【作者】 秦成峰；

【导师】 秦鄂德；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2009， 博士

【摘要】 登革病毒(Dengue virus,DEN)为单股正链RNA病毒,基因组长约11 kb,为黄病毒科黄病毒属重要成员。自然界中存在4个血清型登革病毒,感染人类后均可导致登革热、登革出血热和登革休克综合征等一系列严重疾病,对热带亚热带地区的公共卫生安全造成重要威胁。目前,登革病毒的致病机理仍不十分清楚,临床上也没有疫苗和特效治疗药物。登革病毒感染人类细胞后,可迅速激活宿主的天然免疫反应,在感染早期限制病毒的复制和传播。Toll样受体(Toll-like receptor,TLR)和RIG-I样受体(RIG-I-like receptor,RLR)能够侦测和识别入侵的病毒,诱导I型干扰素和促炎症细胞因子产生,在机体的抗病毒天然免疫反应中具有重要作用。TLRs主要表达于免疫细胞和内含体表面,主要在类浆树突细胞等免疫细胞中发挥作用。RLR是一类新发现的模式识别受体,一般在非免疫细胞中识别细胞质中的病毒RNA,诱导I型干扰素的产生。登革病毒主要通过蚊媒叮咬传播,包括朗格汉斯细胞等上皮细胞等被认为是可能的初级靶细胞。在登革病毒感染的人类上皮细胞中,RLR能否以及如何发挥其生物学功能目前并不清楚。本研究以人肺上皮细胞A549作为模型,首先观察了登革病毒诱导的I型干扰素反应,然后进一步通过RNAi和过表达的方法对RIG-I、MDA5等信号分子在病毒诱导的I型干扰素反应中的作用进行了分析,并初步探讨了RIG-I抑制登革病毒的作用机制。主要实验结果包括以下三个部分:一、登革病毒感染可激活RIG-I信号通路,诱导I型干扰素的产生我们首先观察了登革病毒感染A549细胞后诱导IFN-β产生的能力,结果发现登革病毒感染细胞12 h后即可诱导IFN-β的产生,而热灭活病毒不具备诱导IFN-β产生的能力。同时,利用复制子系统进行研究发现,转染登革病毒复制子后同样能够诱导IFN-β的产生,说明病毒基因组的复制是诱导IFN-β的产生的前提条件。进一步通过RT-PCR和Western blotting的方法对RIG-I及其接头蛋白IPS-1的表达水平进行检测,结果发现登革病毒感染后细胞中RIG-I和IPS-1的表达水平均明显上调。在此基础上,我们利用IRF-3和IFN-β启动子控制的CAT报告质粒(PRDIII-I)-CAT和(-110-IFNβ)-CAT定量分析了病毒感染对RIG-I下游信号分子的激活作用,结果发现登革病毒感染可显著增强IRF-3和IFN-β启动子的转录活性。同时,我们分别使用不同血清型和同一血清型不同分离株登革病毒感染A549细胞,结果发现其诱导IFN-β产生的能力未存在显著差异。此外,西尼罗病毒感染A549细胞后,同样能够激活RIG-I分子,诱导IFN-β的产生。上述结果表明,登革病毒在A549细胞中的复制能够激活RIG-I信号通路,并通过一系列级联信号反应诱导I型干扰素的产生;而且,病毒血清型和毒力对病毒诱导的I型干扰素反应并无显著影响。二、MDA5和RIG-I共同参与登革病毒诱导的IFN-β反应为了进一步明确RIG-I在登革病毒感染过程中的作用,我们首先在A549细胞中瞬时表达RIG-I,结果发现全长RIG-I的过表达可显著上调登革病毒诱导的IFN-β反应,而作为显负性突变体的RIG-IC能够下调IFN-β反应。进一步通过siRNA的方法敲除A549细胞中RIG-I基因,结果发现登革病毒感染上述细胞后,IFN-β启动子转录活性仍可被激活,上清中仍可检测到IFN-β的大量产生。这说明,登革病毒感染后I型干扰素反应可能不仅仅由RIG-I信号通路决定。进一步研究发现,登革病毒感染同样可上调MDA5的表达,MDA5在A549细胞中的过表达同样能够激活IFN-β启动子,诱导IFN-β的产生,并呈剂量依赖性。然而MDA5的过表达对登革病毒诱导IFN-β反应并无显著的促进作用,使用siRNA敲除MDA5后发现,登革病毒感染诱导的IFN-β反应并未受MDA5敲除的影响。在此基础上,我们利用shRNA表达载体筛选获得了稳定干扰RIG-I的工程细胞系,登革病毒感染上述细胞后,MDA5的表达明显上调,进一步通过siRNA敲除MDA5,结果发现登革病毒诱导IFN-β产生的能力显著降低。上述结果说明,登革病毒感染上皮细胞后,可同时激活RIG-I和MDA5,RIG-I或MDA5可能存在一种功能上的替代机制,共同参与登革病毒诱导的IFN-β反应,其中RIG-I信号通路可能发挥更主要的作用。三、RIG-I对登革病毒和西尼罗病毒的抑制作用对构建的稳定干扰RIG-I的工程细胞株进行研究发现,登革病毒感染上述细胞后,致细胞病变效应明显增强,上清中子代病毒的产量明显提高。我们进一步通过过表达的方法观察了RIG-I对登革病毒和西尼罗病毒复制的抑制效果,结果发现RIG-I在A549细胞中的过表达能够显著降低子代病毒的产量,并呈剂量依赖性。为明确RIG-I抑制病毒复制的具体作用机制,我们使用IFN-β抗体阻断JAK-STAT信号通路,结果发现RIG-I的过表达仍然对病毒的产量存在明显的抑制作用,说明IFN-β激活的JAK-STAT通路可能不是RIG-I抑制病毒的主要原因,RIG-I本身可能就具有抗病毒作用。我们进一步在A549细胞中表达RIG-I的不同区段,结果发现RIG-I和RIG-IC的过表达对登革病毒均具有抑制作用,说明RIG-I的抗病毒作用可能主要由其C端解旋酶区决定。在此基础上,通过Western blotting的方法对病毒感染早期的蛋白表达水平进行检测发现,RIG-I和RIG-IC的表达使细胞中登革病毒E蛋白的表达水平明显降低。因此,在登革病毒的感染初期,RIG-I很可能不仅诱导I型干扰素的产生,其C端解旋酶区还可通过某种机制抑制病毒蛋白的合成,限制病毒的复制和传播。总之,我们的研究发现,登革病毒感染人类上皮细胞可迅速上调RIG-I和MDA5的表达,RIG-I和MDA5共同参与,并以RIG-I为主诱导IFN-β的产生;此外,RIG-I羧基端能够抑制病毒蛋白的早期合成,对多种蚊媒黄病毒具有抑制作用。这些结果证实了RIG-I/MDA5在病毒感染过程中的重要作用,初步揭示了上皮细胞中登革病毒感染诱导I型干扰素产生的基本过程,为深入探讨病毒-宿主相互作用和抗病毒药物的研制奠定了基础。更多还原

【Abstract】 Dengue viruses (DENV) type 1-4, together with West Nile virus (WNV), Japanese Encephalitis virus (JEV), etc, belong to the genus Flavivirus family Flavivirida, containing single-stranded, positive-sense genome RNAs of approximately 11 kb in length. DENV infections can result in mild dengue fever (DF), severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In terms of the morbidity and mortality rates, DENV is becoming one of the most important publich health problems in tropical and subtropical countries. The pathogenesis from DF to DHF/DSS has not yet been elucidated, and no vaccine or antiviral drugs are currently available.At the initial stage of virus infection, host pattern recognition recepotors (PRR), including Toll-like receptor (TLRs) and RIG-I-like receptors (RLRs), are responsible for sensing viral RNA and initiates innate antiviral responses, including the activation of proinflammatory cytokines and type I interferon (IFN). Toll-like receptors are abundant on monocytes/macrophages and DCs, and play a key role in regulating the inflammatory response against infectious viruses. RLRs, including RIG-I and MDA5, are supposed to sense cytoplasmic viral RNA mainly in non-immune cells. DENV is transmintted by mosquitoes feed, skin epithelial cells have been supposed to be one of the primary target cells. However, the role of RLRs in human epithelial cells during the induction of IFN by DENV is still unknown. In this study, human lung epithelial cell lines A549 were chosen to observe the IFN response to DENV infection, and then the role of RIG-I and MDA5 during DENV-induced IFN response were analyzed by RNAi and over-expression assays. What’s more, the antiviral effects and mechanism of RIG-I against DENV and West Nlie virus were determined. The main results are described as following in three parts:1. DENV induced the expression of RIG-I and IFN-βin A549 cells. Firstly, to study the IFN response to DENV infection in human epithelial cells, A549 cells were infected with DENV and heat-inactivated DENV, respectively, and IFN-βwere found secreted in culture supertants since 12 h post-infection by a commercial human IFN-βELISA kit, while no IFN-βwas detected in A549 cells treated with heat-inactivated virus. Also, transfection of a DENV replicon was found to be able to induce the secretion of IFN-βin A549 cells. These results suggested that the production of IFN-βwere due to virtual virus infection or viral RNA replication. Further, RT-PCR and Western blotting resuluts showed that the expression of RIG-I and it’s adaptor, IPS-1, were upregulated upon DENV infection in A549 cells. CAT reportor plasmids that waer under the control of the IRF-3 and IFN-βpromoter were used to analyze the effects of DENV infection, and the results indicated that IRF-3 and IFN-βpromoter were activated 24 hrs post infection.Then, different isolates of DENV that differed in serotypes and virulence clearly were used to infect A549 cells, and IFN-βlevels were compared and assayed. While no significant difference was found among those isolates. What’s more, similar RIG-I activation and IFN-βproduction were observed in A549 cells infected with WNV. Together, these results suggested that DENV infection can activate the IRG-I signal pathway molecules, including RIG-I, IPS-1 and IRF-3, finally induce the expression of IFN-βin human epithelial cells. Serotype and virulence of DENV have no effect on the IFN-βresponse to DENV infection in A549 cells.2. RIG-I and MDA5 are both involved in IFN-βresponse to DENV infection.In order to demonstrate the role of RIG-I in the IFN-βresponse to DENV infection, full-length RIG-I and its dominant negative muatant, RIG-IC, were transiently expressed in A549 cells followed by DENV infection. ELISA and CAT assays results showed that IFN-βlevel and promoter activity was induced by RIG-I and further enhanced by DENV infection, and that RIG-IC caused a reduction in IFN-βlevel and promoter activity. Further, siRNAs were used to knockdown the endogenous expression of RIG-I in A549 cells, while no significant difference was found between the DENV-induced IFN-βresponse in RIG-I-konckout cells and control cells. Next, the role of MDA5 in the IFN-βresponse to DENV infection was analyzed by RNAi and over-expression. The expression of MDA5 was up-regulated upon DENV infection, and over-expression of MDA5 in A549 cells also induced the secretion of IFN-β. While MDA5 knockout and over-expression had no significant effects on the IFN-βresponse to DENV infection in A549 cells.Furthermore, stable knockdown cell lines of human RIG-I were developed and MDA5 were subsequently knocked out by siRNA. The IFN-βresponse to DENV infection was almost blocked in this cell line. Together, these results suggest that both RIG-I and MDA5 are involved, and either of the two is essential for the induction of IFN-βresponse to DENV infection, and RIG-I plays a principle role in the IFN-βresponse to DENV infection.3. Antivial effects and mechanism of RIG-I against DENV and WNV.Previous experiments based on the stable RIG-I-knockout cells showed that RIG-I knockout enhanced the cytopathic effects, and increased the yield of progeny DENV, suggesting a potential antiviral effects of RIG-I. To test whether RIG-I? can inhibit replication of DENV and WNV, RIG-I was transiently over-expressed in A549 cells followed by infection with DENV or WNV, and the progeny virus yield decreased sharply in a dose-dependent manner. Further, treatment of A549 cells with human IFN-βantibodies had no effects on the antiviral activity, demonstrating that the IFN-β-provoked JAK-STAT pathways are not essential for the antiviral effect of RIG-I. Next, over-expression of RIG-I and RIG-IC, not RIG-IN, showed similar antiviral effects, suggesting the C-terminal of RIG-I was responsible for the antiviral activity. What’s more, the DENV E protein level decreased in RIG-I and RIG-IC expressing A549 cells compared with that in RIG-IN expressing and control cells. So at the early stage of DENV infection, the activated RIG-I not only induces IFN-β, but also inhibits the synthesis of viral protein through its C-terminal region.In conclusion, our results demonstrate that DENV infection can activate both RIG-I and MDA5, and either of the two is essential for the induction of IFN-βresponse to DENV infection. In addition, the C teiminal region of RIG-I can inhibit the synthsis of viral protein, possessing antiviral activity against multiple flaviviruses. Our findings indicat the critical role of RIG-I and MDA5 during DENV infection, and describe the possible process of type I interferon production in DENV-infected human lung cells for the first time, provide insights for the understanding of virus-host interaction and development of intervention strategy for viral infections.更多还原