稳定表达人IFITM3的猪IPEC-J2细胞系的构建及其对PEDV增殖的影响

doi:10.11843/j.issn.0366-6964.2025.10.037

Abstract

Abstract:

Previous genome-wide CRISPR screening in human hepatocellular carcinoma cells identified human interferon-induced transmembrane protein 3 (IFITM3) as a critical host factor regulating porcine epidemic diarrhea virus (PEDV) infection. However, its functional role in porcine-derived cells remains unexplored. Here, we generated a porcine IPEC-J2 cell line stablely overexpressing hIFITM3 (IPEC-J2^hIFITM3) and systematically evaluated its impact on viral proliferation. The recombinant lentiviral vector encoding hIFITM3 was co-transfected with packaging plasmids pMD2.G/psPAX2 into HEK293T cells to produce replication-incompetent lentiviral particles. Following transduction of IPEC-J2 cells and puromycin selection, monoclonal cells were established via limiting dilution. Robust hIFITM3 overexpression was confirmed by Western blot, indirect immunofluorescence assay (IFA), and RT-qPCR. CCK-8 assays verified no significant cytotoxicity associated with hIFITM3 expression. Functional validation demonstrated that IPEC-J2^hIFITM3 cells potently suppressed vesicular stomatitis virus (VSV-GFP) replication, consistent with IFITM3's canonical antiviral function. Strikingly, challenge with PEDV DR13-GFP revealed a paradoxical pro-viral effect: IPEC-J2^hIFITM3 cells exhibited a 1 000-fold increase in viral titer (TCID₅₀ ·mL^-1) compared to wild-type cells at 24 hpi. This study establishes a porcine intestinal epithelial model stably expressing hIFITM3, which unexpectedly potentiates PEDV replication. These findings not only highlight the species-specific functional divergence of IFITM3 but also provide a unique platform for investigating PEDV-host interactions, optimizing viral cultivation, and developing targeted antiviral therapeutics.

Key words: porcine epidemic diarrhea virus, IPEC-J2 cells, IFITM3, cell line

CLC Number:

S852.659.6

TANG Jinmeng, YU Shunan, YUAN Yixin, MA Yuchen, DU Wenjuan, YU Linyang, LI Yongtao. Construction of Porcine IPEC-J2 Cell Line Stably Expressing Human IFITM3 and Its Effect on PEDV Proliferation[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(10): 5180-5189.

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References 26

1	SUEYOSHIM,TSUDAT,YAMAZAKIK,et al.An immunohistochemical investigation of porcine epidemic diarrhoea[J].J Comp Pathol,1995,113(1):59-67. doi: 10.1016/S0021-9975(05)80069-6
2	ZHAOF,MAX,YANGJ,et al.Investigation of Transmission and Evolution of PEDV Variants and Co-Infections in Northeast China from 2011 to 2022[J].Animals,2024,14(15):2168. doi: 10.3390/ani14152168
3	PENSAERTM B,DE BOUCKP.A new coronavirus-like particle associated with diarrhea in swine[J].Arch Virol,1978,58(3):243-247. doi: 10.1007/BF01317606
4	SONGD,PARKB.Porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines[J].Virus Genes,2012,44(2):167-175. doi: 10.1007/s11262-012-0713-1
5	WANGD,FANGL,XIAOS.Porcine epidemic diarrhea in China[J].Virus Res,2016,226,7-13. doi: 10.1016/j.virusres.2016.05.026
6	LIK,MARKOSYANR M,ZHENGY M,et al.IFITM proteins restrict viral membrane hemifusion[J].PLoS Pathog,2013,9(1):e1003124. doi: 10.1371/journal.ppat.1003124
7	MEISCHELT,FRITZLARS,VILLALON-LETELIERF,et al.IFITM Proteins That Restrict the Early Stages of Respiratory Virus Infection Do Not Influence Late-Stage Replication[J].J Virol,2021,95(20):e0083721. doi: 10.1128/JVI.00837-21
8	BRASSA L,HUANGI C,BENITAY,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. doi: 10.1016/j.cell.2009.12.017
9	LIL,FUF,XUEM,et al.IFN-lambda preferably inhibits PEDV infection of porcine intestinal epithelial cells compared with IFN-alpha[J].Antiviral Research,2017,140,76-82. doi: 10.1016/j.antiviral.2017.01.012
10	RENH,WANGS,XIEZ,et al.Analysis of chicken IFITM3 gene expression and its effect on avian reovirus replication[J].Viruses,2024,16(3):330. doi: 10.3390/v16030330
11	ZHAOX,GUOF,LIUF,et al.Interferon induction of IFITM proteins promotes infection by human coronavirus OC43[J].Proceedings of the National Academy of Sciences,2014,111(18):6756-6761. doi: 10.1073/pnas.1320856111
12	PRELLI BOZZOC,NCHIOUAR,VOLCICM,et al.IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro[J].Nat Commun,2021,12(1):4584. doi: 10.1038/s41467-021-24817-y
13	张格. 两性霉素B抗猪流行性腹泻病毒的作用研究[D]. 郑州: 河南农业大学, 2021.
	ZHANG G. The study of the antiviral effect of amphotericin B against porcine epidemic diarrhea virus[D]. Zhengzhou: Henan Agricultural University, 2021. (in Chinese)
14	CHENGJ,HEJ,FENGS,et al.IFITM1 is a host restriction factor that inhibits porcine epidemic diarrhea virus infection[J].J Nanobiotechnology,2024,22(1):677. doi: 10.1186/s12951-024-02884-9
15	庞照霞,郝鹏飞,屈巧巧,等.干扰素诱导跨膜蛋白3基因敲除Caco2细胞株的构建及其对水泡性口炎病毒复制的影响[J].中国兽医学报,2023,43(3):476-482.
	PANGZ X,HAOP F,QUQ Q,et al.Construction of Caco2 Cell Line with Interferon-Induced Transmembrane Protein 3 (IFITM3) Gene Knockout and Its Impact on Vesicular Stomatitis Virus Replication[J].Chinese Journal of Veterinary Science,2023,43(3):476-482.
16	WEIDNERJ M,JIANGD,PANX B,et al.Interferon-induced cell membrane proteins, IFITM3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms[J].J Virol,2010,84(24):12646-12657. doi: 10.1128/JVI.01328-10
17	FRIEDLOVÁN,ZAVADIL KOKÁŠF,HUPPT R,et al.IFITM protein regulation and functions: Far beyond the fight against viruses[J].Front Immunol,2022,13,1042368. doi: 10.3389/fimmu.2022.1042368
18	LUJ,PANQ,RONGL,et al.The IFITM proteins inhibit HIV-1 infection[J].J Virol,2011,85(5):2126-2137. doi: 10.1128/JVI.01531-10
19	NARAYANAS K,HELBIGK J,MCARTNEYE M,et al.The Interferon-induced Transmembrane Proteins, IFITM1, IFITM2, and IFITM3 Inhibit Hepatitis C Virus Entry[J].J Biol Chem,2015,290(43):25946-25959. doi: 10.1074/jbc.M115.657346
20	HUANGI C,BAILEYC C,WEYERJ L,et al.Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus[J].PLoS Pathog,2011,7(1):e1001258. doi: 10.1371/journal.ppat.1001258
21	LIC,GUOX R,DONGZ M,et al.Novel interacting proteins identified by tandem affinity purification and mass spectrometry associated with IFITM3 protein during PDCoV infection[J].Int J Biol Macromol,2024,277(Pt 2):132755.
22	AMINI-BAVIL-OLYAEES,CHOI YOUNJ,LEE JUNH,et al.The antiviral effector IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry[J].Cell Host & Microbe,2013,13(4):452-464.
23	LEEJ,ROBINSONM E,MAN,et al.IFITM3 functions as a PIP3 scaffold to amplify PI3K signalling in B cells[J].Nature,2020,588(7838):491-497. doi: 10.1038/s41586-020-2884-6
24	BAILEYC C,ZHONGG,HUANGI C,et al.IFITM-family proteins: The cell's first line of antiviral defense[J].Annu Rev Virol,2014,1,261-283. doi: 10.1146/annurev-virology-031413-085537
25	JOHNS P,CHINC R,PERREIRAJ M,et al.The CD225 domain of IFITM3 is required for both IFITM protein association and inhibition of influenza A virus and dengue virus replication[J].J Virol,2013,87(14):7837-7852. doi: 10.1128/JVI.00481-13
26	谭鹏. 猪/牛IFITMs基因的克隆、表达及其抗口蹄疫病毒作用[D]. 长春: 吉林农业大学, 2015.
	TAN P. Cloning and expression of porcine/bovine IFITMs genes and their antiviral effect against foot-and-mouth disease virus[D]. Changchun: Jilin Agricultural University, 2015. (in Chinese)

引物名称 Primer name	引物序列 Primer sequence
Sus-GAPDH：F(5′→3′)	TCGGAGTGAACGGATTTGGC
Sus-GAPDH：R(5′ →3′)	TGCCGTGGGTGGAATCATAC
Sus-IFITM3：F(5′ →3′)	TGCTCTAGAATGAATTGCGCTTCCCAGCC
Sus-IFITM3：R(5′ →3′)	CGGGATCCTTAGTGGTGGTGGTGGTGGTGCTAGTAG
Hu-IFITM3：F(5′ →3′)	TCACACTGTCCAAACCTTCTTC
Hu-IFITM3：R(5′ →3′)	GGATGTGGATCACGGTGGAC
PQCXIP-Flag-hIFITM3质粒：F(5′)	ACGCCATCCACGCTGTTTTGACCT
PQCXIP-Flag-hIFITM3质粒：R(3′)	AAGCGGCTTCGGCCAGTAACGTTA

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Construction of Porcine IPEC-J2 Cell Line Stably Expressing Human IFITM3 and Its Effect on PEDV Proliferation

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