猪 viperin 蛋白可抑制猪瘟病毒（classical swine fever virus，CSFV）在体外的复制。
Porcine Viperin protein inhibits the replication of classical swine fever virus (CSFV) in vitro.
机构信息
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, Nanjing, 210014, China.
College of Veterinary Medicine, Jilin University, Changchun, 130062, China.
出版信息
Virol J. 2017 Oct 23;14(1):202. doi: 10.1186/s12985-017-0868-4.
BACKGROUND
Classical swine fever virus (CSFV) is the causative pathogen of Classical swine fever (CSF), a highly contagious disease of swine. Viperin is one of the hundreds of interferon-stimulated genes (ISGs), and possesses a wide range of antiviral activities. The aim of this study was to explore whether porcine Viperin has the anti-CSFV activity.
METHOD
The influences of CSFV infection on Viperin expression and Newcastle disease virus (NDV)/Pseudorabies virus (PRV)-induced Viperin expression were examined in 3D4/21 cells and porcine peripheral blood mononuclear cells (PBMCs). Porcine Viperin gene was amplified to generate cell line PK-Vi over-expressing Viperin. CSFV was inoculated in the cell lines and viral load was detected by qRT-PCR, virus titration and Western blot. The influence of Viperin expression on CSFV binding, entry and release in the cells was also examined. The co-localization of Viperin with CSFV and its proteins (E2, NS5B) was determined by confocal laser scanning microscopy test. Co-IP assay was performed to check the interaction of Viperin with CSFV proteins.
RESULTS
CSFV infection could not induce Viperin expression in vitro while significantly inhibiting NDV/PRV-induced Viperin expression at 12, 24 and 48 h post infection (hpi; P < 0.05). The proliferation of CSFV in PK-Vi was significantly inhibited at 24, 48 and 72 hpi (P < 0.05), comparing with control cells (PK-C1 expressing EGFP). Virus in both cell culture supernatants and cell pellets were reduced equally. CSFV binding and entry in the cells were not interfered by Viperin expression. These results indicated its anti-CSFV function occurred during the genome and/or protein synthesis step. Confocal laser scanning microscopy test showed the Viperin-EGFP protein co-localized with CSFV E2 protein in CSFV infected PK-Vi cells. Further experiments indicated that Viperin protein co-localized with E2 and NS5B proteins of CSFV in the transfected 293 T cells. Furthermore, Co-IP assay confirmed the interaction of Viperin with E2 protein, but not NS5B.
CONCLUSION
Porcine Viperin effectively inhibited CSFV replication in vitro, potentially via the interaction of Viperin with CSFV E2 protein in cytoplasm. The results provided foundation for further studies of the interaction of Viperin with CSFV and other viruses.
背景
猪瘟病毒（CSFV）是猪瘟（CSF）的病原体，CSF 是一种高度传染性的猪病。Viperin 是成百上千种干扰素刺激基因（ISGs）之一，具有广泛的抗病毒活性。本研究旨在探讨猪 Viperin 是否具有抗 CSFV 活性。
方法
在 3D4/21 细胞和猪外周血单核细胞（PBMCs）中，检测 CSFV 感染对 Viperin 表达的影响以及新城疫病毒（NDV）/伪狂犬病病毒（PRV）诱导的 Viperin 表达。扩增猪 Viperin 基因，构建过表达 Viperin 的细胞系 PK-Vi。将 CSFV 接种于细胞系中，通过 qRT-PCR、病毒滴定和 Western blot 检测病毒载量。还检测了 Viperin 表达对 CSFV 结合、进入和释放的影响。通过共聚焦激光扫描显微镜试验确定 Viperin 与 CSFV 及其蛋白（E2、NS5B）的共定位。通过 Co-IP 测定检查 Viperin 与 CSFV 蛋白的相互作用。
结果
CSFV 感染不能诱导体外 Viperin 表达，但在感染后 12、24 和 48 小时（hpi）时显著抑制 NDV/PRV 诱导的 Viperin 表达（P<0.05）。与对照细胞（表达 EGFP 的 PK-C1）相比，CSFV 在 PK-Vi 中的增殖在 24、48 和 72 hpi 时受到显著抑制（P<0.05）。细胞培养上清液和细胞沉淀中的病毒均等量减少。Viperin 表达不干扰 CSFV 的结合和进入细胞。这些结果表明其抗 CSFV 功能发生在基因组和/或蛋白合成步骤。共聚焦激光扫描显微镜试验显示，在 CSFV 感染的 PK-Vi 细胞中，Viperin-EGFP 蛋白与 CSFV E2 蛋白共定位。进一步的实验表明，在转染的 293T 细胞中，Viperin 蛋白与 CSFV 的 E2 和 NS5B 蛋白共定位。此外，Co-IP 测定证实了 Viperin 与 E2 蛋白的相互作用，但与 NS5B 蛋白没有相互作用。
结论
猪 Viperin 可有效抑制 CSFV 在体外的复制，可能通过 Viperin 与 CSFV E2 蛋白在细胞质中的相互作用。该结果为进一步研究 Viperin 与 CSFV 和其他病毒的相互作用提供了基础。
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