抗病毒蛋白Viperin抑制猪瘟病毒在PK-15细胞上的复制

doi:10.11843/j.issn.0366-6964.2018.01.016

摘要/Abstract

摘要：

Viperin是一种抗病毒蛋白，具有广谱的抗病毒功能。为了研究猪源抗病毒蛋白Viperin对猪瘟病毒（CSFV）的抗病毒作用及机制，通过构建细胞系过表达或通过siRNA转染抑制Viperin表达，采用荧光定量RT-PCR和病毒滴度测定检测CSFV增殖水平的变化；检测培养上清和细胞中病毒含量的变化趋势，评价其对病毒释放的影响；进而通过共聚焦试验和免疫共沉淀（co-immunoprecipitation，Co-IP）试验检测Viperin与病毒E2蛋白的共定位与相互作用。与对照细胞相比，过表达Viperin蛋白可以显著抑制CSFV在PK-15细胞上的复制，在感染后24、48、72 h，病毒基因水平和病毒滴度分别下降68.75%、83.61%、77.27%和68.75%、87.5%、80.39%。通过RNA干扰技术抑制Viperin在PK-Vi细胞中的表达后CSFV复制显著恢复（仍低于对照组）。培养上清和细胞中病毒含量均同等程度受到抑制，表明Viperin表达对病毒的释放没有影响。共聚焦试验证明Viperin蛋白与E2蛋白在细胞内存在共定位现象。免疫共沉淀试验证明Viperin蛋白与E2蛋白存在相互作用。该研究证实Viperin具有抗CSFV作用，该作用可能是通过与病毒E2蛋白相互作用实现的，这为CSFV与宿主免疫反应相互作用研究提供了理论基础。

Abstract:

Viperin is an antiviral protein which could inhibit the replication of a wide range of viruses. The aim of this study was to explore the anti-CSFV activity of porcine Viperin protein. CSFV was inoculated in the cell line of PK-Vi over-expressing Viperin, viral load was detected by real-time qRT-PCR and virus titration. Knockdown of Viperin expression in PK-Vi by siRNA (siVi) was performed and CSFV replication was detected. Viral load in cell culture supernatants and cell lysates were examined to analyze the influence of Viperin on virus release. The co-localization and interaction of Viperin with CSFV E2 protein was determined by confocal laser scanning microscopy test and co-immunoprecipitation (Co-IP) assay. The genome copy numbers and viral titers of CSFV in PK-Vi was significantly decreased by 68.75%, 83.61%, 77.27% and 68.75%, 87.5%, 80.39% at 24, 48 and 72 hpi (P<0.05), comparing with control cells (PK-C1 expressing EGFP). Knockdown of Viperin expression retrieved the replication of CSFV, which was significantly higher than those in PK-Vi and siNC transfected PK-Vi (P<0.05) although it was still lower than those of PK-C1 and siRNA treated PK-C1 groups. Viperin expression had no effect on virus release from PK-15 cells. Confocal laser scanning microscopy test showed Viperin protein co-localized with E2 protein in CSFV infected cells and plasmid transfected 293T cells. Co-IP assay indicated that Viperin could interact with E2 protein. Porcine Viperin protein effectively inhibited CSFV replication in vitro, potentially via interaction of Viperin with CSFV proteins. The results provide foundation for further studies of the interaction of CSFV infection with host immune response.

中图分类号:

S852.651

李文良, 毛立, 邓加武, 郝飞, 李基棕, 杨蕾蕾, 张纹纹, 江杰元. 抗病毒蛋白Viperin抑制猪瘟病毒在PK-15细胞上的复制[J]. 畜牧兽医学报, 2018, 49(1): 139-146.

LI Wen-liang, MAO Li, DENG Jia-wu, HAO Fei, LI Ji-zong, YANG Lei-lei, ZHANG Wen-wen, JIANG Jie-yuan. Porcine Anti-viral Protein Viperin Inhibits the Replication of Classical Swine Fever Virus in PK-15 Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(1): 139-146.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2018.01.016

https://www.xmsyxb.com/CN/Y2018/V49/I1/139

参考文献

[1] CHIN K C, CRESSWELL P. Viperin (cig5), an IFN-inducible antiviral protein directly induced by human cytomegalovirus[J]. Proc Natl Acad Sci U S A, 2001, 98(26):15125-15130.
[2] MATTIJSSEN S, PRUIJN G J M. Viperin, a key player in the antiviral response[J]. Microbes Infect, 2012, 14(5):419-426.
[3] SEO J Y, YANEVA R, CRESSWELL P, et al. Viperin:a multifunctional, interferon-inducible protein that regulates virus replication[J]. Cell Host Microbe, 2011, 10(6):534-539.
[4] SHEN G H, WANG K Z, WANG S, et al. Herpes simplex virus 1 counteracts viperin via its virion host shutoff protein UL41[J]. J Virol, 2014, 88(20):12163-12166.
[5] TAN K S, OLFAT F, PHOON M C, et al. In vivo and in vitro studies on the antiviral activities of viperin against influenza H1N1 virus infection[J]. J Gen Virol, 2012, 93(Pt 6):1269-1277.
[6] BAI X X, YANG H Y, WAN L X, et al. Involvement of viperin in prevention of intrauterine transmission of hepatitis B virus[J]. APMIS, 2017, 125(2):170-175.
[7] TANG H B, LU Z L, WEI X K, et al. Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4[J]. Sci Rep, 2016, 6:30529.
[8] RABBANI M A G, RIBAUDO M, GUO J T, et al. Identification of interferon-stimulated gene proteins that inhibit human parainfluenza virus type 3[J]. J Virol, 2016, 90(24):11145-11156.
[9] TANG Y D, NA L, ZHU C H, et al. Equine viperin restricts equine infectious anemia virus replication by inhibiting the production and/or release of viral Gag, Env, and receptor via distortion of the endoplasmic reticulum[J]. J Virol, 2014, 88(21):12296-12310.
[10] FANG J Y, WANG H Y, BAI J, et al. Monkey viperin restricts porcine reproductive and respiratory syndrome virus replication[J]. PLoS One, 2016, 11(5):e0156513.
[11] 方剑玉, 白娟, 郭小参, 等. 猪源Viperin蛋白抑制猪繁殖与呼吸综合征病毒感染MARC-145细胞[J]. 病毒学报, 2017, 33(2):216-223.
FANG J Y, BAI J, GUO X C, et al. Swine Viperin restricts replication of the porcine reproductive and respiratory syndrome virus[J]. Chinese Journal of Virology, 2017, 33(2):216-223. (in Chinese)
[12] LUO Y Z, LI S, SUN Y, et al. Classical swine fever in China:a minireview[J]. Vet Microbiol, 2014, 172(1-2):1-6.
[13] ZHANG X M, JING J, LI W L, et al. Porcine Mx1 fused to HIV Tat protein transduction domain (PTD) inhibits classical swine fever virus infection in vitro and in vivo[J]. BMC Vet Res, 2015, 11:264.
[14] LI L F, YU J H, LI Y F, et al. Guanylate-binding protein 1, an interferon-induced GTPase, exerts an antiviral activity against classical swine fever virus depending on its GTPase activity[J]. J Virol, 2016, 90(9):4412-4426.
[15] 李文良, 毛立, 杨蕾蕾, 等. 稳定表达猪Viperin的PK-15细胞系的构建与鉴定[J]. 江苏农业学报, 2016, 32(1):128-132.
LI W L, MAO L, YANG L L, et al. Construction and identification of PK-15 cell line stably expressing porcine Viperin[J]. Jiangsu Journal of Agricultural Science, 2016, 32(1):128-132. (in Chinese)
[16] BAUHOFER O, SUMMERFIELD A, SAKODA Y, et al. Classical swine fever virus N^pro interacts with interferon regulatory factor 3 and induces its proteasomal degradation[J]. J Virol, 2007, 81(7):3087-3096.
[17] XIAY H, CHEN L, PAN Z S, et al. A novel role of classical swine fever virus E^rns glycoprotein in counteracting the Newcastle disease virus (NDV)-mediated IFN-β induction[J]. J Biochem Mol Biol, 2007, 40(5):611-616.
[18] ZHAO Y C, PANG D X, WANG T D, et al. Human MxA protein inhibits the replication of classical swine fever virus[J]. Virus Res, 2011, 156(1-2):151-155.
[19] CHAN Y L, CHANG T H, LIAO C L, et al. The cellular antiviral protein viperin is attenuated by proteasome-mediated protein degradation in Japanese encephalitis virus-infected cells[J]. J Virol, 2008, 82(21):10455-10464.
[20] JIANG D, WEIDNER J M, QING M, et al. Identification of five interferon-induced cellular proteins that inhibit west nile virus and dengue virus infections[J]. J Virol, 2010, 84(16):8332-8341.
[21] HELBIGK J, CARR J M, CALVERT J K, et al. Viperin is induced following dengue virus type-2(DENV-2) infection and has anti-viral actions requiring the C-terminal end of Viperin[J]. PLoS Negl Trop Dis, 2013, 7(4):e2178.
[22] UPADHYAY A S, VONDERSTEIN K, PICHLMAIR A, et al. Viperin is an iron-sulfur protein that inhibits genome synthesis of tick-borne encephalitis virus via radical SAM domain activity[J]. Cell Microbiol, 2014, 16(6):834-848.
[23] VAN DER HOEK K H, EYRE N S, SHUE B, et al. Viperin is an important host restriction factor in control of Zika virus infection[J]. Sci Rep, 2017, 7:4475.
[24] WANG X Y, HINSON E R, CRESSWELL P. The interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts[J]. Cell Host Microbe, 2007, 2(2):96-105.
[25] FENWICK M K, LI Y, CRESSWELL P, et al. Structural studies of viperin, an antiviral radical SAM enzyme[J]. Proc Natl Acad Sci U S A, 2017, 114(26):6806-6811.
[26] HELBIG K J, EYRE N S, YIP E, et al. The antiviral protein viperin inhibits hepatitis C virus replication via interaction with nonstructural protein 5A[J]. Hepatology, 2011, 54(5):1506-1517.
[27] JUMAT M R, HUONG T N, RAVI L I, et al. Viperin protein expression inhibits the late stage of respiratory syncytial virus morphogenesis[J]. Antiviral Res, 2015, 114:11-20.