脑心肌炎病毒VP1蛋白抑制Ⅰ型IFN信号通路和促进病毒体外增殖

doi:10.3969/j.issn.1004-1524.2021.01.03

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (1): 18-26.DOI: 10.3969/j.issn.1004-1524.2021.01.03

脑心肌炎病毒VP1蛋白抑制Ⅰ型IFN信号通路和促进病毒体外增殖

韩玉梅¹^,²(), 谢晶莹³, 毕英杰¹^,², 许淑娟¹^,², 冯若飞¹^,^*()

1.西北民族大学生物医学研究中心生物工程与技术国家民委重点实验室,甘肃兰州 730030
2.西北民族大学生命科学与工程学院,甘肃兰州 730030
3.甘肃农业大学动物医学院,甘肃兰州 730070

收稿日期:2020-04-23 出版日期:2021-01-25 发布日期:2021-01-25
通讯作者: 冯若飞
作者简介:*冯若飞,E-mail:fengruofei@xbmu.edu.cn
韩玉梅(1988—),女,青海民和人,硕士研究生,主要研究分子病毒学与动物疫病防治。E-mail:2397695720@qq.com
基金资助:
国家自然科学基金(31460665);2019年中央专项研究生科研创新项目(Yxm2019148);国家民委中青年英才计划((2018)98);教育部“创新团队发展计划”(IRT_17R88)

VP1 protein of encephalomyocarditis virus inhibits type I IFN signaling pathway and promotes virus proliferation in vitro

HAN Yumei¹^,²(), XIE Jingying³, BI Yingjie¹^,², XU Shujuan¹^,², FENG Ruofei¹^,^*()

1. Key Laboratory of Biotechnology and Bioengineering of State Ethnic Affairs Commission, Biomedical Research Center, Northwest Minzu University, Lanzhou 730030, China
2. College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030, China
3. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China

Received:2020-04-23 Online:2021-01-25 Published:2021-01-25
Contact: FENG Ruofei

摘要/Abstract

摘要：

脑心肌炎病毒是一种重要的人畜共患病病原,为了初步研究EMCV介导的天然免疫反应的分子机制,将VP1蛋白克隆至真核表达载体pCMV-HA,转染HEK293细胞后,使其在细胞内过表达,通过ELISA和实时荧光定量PCR检测VP1过表达对IFN-β表达和RLRs信号通路相关因子,以及下游刺激基因表达的影响。结果表明,VP1蛋白可显著抑制IFN-β的表达。进一步研究发现,VP1可显著抑制EMCV引起的RLRs信号通路相关因子mRNA水平(P<0.01),并且随着VP1的表达,MAVS蛋白的表达明显减少,但对MDA5表达无影响。实验数据初步表明,VP1蛋白可以通过抑制MAVS的表达,进而抑制Ⅰ型IFN基因的表达并阻断其下游信号通路发挥作用。

关键词: 脑心肌炎病毒, 结构蛋白VP1, Ⅰ型IFN信号通路;

Abstract:

Encephalomyocarditis virus (EMCV) is an important zoonotic pathogen. In order to preliminarily study the molecular mechanism of EMCV mediated innate immune response, this study cloned VP1 protein into eukaryotic expression vector pCMV-HA, transfected it into HEK293 cells to make it overexpress in cells. The effects of VP1 overexpression on IFN-β expression and RLRs signaling pathway-related factors and downstream stimulation gene expression were detected by ELISA and real time fluorescence quantitative PCR. Results showed VP1 could significantly inhibit IFN-β expression. Further study found that VP1 could significantly inhibit the mRNA level of EMCV-induced RLRs signaling pathway-related factors, and with the expression of VP1, MAVS protein expression was significantly reduced, but had no effect on the expression of MDA5. Therefore, the data of this experiment preliminarily showed that VP1 protein could inhibit the expression of type Ⅰ IFN gene expression and block its downstream signaling pathway by inhibiting the expression of MAVS.

Key words: encephalomyocarditis virus, structural protein VP1, Type Ⅰ IFN signaling pathway

中图分类号:

S855.99

韩玉梅, 谢晶莹, 毕英杰, 许淑娟, 冯若飞. 脑心肌炎病毒VP1蛋白抑制Ⅰ型IFN信号通路和促进病毒体外增殖[J]. 浙江农业学报, 2021, 33(1): 18-26.

HAN Yumei, XIE Jingying, BI Yingjie, XU Shujuan, FENG Ruofei. VP1 protein of encephalomyocarditis virus inhibits type I IFN signaling pathway and promotes virus proliferation in vitro[J]. Acta Agriculturae Zhejiangensis, 2021, 33(1): 18-26.

图/表 9

表1 EMCV TaqMan探针法实时荧光定量PCR引物及探针

Table 1 TaqMan real time fluorescence quantitative PCR primers and probe for EMCV detection

引物 Primer	引物序列Primer sequence(5'-3')
EMCV-probe	(FAM)CACTTCGATCACTATGCTTGCCGTT(Eclipse)
EMCV-qF	GTCATACTATCGTCCAGGGACTCTAT
EMCV-qR	CATCTGTACTCCACACTCTCGAATG

表2 实时荧光定量PCR引物序列

Table 2 Primer sequences for real time fluorescence quantitative PCR

引物名称Primer name	引物序列Primer sequence(5'-3')
IFN-β-F	TTGTTGAGAACCTCCTGGCT
IFN-β-R	TGACTATGGTCCAGGCACAG
RIG-I-F	CACCTCAGTTGCTGATGAAGGC
RIG-I-R	GTCAGAAGGAAGCACTTGCTACC
MDA5-F	GCTGAAGTAGGAGTCAAAGCCC
MDA5-R	CCACTGTGGTAGCGATAAGCAG
MAVS-F	ATGGTGCTCACCAAGGTGTCTG
MAVS-R	TCTCAGAGCTGCTGTCTAGCCA
TBK1-F	CAACCTGGAAGCGGCAGAGTTA
TBK1-R	ACCTGGAGATAATCTGCTGTCGA
IRF3-F	TCTGCCCTCAACCGCAAAGAAG
IRF3-R	TACTGCCTCCACCATTGGTGTC

图1 EMCV感染对Ⅰ型IFN信号通路相关因子mRNA表达的实时荧光定量PCR检测

Fig.1 Real time fluorescence quantitative PCR detected the mRNA level of type Ⅰ IFN signaling pathway related factor during EMCV infection *,P<0.05;**,P<0.01;***,P<0.001.

图2 VP1在不同细胞中的表达与细胞活力检测

Fig.2 Expression of VP1 in different cell lines and cell viability detection

图3 实时荧光定量PCR和TCID50检测EMCV病毒拷贝数和病毒感染力

Fig.3 EMCV virus copy number and virus titer were detected by real time fluorescence quantitative PCR and TCID50 assay

图4 VP1对Ⅰ型IFN信号通路相关因子mRNA的影响

Fig.4 Effect of VP1 on type Ⅰ IFN signaling pathway related factors mRNA expression

图5 VP1对EMCV诱导的Ⅰ型IFN信号通路相关因子mRNA的影响

Fig.5 Effect of VP1 on EMCV induced type Ⅰ IFN signaling pathway related factors mRNA expression

图6 Western-blot检测VP1对MDA5和MAVS表达的影响

Fig.6 Effects of VP1 on the expression of MDA5 and MAVS by Western-blot

图7 EMCV VP1抑制IFN-β信号通路模式图

Fig.7 Pattern of EMCV VP1 suppressing IFN-β signaling pathway

参考文献 32

[1]	STRAW BE, ALLAIRES D, WILLIAM L, et al. Diseases of swine [J]. Ames: Iowa State University Press, 1999: 139-144.
[2]	WANG Z, LIU Y B, LIN W C, et al. A real-time PCR to detect and analyze virulent EMCV loads in sows and piglets[J]. Molecular Biology Reports, 2012,39(12):10013-10017. DOI URL PMID
[3]	HUNTER P, SWANEPOEL S P, ESTERHUYSEN J J, et al. The efficacy of an experimental oil-adjuvanted encephalomyocarditis vaccine in elephants, mice and pigs[J]. Vaccine, 1998,16(1):55-61. URL PMID
[4]	ZIMMERMANN A, NELSEN-SALZ B, KRUPPENBACHER J P, et al. The complete nucleotide sequence and construction of an infectious cDNA clone of a highly virulent encephalomyocarditis virus[J]. Virology, 1994,203(2):366-372. URL PMID
[5]	TESH R B, WALLACE G D. Observations on the natural history of encephalomyocarditis virus[J]. The American Journal of Tropical Medicine and Hygiene, 1978,27(1):133-143. DOI URL
[6]	张元峰, 张昆丽, 薛娟, 等. 猪脑心肌炎病毒VP1蛋白与宿主RPS20蛋白相互作用研究[J]. 中国预防兽医学报, 2018,40(8):715-719.
	ZHANG Y F, ZHANG K L, XUE J, et al. Encephalomyocarditis virus VP1 interacts with ribosomal protein 20[J]. Chinese Journal of Preventive Veterinary Medicine, 2018,40(8):715-719.(in Chinese with English abstract)
[7]	BAI J, CHEN X H, JIANG K F, et al. Identification of VP1 peptides diagnostic of encephalomyocarditis virus from swine[J]. Virology Journal, 2014,11:226. DOI URL PMID
[8]	KATO H, TAKEUCHI O, SATO S, et al. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses[J]. Nature, 2006,441(7089):101-105. URL PMID
[9]	LOO Y M, FORNEK J, CROCHET N, et al. Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity[J]. Journal of Virology, 2008,82(1):335-345. DOI URL PMID
[10]	KAWAI T, AKIRA S. Signaling to NF-κB by Toll-like receptors[J]. Trends in Molecular Medicine, 2007,13(11):460-469. DOI URL PMID
[11]	LOO Y M, GALE M Jr. Immune signaling by RIG-I-like receptors[J]. Immunity, 2011,34(5):680-692. DOI URL PMID
[12]	SETH R B, SUN L J, EA C K, et al. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-κB and IRF3[J]. Cell, 2005,122(5):669-682. URL PMID
[13]	VAZQUEZ C, HORNER S M. MAVS coordination of antiviral innate immunity[J]. Journal of Virology, 2015,89(14):6974-6977. DOI URL PMID
[14]	SCHOGGINS J W, RICE C M. Interferon-stimulated genes and their antiviral effector functions[J]. Current Opinion in Virology, 2011,1(6):519-525. DOI URL PMID
[15]	SCHNEIDER W M, CHEVILLOTTE M D, RICE C M. Interferon-stimulated genes: a complex web of host defenses[J]. Annual Review of Immunology, 2014,32(1):513-545.
[16]	TAKEUCHI O, AKIRA S. Pattern recognition receptors and inflammation[J]. Cell, 2010,140(6):805-820. URL PMID
[17]	VANCE R E, ISBERG R R, PORTNOY D A. Patterns of pathogenesis: discrimination of pathogenic and nonpathogenic microbes by the innate immune system[J]. Cell Host & Microbe, 2009,6(1):10-21. DOI URL PMID
[18]	LI X Y, WANG J C, LIU J, et al. Engagement of soluble resistance-related calcium binding protein (sorcin) with foot-and-mouth disease virus (FMDV) VP1 inhibits type I interferon response in cells[J]. Veterinary Microbiology, 2013,166(1/2):35-46. DOI URL
[19]	张海霞, 冯若飞, 王丹, 等. 脑心肌炎病毒 TaqMan real-time PCR检测方法的建立及应用[J]. 中华微生物学和免疫学杂志, 2013,33(9):706-710.
	ZHANG H X, FENG R F, WANG D, et al. Establishment and application of a TaqMan real-time PCR assay for the detection of encephalomyo-carditis virus[J]. Chinese Journal of Microbiology and Immunology, 2013,33(9):706-710.(in Chinese with English abstract)
[20]	BOWIE A G, UNTERHOLZNER L. Viral evasion and subversion of pattern-recognition receptor signalling[J]. Nature Reviews. Immunology, 2008,8(12):911-922. URL PMID
[21]	KUMAR H, KAWAI T, KATO H, et al. Essential role of IPS-1 in innate immune responses against RNA viruses[J]. Journal of Experimental Medicine, 2006,203(7):1795-1803. DOI URL
[22]	LUO H L, WINKELMANN E, XIE G R, et al. MAVS is essential for primary CD4+T cell immunity but not for recall T cell responses following an attenuated west Nile virus infection[J]. Journal of Virology, 2017,91(6):e02097-16. DOI URL PMID
[23]	WANG J Y, LEI C Q, JI Y H, et al. Duck tembusu virus nonstructural protein 1 antagonizes IFN-β signaling pathways by targeting VISA[J]. Journal of Immunology, 2016,197(12):4704-4713. DOI URL
[24]	WANG X M, LI Y, MAO A P, et al. Hepatitis B virus X protein suppresses virus-triggered IRF3 activation and IFN-β induction by disrupting the VISA-associated complex[J]. Cellular & Molecular Immunology, 2010,7(5):341-348. DOI URL PMID
[25]	CHEN Z H, BENUREAU Y, RIJNBRAND R, et al. GB virus B disrupts RIG-I signaling by NS₃/4A-mediated cleavage of the adaptor protein MAVS[J]. ournal of Virology, 2007,81(2):964-976.
[26]	LI D, YANG W P, YANG F, et al. The VP3 structural protein of foot-and-mouth disease virus inhibits the IFN-β signaling pathway[J]. The FASEB Journal, 2016,30(5):1757-1766. DOI URL PMID
[27]	LI D, WEI J, YANG F, et al. Foot-and-mouth disease virus structural protein VP3 degrades Janus kinase 1 to inhibit IFN-γ signal transduction pathways[J]. Cell Cycle (Georgetown, Tex.), 2016,15(6):850-860. DOI URL PMID
[28]	MORELLI M, OGDEN K M, PATTON J T. Silencing the alarms: Innate immune antagonism by Rotavirus NSP1 and VP3[J]. Virology, 2015,479/480:75-84.
[29]	LI L, FAN H, SONG Z B, et al. Encephalomyocarditis virus 2C protein antagonizes interferon-β signaling pathway through interaction with MDA5[J]. Antiviral Research, 2019,161:70-84. DOI URL PMID
[30]	ITO M, YANAGI Y, ICHINOHE T. Encephalomyocarditis virus viroporin 2B activates NLRP3 inflammasome[J]. PLoS Pathogens, 2012,8(8):e1002857. DOI URL PMID
[31]	HUANG L, XIONG T, YU H B, et al. Encephalomyocarditis virus 3C protease attenuates type I interferon production through disrupting the TANK-TBK1-IKKε-IRF3 complex[J]. Biochemical Journal, 2017,474(12):2051-2065.
[32]	LUO H, WONG J, WONG B. Protein degradation systems in viral myocarditis leading to dilated cardiomyopathy[J]. Cardiovascular Research, 2010,85(2):347-356. DOI URL PMID

脑心肌炎病毒VP1蛋白抑制Ⅰ型IFN信号通路和促进病毒体外增殖

VP1 protein of encephalomyocarditis virus inhibits type I IFN signaling pathway and promotes virus proliferation in vitro

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 32

相关文章 1

编辑推荐

Metrics

本文评价