT-PCR-R	5′-AGGCGGCTGTTTGTTTCG-3′
vp39-qRT-PCR-F	5′-CTAATGCCCGTGGGTATGG-3′
vp39-qRT-PCR-R	5′-TTGATGAGGTGGCTGTTGC-3′
sw22934-qRT-PCR-F	5′-TTCGTACTGGCTCTTCTCGT-3′
sw22934-qRT-PCR-R	5′-CAAAGTTGATAGCAATTCCCT-3′

蛋白编号 Protein ID	蛋白分子量 Protein molecular weight (kD)	蛋白描述 Protein description
BGIBMGA003212	43633.63	26S蛋白酶体非ATP酶调节亚基6 26S proteasome non-ATPase regulatory subunit 6
BGIBMGA011237	46381.43	26S蛋白酶体非ATP酶调节亚基13 26S proteasome non-ATPase regulatory subunit 13
BGIBMGA003587	48985.52	蛋白质二硫键异构酶类蛋白ERp57前体 Protein disulfide-isomerase like protein ERp57 precursor
BGIBMGA014181	53335.57	线粒体三功能酶β亚基 Trifunctional enzyme subunit beta, mitochondrial
BGIBMGA008046	55096.73	液泡蛋白分选蛋白33A Vacuolar protein sorting-associated protein 33A-like
BGIBMGA012549	55160.29	H⁺转运ATP合成酶β亚基亚型 1 H⁺ transporting ATP synthase beta subunit isoform 1
BGIBMGA004807	56617.87	丝氨酸/苏氨酸蛋白磷酸酶 Serine/threonine-protein phosphatase 5
BmNPV ORF76	18385.21	家蚕核型多角体病毒ORF76 BmNPV ORF76

Screening and Identification of Proteins Interacting with Bombyx mori IAP and Their Effects on BmNPV Proliferation

RichHTML

PDF

Abstract

Cite this article

share this article

Figures/Tables 7

References 38

Related Articles 15

Metrics

Comments

Recommended 0

[1]	LONG YanBi,WU YunFei,ZHANG Qian,CHEN Peng,PAN MinHui. Screening and Identification of HSP90 Interacting Proteins in Silkworm (Bombyx mori) [J]. Scientia Agricultura Sinica, 2022, 55(6): 1253-1262.
[2]	YUAN GuiBo,MO ShuangRong,QIAN Ying,ZANG DongNan,YANG Fan,JIANG HongLiang,WU Yuan,DING HaiDong. Screening of Interacting Protein of Tomato SIVQ6 by GST Pull-Down [J]. Scientia Agricultura Sinica, 2020, 53(15): 3146-3157.
[3]	DONG ZhanQi,JIANG YaMing,PAN MinHui. Screening and Identification of Candidate Proteins Interacting with BmHSP60 in the Silkworm (Bombyx mori) [J]. Scientia Agricultura Sinica, 2019, 52(2): 376-384.
[4]	YI Min,LÜ Qing,LIU KeKe,WANG LiJun,WU YuJiao,ZHOU ZeYang,LONG MengXian. Expression, Purification and Localization Analysis of Polar Tube Protein 2 (NbPTP2) from Nosema bombycis [J]. Scientia Agricultura Sinica, 2019, 52(10): 1830-1838.
[5]	ZHANG Kui,LI ChongYang,SU JingJing,TAN Juan,XU Man,CUI HongJuan. Expression, Purification and Immunologic Function of Integrin β2 in the Silkworm (Bombyx mori) [J]. Scientia Agricultura Sinica, 2019, 52(1): 181-190.
[6]	WANG Fei, LI XianYang, HUA XiaoTing, XIA QingYou. Screening and Analysis of Anti-BmNPV Cytokines in Silkworm (Bombyx mori) [J]. Scientia Agricultura Sinica, 2018, 51(4): 789-799.
[7]	Jie HU,XinYi WANG,Fei WANG. Functional Characterization of BmCaspase-8-Like (BmCasp8L) as an Immune Negative Regulatory Molecule in Silkworm (Bombyx mori) [J]. Scientia Agricultura Sinica, 2018, 51(21): 4188-4196.
[8]	JIANG YaMing, DONG ZhanQi, CHEN TingTing, HU Nan, DONG FeiFan, HUANG Liang, TANG LiangTong, PAN MinHui. Identification the key areas of Bombyx mori Nucleopolyhedrovirus LEF-11 self-interaction [J]. Scientia Agricultura Sinica, 2017, 50(20): 4028-4035.
[9]	GAO Rui, LI ChunLin, TONG XiaoLing, CAO MingYa, SHI MeiNing, XU AnYing, LU Cheng, DAI FangYin. Insight into Genetic Basis of Bombyx mori Resistant Strains with Resistance to BmNPV by Molecular Linkage Analysis [J]. Scientia Agricultura Sinica, 2017, 50(1): 195-204.
[10]	YU Yi-he, LI Xiu-zhen, GUO Da-long, YANG Ying-jun, LI Xue-qiang, ZHANG Guo-hai. Screening and Identification of the Interacting Protein of Cytokinin Response Regulator VvRR2 in Grapevine [J]. Scientia Agricultura Sinica, 2016, 49(6): 1097-1105.
[11]	FAN Jin-tao, JIA Jiao, JIANG Chen-xi, WANG Guan-yu, ZHANG Jing, XING Ji-hong, DONG Jin-gao. Regional Analysis of Transcription Activity and Screening of Interaction Proteins of AtMYB73 Transcription Factor in Arabidopsis [J]. Scientia Agricultura Sinica, 2014, 47(23): 4754-4762.
[12]	GUO Rui, LIU Wei-Xing, PAN Zhong-Hua, XUE Ren-Yu, CAO Guang-Li, ZHU Yue-Xiong, GONG Cheng-Liang. Construction of Recombinant BmNPV Infecting Ecotropis oblique [J]. Scientia Agricultura Sinica, 2012, 45(16): 3288-3296.
[13]	SUN Hai-Tao, XU Zhao-Shi, HOU Jian-Hua, YU Zhuo, ZHAO Yue, LI Lian-Cheng, CHEN Ming, MA You-Zhi. Screening of the Interaction Proteins of TaDREB6 Transcription Factor in Wheat [J]. Scientia Agricultura Sinica, 2011, 44(22): 4740-4747.
[14]	Ke-Ping CHEN . Studies on protein of the occlusion-derived virus of Bombyx mori L. Nucleopolyhedrovirus using two-dimensional gel electrophoresis and mass spectrometry [J]. Scientia Agricultura Sinica, 2008, 41(7): 2215-2218 .
[15]	. The inhibitory effects of the corresponding dsRNA of gp64 gene on the replication and multiplication of BmNPV [J]. Scientia Agricultura Sinica, 2007, 40(12): 2882-2887 .