IRF1 通过靶向 5' 侧翼区的-487 到-325 核苷酸上调 dsRNA 刺激或 CSFV 感染时的 isg15 基因表达。

IRF1 up-regulates isg15 gene expression in dsRNA stimulation or CSFV infection by targeting nucleotides -487 to -325 in the 5' flanking region.

机构信息

State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning 530004, Guangxi, China; Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China.

Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China.

出版信息

Mol Immunol. 2018 Feb;94:153-165. doi: 10.1016/j.molimm.2017.12.025. Epub 2018 Jan 8.

DOI:10.1016/j.molimm.2017.12.025

PMID:29324236

Abstract

Interferon (IFN)-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that is heavily involved in immune response elicitation. As an important member of interferon regulatory factor (IRF) family, IRF1 can activate the expression of multiple genes, including the human optineurin gene (Sudhakar et al., 2013). In this study, a sequence in the promoter region of the optineurin gene was compared to the 5' flanking region of the porcine isg15 gene. Porcine IRF1 also possesses antiviral activity against several swine viruses (Li et al., 2015), but the mechanism is not well understood. Herein, we report that porcine IRF1 and ISG15 were up-regulated in porcine kidney (PK-15) cells following stimulation with double-stranded RNA (dsRNA) or classical swine fever virus (CSFV) infection. We also found that siRNA-mediated knockdown of IRF1 expression resulted in lower ISG15 expression in response to polyinosinic:polycytidylic acid [poly(I:C)] or CSFV infection. The overexpression of IRF1 resulted in ISG15 up-regulation. IRF1 was shown to translocate to the nucleus in response to dsRNA stimulation. To further identify the functional domain of the isg15 gene that promotes IRF1 transcriptional activity, firefly luciferase and ISG15 reporter systems were constructed. The results of the firefly luciferase and ISG15 reporter assay suggested that IRF1 mediates the up-regulation of ISG15. Nucleotides -487 to -325, located in the 5' flanking region of the isg15 gene, constituted the promoter region of IRF1. ChIP assay indicated that IRF1 protein was able to interact with the DNA in the 5'fr of isg15 gene in cells. As an innate immune response protein with broad-spectrum antiviral activity, the up-regulation of ISG15 mediated by IRF1 in porcine cells is reported for the first time. These results warrant further investigation into the antiviral activity of porcine IRF1 against reported swine viruses.

摘要

干扰素刺激基因 15（ISG15）编码一种泛素样蛋白，在免疫反应诱导中起着重要作用。作为干扰素调节因子（IRF）家族的重要成员，IRF1 可以激活多种基因的表达，包括人类视神经病变基因（Sudhakar 等人，2013 年）。在这项研究中，比较了视神经病变基因启动子区域的序列与猪 isg15 基因的 5'侧翼区域。猪 IRF1 还具有针对几种猪病毒的抗病毒活性（Li 等人，2015 年），但机制尚不清楚。在此，我们报告在双链 RNA（dsRNA）或经典猪瘟病毒（CSFV）感染后，猪肾（PK-15）细胞中猪 IRF1 和 ISG15 上调。我们还发现，siRNA 介导的 IRF1 表达下调导致多聚肌苷酸：多聚胞苷酸[poly（I：C）]或 CSFV 感染时 ISG15 表达降低。IRF1 的过表达导致 ISG15 上调。IRF1 被证明在 dsRNA 刺激下易位到核内。为了进一步鉴定促进 IRF1 转录活性的 isg15 基因的功能结构域，构建了萤火虫荧光素酶和 ISG15 报告系统。萤火虫荧光素酶和 ISG15 报告测定的结果表明，IRF1 介导 ISG15 的上调。位于 isg15 基因 5'侧翼的核苷酸 -487 至 -325 构成了 IRF1 的启动子区域。ChIP 测定表明，IRF1 蛋白能够在细胞中与 isg15 基因 5'fr 的 DNA 相互作用。作为一种具有广谱抗病毒活性的先天免疫反应蛋白，首次报道了猪细胞中由 IRF1 介导的 ISG15 上调。这些结果进一步证明了猪 IRF1 对报道的猪病毒的抗病毒活性。