干扰素诱导跨膜蛋白1对猪源冠状病毒吸附宿主细胞的影响

doi:10.16431/j.cnki.1671-7236.2021.04.024

摘要/Abstract

摘要： 为探索干扰素诱导跨膜蛋白1（interferon-inducible transmembrane protein 1，IFITM1）对猪源冠状病毒复制的影响，本研究选用猪传染性胃肠炎病毒（Transmissible gastroenteritis virus，TGEV）高致病毒株及其宿主细胞PK15作为研究对象。正常PK15细胞接种TGEV后，实时荧光定量PCR检测感染后不同时间点PK15细胞中一些干扰素刺激基因（interferon-stimulated genes，ISGs）的mRNA表达水平；利用慢病毒表达系统构建稳定表达和稳定干扰IFITM1表达的PK15细胞系。将一段靶向干扰猪源IFITM1的shRNA序列及猪源IFITM1全长，分别插入pLKO.1-EGFP-Puro载体及pLVML-Myc-MCS-IRES-Puro载体中，分别构建出pLKO.1-IFITM1shRNA-EGFP-Puro及pLVML-Myc-IFITM1-IRES-Puro重组质粒。将重组质粒与慢病毒包装质粒共转染293FT细胞后获得带有目的基因的重组慢病毒，慢病毒侵染PK15细胞后用嘌呤霉素进行筛选，获得稳定表达及稳定干扰IFITM1表达的PK15细胞系，分别命名为PK15-IFITM1及PK15-IFITM1^-/-，并分别用实时荧光定量PCR、间接免疫荧光试验（IFA）及Western blotting检测IFITM1干扰效率及表达情况；TGEV接种PK15-IFITM1^-/-和PK15-IFITM1，实时荧光定量PCR测定细胞中TGEV的拷贝数。结果显示，PK15细胞接种TGEV后的48 h内，一些ISGs的mRNA水平均有所上升；PK15-IFITM1^-/-细胞系的干扰效率为70%，PK15-IFITM1细胞系表达成功；在PK15-IFITM1^-/-细胞系中，IFITM1的mRNA水平显著下调，促进了TGEV的复制。反之，在PK15-IFITM1细胞系中，TGEV的复制受到了抑制。但IFITM1的表达或缺失却不影响TGEV对PK15的吸附作用。总之，IFITM1对TGEV有显著的抗病毒作用，IFITM1不影响TGEV对PK15细胞的早期吸附，这为后续IFITM1抗冠状病毒机制的研究奠定了基础。

关键词: 干扰素诱导跨膜蛋白1(IFITM1); 冠状病毒; 传染性胃肠炎病毒(TGEV)

Abstract: In order to explore the effect of interferon-inducible transmembrane protein 1 (IFITM1) on the replication of porcine coronaviruses,the highly pathogenic virus strain of Transmissible gastroenteritis virus (TGEV) and its host cell PK15 were selected as the research objects.After normal PK15 cells were inoculated with TGEV,Real-time PCR was used to detect the mRNA expression levels of various interferon-stimulated genes (ISGs) in PK15 cells at different time points post infection.PK15 cell lines that stably expressed and stably interfered with the expression of IFITM1 were constructed through the lentiviral expression system.A shRNA sequence targeted to interfere with porcine IFITM1 and a full-length porcine IFITM1 sequence were inserted into pLKO.1-EGFP-Puro vector and pLVML-Myc-MCS-IRES-Puro vector to construct recombinant plasmids pLKO.1-IFITM1shRNA-EGFP-Puro and pLVML-Myc-IFITM1-IRES-Puro,respectively.The recombinant plasmids and the lentiviral packaging plasmids were co-transfected into 293FT cells to obtain a recombinant lentivirus with the target gene.After the lentivirus infected PK15 cells,it was screened with puromycin to obtain a PK15 cell line that stably expressed and stably interfered with the expression of IFITM1,named PK15-IFITM1 and PK15-IFITM1^-/-,respectively,and Real-time PCR,IFA and Western blotting were used to detect the interference efficiency and expression of IFITM1.TGEV was inoculated with PK15-IFITM1^-/- and PK15-IFITM1,and Real-time PCR was used to determine the copies of TGEV in the cells.The results showed that the mRNA level of some ISGs increased within 48 h after PK15 cells were inoculated with TGEV.The interference efficiency of PK15-IFITM1^-/-cell line was 70%,and the expression of PK15-IFITM1 cell line was successful.In the PK15-IFITM1^-/- cell line,the mRNA level of IFITM1 was significantly down-regulated,which promoted the replication of TGEV.Conversely,in the PK15-IFITM1 cell line,the replication of TGEV was inhibited.However,the expression or absence of IFITM1 did not affect the adsorption of TGEV to PK15.In short,IFITM1 had a significant antiviral effect on TGEV,IFITM1 had no effect on the adsorption of TGEV to PK15 cells,which laid the foundation for subsequent research on the anti-coronavirus mechanism of IFITM1.

Key words: interferon-inducible transmembrane protein 1 (IFITM1); coronavirus; Transmissible gastroenteritis virus (TGEV)

中图分类号:

S852.4

颜可欣, 邓治邦, 贺佳怡, 袁琦超, 王教顺, 袁晓民. 干扰素诱导跨膜蛋白1对猪源冠状病毒吸附宿主细胞的影响[J]. 中国畜牧兽医, 2021, 48(4): 1370-1380.

YAN Kexin, DENG Zhibang, HE Jiayi, YUAN Qichao, WANG Jiaoshun, YUAN Xiaomin. Effect of Interferon-inducible Transmembrane Protein 1 on the Adsorption of Porcine Coronavirus to Host Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(4): 1370-1380.

参考文献

[1] 王常乐,王磊,郝冉,等.SARS-CoV-2的研究进展[J].中国人兽共患病学报,2020,36(10):780-785. WANG C L,WANG L,HAO R,et al.What we know about SARS-CoV-2:Recent research progress[J].Chinese Journal of Zoonoses,2020,36(10):780-785.(in Chinese)
[2] 王艳丰,张丁华,李爱心,等.PEDV、TGEV、PoRV 3种猪病毒性腹泻病毒的流行现状及控制策略[J].中国畜牧兽医,2018,45(2):518-527. WANG Y F,ZHANG D H,LI A X,et al.The prevalence and prevention of PEDV,TGEV and PoRV in porcine viral diarrhea[J].China Animal Husbandry & Veterinary Medicine,2018,45(2):518-527.(in Chinese)
[3] HANSEN G H,DELMAS B,BESNARDEAU L,et al.The coronavirus Transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment[J].Journal of Virology,1998,72(1):527-534.
[4] HUANG I C,BAILEY C C,WEYER J L,et al.Distinct patterns of IFITM-mediated restriction of filoviruses,SARS coronavirus,and influenza A virus[J].PLoS Pathogens,2011,7(1):e1001258.
[5] 林苗,黄运生,高彦生.SARS病毒与动物冠状病毒相关性的研究进展[J].中国畜牧兽医,2003,30(3):3-8. LIN M,HUANG Y S,GAO Y S.Research progress on the correlation between SARS-Co1 and animal coronaviruses[J].China Animal Husbandry & Veterinary Medicine,2003,30(3):3-8.(in Chinese)
[6] 段群棚,李晓玉,赵硕,等.2016-2019年广西部分猪场仔猪病毒性腹泻的流行病学调查[J].中国畜牧兽医,2020,47(2):564-574. DUAN Q P,LI X Y,ZHAO S,et al.Epidemiological investigation on diarrhea viral disease of piglets in some pig farms in Guangxi during 2016 to 2019[J].China Animal Husbandry & Veterinary Medicine,2020,47(2):564-574.(in Chinese)
[7] 王淞,曾昊,陈智,等.山东省猪腹泻病例中PEDV、TGEV和PRV的感染调查与分析[J].中国畜牧兽医,2017,44(7):2165-2170. WANG S,ZENG H,CHEN Z,et al.Investigation and analysis of Porcine epidemic diarrhea virus,Transmissible gastroenteritis virus and Pseudorabies virus of swine diarrhea cases in Shandong province[J].China Animal Husbandry & Veterinary Medicine,2017,44(7):2165-2170.(in Chinese)
[8] SCHINDLER C,DARNELL J E.Transcriptional responses to polypeptide ligands:The JAK-STAT pathway[J].Annual Review of Biochemistry,1995,64:621-651.
[9] FRIEDMAN R L,MANLY S P,MCMAHON M,et al.Transcriptional and posttranscriptional regulation of interferon-induced gene expression in human cells[J].Cell,1984,38(3):745-755.
[10] SÄLLMAN A M,BRINGELAND N,FREDRIKSSON R,et al.The dispanins:A novel gene family of ancient origin that contains 14 human members[J].PLoS One,2012,7(2):e31961.
[11] REID L E,BRASNETT A H,GILBERT C S,et al.A single DNA response element can confer inducibility by both alpha- and gamma-interferons[J].Proceedings of the National Academy of Sciences of the United States of America,1989,86(3):840-844.
[12] TANAKA S S,YAMAGUCHI Y L,TSOI B,et al.IFITM/Mil/fragilis family proteins IFITM1 and IFITM3 play distinct roles in mouse primordial germ cell homing and repulsion[J].Developmental Cell,2005,9(6):745-756.
[13] BENFIELD C T O,SMITH S E,WRIGHT E,et al.Bat and pig IFN-induced transmembrane protein 3 restrict cell entry by Influenza virus and Lyssaviruses[J].The Journal of General Virology,2015,96(Pt 5):991-1005.
[14] LANZ C,ANDENMATTEN D,STERTZ S,et al.Swine interferon-inducible transmembrane proteins potently inhibit Influenza A virus replication[J].Journal of Virology,2015,89(1):863-869.
[15] SMITH S E,GIBSON M S,WASH R S,et al.Chicken interferon-inducible transmembrane protein 3 restricts Influenza viruses and Lyssaviruses in vitro[J].Journal of Virology,2013,87(23):12957-12966.
[16] BRASS A L,HUANG I C,BENITA Y,et al.The IFITM proteins mediate cellular resistance to Influenza A H1N1 virus,West Nile virus,and Dengue virus[J].Cell,2009,139(7):1243-1254.
[17] WILKINS C,WOODWARD J,LAU D T Y,et al.IFITM1 is a tight junction protein that inhibits Hepatitis C virus entry[J].Hepatology (Baltimore, Md.),2013,57(2):461-469.
[18] FEELEY E M,SIMS J S,JOHN S P,et al.IFITM3 inhibits Influenza A virus infection by preventing cytosolic entry[J].PLoS Pathogens,2011,7(10):e1002337.
[19] SMITH S E,WESTON S,KELLAM P,et al.IFITM proteins——Cellular inhibitors of viral entry[J].Current Opinion in Virology,2014,4:71-77.
[20] LEE W J,FU R M,LIANG C,et al.IFITM proteins inhibit HIV-1 protein synthesis[J].Scientific Reports,2018,8(1):14551.
[21] COMPTON A A,BRUEL T,PORROT F,et al.IFITM proteins incorporated into HIV-1 virions impair viral fusion and spread[J].Cell Host & Microbe,2014,16(6):736-747.
[22] SMITH R A,YOUNG J,WEIS J J,et al.Expression of the mouse fragilis gene products in immune cells and association with receptor signaling complexes[J].Genes & Immunity,2006,7(2):113-121.
[23] WESTON S,CZIESO S,WHITE I J,et al.A membrane topology model for human interferon inducible transmembrane protein 1[J].PLoS One,2014,9(8):e104341.
[24] XU Y,YANG G,HU G.Binding of IFITM1 enhances the inhibiting effect of caveolin-1 on ERK activation[J].Acta Biochimica et Biophysica Sinica,2009,41(6):488-494.
[25] LI K,MARKOSYAN R M,ZHENG Y M,et al.IFITM proteins restrict viral membrane hemifusion[J].PLoS Pathogens,2013,9(1):e1003124.
[26] WANG J,WANG C F,MING S L,et al.Porcine IFITM1 is a host restriction factor that inhibits Pseudorabies virus infection[J].International Journal of Biological Macromolecules,2020,151:1181-1193.
[27] LIAO Y,GORAYA M U,YUAN X,et al.Functional involvement of interferon-inducible transmembrane proteins in antiviral immunity[J].Frontiers in Microbiology,2019,10:1097.