抗猪瘟病毒干扰素刺激基因的筛选及GBP1抗病毒分子机制的研究

发布时间：2018-11-17 12:07

【摘要】：病毒感染和宿主抗病毒是一个博弈的过程:许多病毒感染宿主后,病毒的病原相关分子模式被宿主的模式识别受体所识别,从而激活宿主I型干扰素(IFN)信号通路,导致数以百计的干扰素刺激基因(ISGs)转录,为机体提供抗病毒防御;与此同时,病毒也进化出多种高效的逃逸策略来抵抗宿主合成IFN、信号传导以及ISGs的抗病毒功能。ISGs作为重要的抗病毒效应分子,其在宿主防御和清除外源病原感染方面发挥重要作用。因此,研究抗病毒ISGs对于控制和治疗病毒感染具有重要意义。猪瘟(CSF)是一种急性、烈性传染病,在多个国家流行,给我国乃至世界养猪业造成巨大的经济损失。猪瘟病毒(CSFV)是CSF的病原,其属于黄病毒科瘟病毒属成员。CSFV感染宿主后,可激活I型IFN信号通路,产生抗病毒ISGs。与黄病毒科其它成员的抗病毒ISGs研究相比较,抗CSFV特异性ISGs研究报道很少,特别是ISGs抗CSFV感染的分子机制研究尚属空白。研究抗CSFV ISGs不仅有助于深入了解宿主抗病毒机理,同时也为揭示CSFV逃避宿主抗病毒策略提供线索。因此,研究抗CSFV ISGs既具有理论意义又具有重大的应用价值。本研究中,我们构建了20种慢病毒介导的过表达猪源ISGs的PK-15细胞系。利用过表达细胞系结合表达萤火虫荧光素酶的报告猪瘟病毒(rCSFV-Fluc)筛选抗CSFV的ISGs,成功地筛选到6个ISGs显著地抑制rCSFV-Fluc复制,分别是GBP1、GBP2、CD47、ZNF313、OASL和OAS1。将筛选到的过表达GBP1、CD47、ZNF313和OASL细胞系感染亲本病毒CSFV Shimen株,结果表明,过表达这些分子可以抑制亲本病毒复制,说明这4个ISGs分子是抗CSFV的ISGs。我们优先选取鸟苷酸结合蛋白1(GBP1)进行深入研究。研究结果表明,过表达GBP1抑制CSFV复制,相反利用针对GBP1的小干扰RNA沉默内源性的GBP1可以促进CSFV复制。此外,GBP1发挥抗CSFV作用主要发生在病毒复制早期,及剂量依赖性的抑制CSFV内部核糖体进入位点(IRES)的翻译效率。CSFV感染猪或PK-15细胞后,可以引起GBP1在转录水平上调表达。在GBP1抗CSFV分子机制方面,研究证实GBP1不能激活IFN-β和NF-κB信号通路,其抗CSFV是依赖于自身的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。有趣的是,我们的研究还发现,GBP1(K51A)突变体丧失了GTPase活性,且不能抑制CSFV复制。免疫共沉淀试验和GST pulldown试验结果表明,GBP1 N端的GTPase结构域和NS5A的C端功能区是GBP1-NS5A相互作用的关键区域。总之,本研究利用报告病毒并结合过表达细胞系的高通量筛选策略,获得了6个抗CSFV ISGs候选分子。经过系统的鉴定,证实GBP1为抗CSFV的ISG,并阐明了该分子发挥抗病毒作用依赖于其自身的GTPase活性,而CSFV则利用其NS5A蛋白与GBP1相互作用抑制GBP1的GTPase活性,进而拮抗GBP1的抗病毒功能。
[Abstract]:Viral infection and host versus virus are a game process: after many viruses infect the host, the pathogen-associated molecular patterns of the virus are recognized by the host's pattern recognition receptor, thus activating the host type I interferon (IFN) signaling pathway. It results in hundreds of interferon stimulating gene (ISGs) transcription, providing antiviral defense for the body; At the same time, the virus has evolved a variety of efficient escape strategies to resist the host synthesis of IFN, signal transduction and the antiviral function of ISGs. ISGs is an important antiviral effector. It plays an important role in host defense and elimination of exogenous pathogens infection. Therefore, the study of antiviral ISGs is of great significance in the control and treatment of viral infection. Swine fever (CSF) is an acute and acute infectious disease, which is prevalent in many countries and has caused huge economic losses to the pig industry in China and the world. Classical swine fever virus (CSFV) is the pathogen of CSF, which belongs to the family Flavovirus.After CSFV infects the host, it activates the type I IFN signaling pathway and produces anti-virus ISGs.. Compared with other members of the yellow virus family, there are few studies on anti-virus ISGs, especially on the molecular mechanism of ISGs anti-CSFV infection. The study of anti-CSFV ISGs not only helps to understand the mechanism of host anti-virus, but also provides clues for CSFV to escape from host anti-virus. Therefore, the study of anti-CSFV ISGs has both theoretical significance and great application value. In this study, we constructed 20 lentivirus-mediated PK-15 cell lines that overexpression porcine ISGs. Screening of ISGs, resistant to CSFV by overexpression Cell Line and expression of Fluorworm luciferase Reporter Swine Fever virus (rCSFV-Fluc) six ISGs significantly inhibited rCSFV-Fluc replication, GBP1,GBP2,CD47,ZNF313,OASL and OAS1., respectively. The selected overexpression GBP1,CD47,ZNF313 and OASL cell lines were infected with parental virus CSFV Shimen strain. The results showed that the overexpression of these molecules could inhibit the replication of parental virus, indicating that the four ISGs molecules were ISGs. resistant to CSFV. We preferentially selected guanylate binding protein 1 (GBP1) for further study. The results show that overexpression of GBP1 inhibits CSFV replication, whereas RNA silencing endogenous GBP1 against GBP1 can promote CSFV replication. In addition, the anti- CSFV effect of GBP1 mainly occurred in the early stage of viral replication, and in a dose-dependent manner inhibited the translation efficiency of (IRES), the ribosomal entry site of CSFV. CSFV infection with porcine or PK-15 cells could induce the up-regulation of GBP1 expression at the transcriptional level. In the aspect of the molecular mechanism of GBP1 anti-CSFV, it has been proved that GBP1 can not activate IFN- 尾 and NF- 魏 B signaling pathway, and its anti-CSFV is dependent on its GTPase activity. CSFV has antiviral effect on host GBP1, and it interacts with GBP1 by using self-synthesized NS5A protein. By inhibiting the GTPase activity of GBP1 and then antagonizing the CSFV activity of GBP1. Interestingly, our study also found that GBP1 (K51A) mutants lost GTPase activity and did not inhibit CSFV replication. The results of immunoprecipitation and GST pulldown tests showed that the N-terminal GTPase domain of GBP1 and the C-terminal functional region of NS5A were the key regions of GBP1-NS5A interaction. In conclusion, in this study, six candidate molecules against CSFV ISGs were obtained by using the high throughput screening strategy of report virus and overexpression cell lines. It was confirmed that GBP1 was an antiviral ISG, of CSFV and that its antiviral action depended on its own GTPase activity, while CSFV used its NS5A protein to interact with GBP1 to inhibit GTPase activity of GBP1, thus antagonizing the antiviral function of GBP1.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.28

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