抗猪瘟病毒干扰素刺激基因的鉴定及其抗病毒分子机制的研究

Viral infections can regulate type I interferon (IFN) response in the host, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which in turn regulate viral replication. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which is a highly contagious disease that endangers the pig industry in most countries. We have previously demonstrated that several cellular proteins (such as HB) inhibit CSFV replication via the IFN signaling pathway. However, little is known about the anti-CSFV potential of ISGs and their antiviral molecular mechanisms. In this proposal, firstly, we will screen the anti-CSFV ISGs using Flow cytometry and luciferase reporter CSFV, and subsequently validate the antiviral activity of the screened ISGs in PK-15 cells using the parent CSFV. Secondly, we will focus on the interactions between the ISGs and the CSFV proteins, the regulation of the IFN signaling pathway and the specific stage(s) in the CSFV life cycle exerted by the anti-CSFV ISGs to clarify the antiviral mechanisms of the ISGs. Finally, we will testify if the anti-CSFV ISGs are able to antagonize other members within the Flaviviridae family or other important swine viruses. The project is expected to elucidate the antiviral molecular mechanisms of anti-CSFV ISGs, which will facilitate the development of anti-CSFV agents derived from the antiviral ISGs and provide new ideas and strategies for the control of CSF.

病毒感染可激活宿主的I型干扰素（IFN）应答，引起数以百计的干扰素刺激基因（ISGs）的转录，而ISGs可调控病毒复制的不同阶段。猪瘟（CSF）是由猪瘟病毒（CSFV）引起的一种严重危害养猪业的烈性传染病。此前我们证实，宿主细胞HB蛋白可通过激活IFN信号通路抑制CSFV的复制，但目前对ISGs的抗CSFV作用及其分子机制尚不清楚。本项目拟利用流式细胞术、荧光素酶报告病毒筛选抗CSFV ISGs；然后，通过siRNA下调表达和慢病毒介导过表达ISGs，用亲本病毒验证筛选的ISGs的抗病毒活性；之后，从ISGs与CSFV蛋白的相互作用、ISGs对IFN信号通路的调控以及抗病毒ISGs参与病毒复制周期的具体阶段等方面，揭示ISGs抗CSFV的分子机制；最后，通过检测其对其它病毒的抗病毒活性，明确其抗病毒广谱性或专一性。本项目将揭示ISGs抗CSFV的分子机制，为开发新型抗CSF策略奠定基础。

本研究中，我们利用过表达猪源ISGs的细胞系结合表达萤火虫荧光素酶的猪瘟病毒（CSFV）报告病毒（rCSFV-Fluc）成功地筛选到GBP1、GBP2、CD47、ZNF313、OASL和OAS1显著地抑制rCSFV-Fluc复制。感染亲本病毒CSFV Shimen株进行验证，结果证实过表达GBP1、GBP2、ZNF313和OASL显著地抑制亲本病毒复制，说明这4个ISGs分子是抗CSFV的ISGs。.1).GBP1抑制CSFV的分子机制.双荧光素酶报告试验结果表明GBP1不能激活IFN-β和NF-κB信号通路。我们构建了GBP1的不同突变体，利用GTPase活性检测试剂盒检测GBP1及突变体的GTPase活性，结果表明GBP1(K51A)突变体丧失了GTPase活性，且其不能抑制CSFV复制，说明GBP1发挥抗病毒作用依赖于其自身的GTPase活性。CSFV面对宿主GBP1的抗病毒效应，其利用自身合成的NS5A蛋白与GBP1相互作用，通过抑制GBP1的GTPase活性进而拮抗GBP1的抗CSFV活性。.2).OASL抑制CSFV的分子机制。.利用siRNA下调内源性RNaseL后，pOASL仍具有抑制CSFV复制的活性，说明pOASL抑制CSFV复制不依赖经典的OAS/RNaseL通路；其次，通过双荧光素酶报告试验证实过表达pOASL能够增强IFN-β信号通路。通过免疫共沉淀（Co-IP）试验筛选到pOASL与MDA5蛋白存在直接的相互作用，且证实pOASL能够增强MDA5介导的I型IFN信号通路；最后，我们通过siRNA下调内源性MDA5和RIG-I表达，进一步证实pOASL发挥抗CSFV效应依赖于MDA5介导的I型IFN信号通路。.本研究深入研究了GBP1和OASL抑制CSFV复制的分子机制，不仅有助于深入了解宿主的抗病毒机理，同时也为揭示CSFV逃避宿主抗病毒策略提供线索。