mTOR信号通路调控猪瘟病毒复制的机制研究

发布时间：2018-09-10 08:20

【摘要】：猪瘟(Classical swine fever,CSF)是由猪瘟病毒(Classical swine fever virus,CSFV)引起的一种以高热、出血和免疫抑制为典型特征的严重危害养猪业的重大传染病,是世界动物卫生组织(OIE)规定的必须报告的疫病,对我国乃至全球的养猪业造成巨大的经济损失。CSFV属于黄病毒科(Flaviviridae)瘟病毒属(Pestivirus),是一种单股正链RNA病毒。目前对CSFV的致病机制虽有一定的研究,但对于CSFV在宿主细胞中感染复制的机制,以及免疫逃逸的致病机理仍缺乏全面深入的了解。由于病毒要建立持续性感染,必须利用宿主细胞中可调控蛋白合成、能量代谢、细胞存活生长的关键信号通路。因此,探讨CSFV感染的宿主细胞中参与调控细胞存活、蛋白合成和细胞自噬等过程的关键信号通路,阐明其参与CSFV复制增殖的机制,为猪瘟的防治提供新的靶点和理论依据。哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)信号通路连接着从感应能量和生长因子到调控细胞存活生长及大分子合成代谢的过程,除了在肿瘤发生发展中发挥重要的作用,也参与多种DNA、RNA病毒的感染复制,在病毒复制中扮演重要的角色。但对于CSFV与mTOR信号通路的关系,目前尚没有明确报道。因此,本研究拟阐明CSFV对mTOR信号通路的调控,以及mTOR信号通路调控CSFV复制的分子机制,从mTOR的角度为解释CSFV的复制和持续性感染提供新的理论依据。本研究首先利用细胞内信号通路蛋白芯片技术以及western-blot等方法证实与未感染CSFV的细胞相比较,CSFV(石门强毒株)感染ST细胞能够显著抑制Akt/mTOR信号通路的激活,该抑制在6-24 h最为显著,随后逐渐恢复,至48 h恢复至处理前水平。而CSFV结构蛋白Ems、非结构蛋白NS3、NS5A在抑制Akt/mTOR信号通路的激活中发挥重要的作用。为了探讨Akt/mTOR信号通路对CSFV自身复制的影响,利用mTOR抑制剂rapamycin和激活剂insulin改变宿主细胞mTOR通路活性后感染CSFV,western-blot和荧光定量PCR等结果表明在24 h内,rapamycin虽抑制细胞增殖但显著促进CSFV的复制;而insulin促进细胞增殖但显著抑制CSFV复制;值得注意的是在48 h时这一调控趋势并不存在;我们推测:CSFV感染抑制mTOR/S6K1,导致S6K1通过IRS(胰岛素受体)诱导Akt负反馈激活,进而抵消了由mTOR抑制引起的病毒复制。随后,我们进一步探讨mTOR上游Akt对CSFV复制的影响;分别应用Akt特异性抑制剂(LY294002)抑制Akt活性,以及Akt激活剂(SC79)激活Akt后感染CSFV,荧光定量PCR结果表明,与未经处理的ST细胞相比较,抑制Akt活性能够在72h内持续上调CSFV基因组拷贝数;而激活Akt则持续降低CSFV基因组拷贝数。与单独改变mTOR活性引起的只在24 h内调控病毒复制有所不同,这很有可能是由于CSFV感染引起mTOR抑制诱导Akt负反馈激活以及Akt作为mTOR上游引起比mTOR更强的作用导致。上述研究结果表明Akt/mTOR信号通路可负调控CSFV的复制,也提示CSFV感染ST细胞可能通过诱导Akt负反馈激活来调控病毒复制和维持细胞稳态。为了深入探究Akt/mTOR信号通路负调控CSFV复制的分子机制,本研究以mTOR信号通路下游调控自噬及蛋白合成的两个效应分子ULK1和S6K1为切入点,应用透射电镜、western-blot、共聚焦显微镜、LC3双荧光自噬慢病毒、IFA检测病毒滴度以及荧光定量PCR等方法证明CSFV感染能够增强ST细胞的自噬流,随后应用rapamycin和insulin以及ULK1抑制剂抑制或激活mTOR/ULK1活性,结果显示自噬被显著促进或抑制,而病毒的复制被相应的促进或抑制。证明CSFV通过mTOR/ULK1依赖的信号通路诱导细胞自噬进而促进病毒复制。另一方面,应用双荧光素酶报告基因系统,通过构建含CSFV内部核糖体进入位点(IRES)的萤火虫荧光素酶重组载体(CSFV-IRES),应用过表达和shRNA干扰等方法证明过表达和干扰表达S6K1能够抑制和促进CSFV-IRES蛋白驱动活性,随后病毒滴度检测和荧光定量PCR结果显示,过表达和干扰表达S6K1能抑制和促进CSFV复制。接着,利用免疫共沉淀、核糖体分离技术以及荧光定量PCR等方法证实,CSFV感染ST细胞能抑制mTOR/S6K1磷酸化激活,进而促进S6K1与真核起始因子3(elF3A)的结合,释放CSFV-IRES与eIF3竞争结合40S核糖体,促进病毒mRNA翻译。Akt/mTOR信号通路抑制能够导致细胞增殖抑制和细胞凋亡,而在体外,CSFV感染宿主细胞通常不抑制细胞增殖和凋亡。因此结合以上的研究,我们探讨CSFV感染抑制Akt/mTOR信号通路是否通过诱导Akt负反馈维持细胞增殖和抗凋亡,以便于为病毒复制提供稳定的细胞内环境。通过western-blot检测CSFV感染后Akt磷酸化水平,结果表明Akt磷酸化水平先降低后恢复,进一步证实CSFV感染ST细胞可诱导Akt负反馈激活。随后流式细胞检测发现CSFV感染初期能够将细胞周期阻滞于G0/G1期,而48 h后没有阻滞作用;另外使用Akt抑制剂LY294002单独或与CSFV共同作用ST细胞,SRB实验检测细胞增殖,结果显示抑制Akt活性同时感染CSFV较单独抑制Akt或单独感染CSFV时,对细胞增殖的抑制显著增强,间接提示CSFV感染诱导宿主细胞Akt负反馈激活,维持宿主细胞增殖,而一旦Akt被抑制剂持续抑制,感染CSFV诱导的Akt负反馈激活也被相应地抑制,因此不能维持细胞增殖。接着,细胞凋亡检测结果显示,抑制Akt活性同时感染CSFV较单独抑制Akt或单独感染CSFV时对细胞引起的凋亡率更大,证明CSFV诱导的负反馈激活能够维持细胞存活。最后,以Akt抑制剂与CSFV共同作用ST细胞至24 h、48 h、72 h,荧光定量PCR检测结果表明,与单独感染CSFV细胞相比较,在72 h内持续阻断Akt能够持续上调病毒基因组拷贝数,进一步提示CSFV感染引起Akt负反馈激活,进而维持病毒的复制。以上结果充分证实CSFV感染ST细胞诱导Akt负反馈激活的存在并且能够维持细胞存活和病毒自身复制。综上,本研究全面证实CSFV感染宿主细胞能够通过劫持mTOR信号通路以维持病毒复制和细胞存活,为以Akt/mTOR激活防治猪瘟提供新的靶点和思路。
[Abstract]:Classical swine fever (CSF) is a serious infectious disease caused by classical swine fever virus (CSFV), which is characterized by high fever, hemorrhage and immunosuppression. It is an epidemic disease that must be reported by the World Organization for Animal Health (OIE). It has caused tremendous damage to the pig industry in China and even the world. CSFV belongs to the genus Pestivirus of Flaviviridae. It is a single stranded positive-stranded RNA virus. Although the pathogenesis of CSFV has been studied, the mechanism of infection and replication of CSFV in host cells and the pathogenesis of immune escape are still poorly understood. Continuous infection requires the use of key signaling pathways that regulate protein synthesis, energy metabolism, and cell survival and growth in host cells. The mammalian target of rapamycin (mTOR) signaling pathway links the processes from induction energy and growth factors to regulation of cell survival, growth and macromolecule synthesis and metabolism. Infection replication plays an important role in viral replication. However, the relationship between CSFV and mTOR signaling pathway has not been clearly reported. Therefore, this study aims to elucidate the regulation of CSFV on mTOR signaling pathway and the molecular mechanism of mTOR signaling pathway on CSFV replication, providing an explanation for CSFV replication and persistent infection from the perspective of mTOR. In this study, compared with non-CSFV-infected cells, CSFV-infected ST cells significantly inhibited the activation of Akt/mTOR signaling pathway. The inhibition was most significant at 6-24 h, then gradually recovered until 48 h. In order to investigate the effect of Akt/mTOR signaling pathway on CSFV self-replication, we used mTOR inhibitor rapamycin and activator insulin to change the activity of host cell mTOR pathway and infected CSFV, Western-blot and fluorescence. Quantitative PCR and other results showed that rapamycin inhibited cell proliferation but significantly promoted CSFV replication within 24 hours; insulin promoted cell proliferation but significantly inhibited CSFV replication; it is noteworthy that this regulatory trend did not exist at 48 hours; we speculated that CSFV infection inhibited mTOR/S6K1, resulting in S6K1 inducing Akt negative reaction via IRS (insulin receptor). Subsequently, we further investigated the effect of Akt upstream of mTOR on CSFV replication; Akt specific inhibitor (LY294002) was used to inhibit Akt activity; and Akt activator (SC79) was used to activate Akt to infect CSFV. Fluorescence quantitative PCR results showed that it was inhibited compared with untreated ST cells. Akt-producing activity could continuously up-regulate the copy number of CSFV genome within 72 hours, while activation of Akt could continuously decrease the copy number of CSFV genome. These results suggest that the Akt/mTOR signaling pathway can negatively regulate the replication of CSFV, and that ST cells infected with CSFV may regulate virus replication and maintain cell homeostasis by inducing Akt negative feedback activation. Two effector molecules ULK1 and S6K1, which regulate autophagy and protein synthesis downstream of the pathway, were used as entry points. TEM, Western-blot, confocal microscopy, LC3 double fluorescent autophagy lentivirus, IFA detection of virus titer and fluorescence quantitative PCR proved that CSFV infection could enhance the autophagy of ST cells. K1 inhibitors inhibit or activate mTOR/ULK1 activity, indicating that autophagy is significantly promoted or inhibited, whereas virus replication is correspondingly promoted or inhibited. It is demonstrated that CSFV induces cell autophagy through mTOR/ULK1-dependent signaling pathway and promotes virus replication. On the other hand, double luciferase reporter gene system is used to construct CSFV-containing interior. The fluorescein enzyme recombinant vector (CSFV-IRES) at the ribosomal entry site (IRES) was used to demonstrate that the overexpression and interference of S6K1 could inhibit and promote the drive activity of CSFV-IRES protein. The results of viral titer detection and fluorescence quantitative PCR showed that overexpression and interference of S6K1 could inhibit and promote CSFV-IRES protein. FV replication. Immunocoprecipitation, ribosome isolation and fluorescence quantitative PCR confirmed that CSFV-infected ST cells inhibited the phosphorylation of mTOR/S6K1, thereby facilitating the binding of S6K1 to eukaryotic initiation factor 3 (elF3A), releasing CSFV-IRES to compete with eIF3 to bind to 40S ribosomes and promoting the translation of viral mRNA. Akt/mTOR signaling pathway was inhibited. CSFV infection usually does not inhibit cell proliferation and apoptosis in vitro. Therefore, in combination with the above studies, we explore whether CSFV infection inhibits Akt/mTOR signaling pathway by inducing Akt negative feedback to maintain cell proliferation and anti-apoptosis in order to provide stable details for virus replication. Akt phosphorylation was detected by Western-blot. The results showed that Akt phosphorylation decreased first and then recovered. It was further confirmed that CSFV-infected ST cells could induce Akt negative feedback activation. Using Akt inhibitor LY294002 alone or in conjunction with CSFV, the proliferation of ST cells was detected by SRB assay. The results showed that inhibition of Akt activity and infection with CSFV increased significantly compared with inhibition of Akt alone or infection with CSFV alone. Once Akt was inhibited by the inhibitor, the negative feedback activation of Akt induced by CSFV was inhibited accordingly, so the cell proliferation could not be maintained. Next, the results of cell apoptosis detection showed that the rate of cell apoptosis induced by inhibiting Akt activity and infecting CSFV at the same time was higher than that induced by inhibiting Akt alone or CSFV alone. Finally, the results of fluorescence quantitative PCR assay showed that continuous blockade of Akt for 72 hours could continuously increase the number of viral genomic copies compared with single infection of CSFV cells, suggesting that CSFV infection could cause negative feedback activation of Akt and consequently maintain the disease. These results fully confirm that CSFV-infected ST cells induce Akt negative feedback activation and can maintain cell survival and virus replication. In conclusion, this study fully confirms that CSFV-infected host cells can maintain virus replication and cell survival by hijacking the mTOR signaling pathway, providing a basis for the prevention and treatment of swine fever with Akt/mTOR activation. New targets and ideas.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651

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