【作者】 秦岚；

【导师】 洪涛； 王健伟；

【作者基本信息】 北京协和医学院 ， 病原生物学， 2010， 博士

【摘要】 轮状病毒(Rotavirus, RV)是导致5岁以下婴幼儿急性腹泻的最主要病原,每年导致60万人死亡,严重危害人类健康,目前其致病机制尚不清楚。阐明RV的致病机制,尤其是其逃逸宿主免疫防御机理,将为新型抗RV策略及疫苗的研发提供依据。RV非结构蛋白1(NSP1)在RV拮抗宿主天然免疫应答中起重要作用,其功能尚不完全清楚。目前的研究表明,NSP1蛋白可以通过介导干扰素调控因子(IRF)家族蛋白的降解,以及通过抑制核转录因子(NF-κB)的活化来阻断干扰素(IFN)信号通路,从而拮抗宿主抗病毒应答。然而,有证据表明不同种属来源RV NSP1同源性较低,而且不同NSP1蛋白与天然免疫的作用方式可能存在差异,如猪RVOSU株NSP1蛋白的IRF结合结构域并不能介导IRF蛋白的降解。这提示不同来源NSP1可能通过多种途径拮抗宿主天然免疫。鉴此,本研究拟进一步探索NSP1与IFN信号通路相互作用的靶点,深入探讨NSP1拮抗宿主天然免疫机理,并通过差异蛋白质组学技术,分析NSP1的表达对宿主细胞蛋白的调控,以期深入了解NSP1的功能。一、NSP1拮抗宿主天然免疫机理研究首先我们观察到已知通过降解IRF抑制IFN应答的RV毒株的NSP1在缺失IRF结合功能结构域后,仍可以抑制IFN-β启动子活性,提示NSP1存在其它拮抗IFN作用靶点。免疫沉淀分析发现NSP1可以与维甲酸诱导基因I (RIG-I)结合,并降低RIG-I蛋白表达,而且人、猴、猪等不同种属来源的RV NSP1均可以下调RIG-I.在RV感染时,我们发现随着病毒复制增加,RIG-I也出现表达量的变化,感染后4h RIG-I表达增加,而从感染后12h开始RIG-I表达逐渐减少,提示RIG-I在RV感染中确实被抑制。NSP1拥有RING结构域,但是否具有泛素连接酶活性尚未被直接证实。我们证实NSP1为泛素E3连接酶,可以增强细胞泛素化及自身泛素化,并且泛素连接酶功能与N端RING结构域相关。但是NSP1对RIG-I的抑制作用可能不是通过泛素-蛋白酶体途径。生物信息学预测表明,RIG-I中拥有Caspase1的切割位点,RIG-I是否通过激活Caspasel切割RIG-I拮抗其作用尚需要进一步研究。作为模式识别受体,RIG-I在机体监测入侵RV并启动天然免疫应答中发挥了极其重要的作用,本研究首次发现了NSP1可以减少RIG-I蛋白表达,以拮抗宿主IFN应答,该研究结果揭示RIG-I为NSP1作用新靶点,进一步丰富了对于RV逃逸宿主天然免疫机制的认识。二、NSP1对宿主细胞蛋白质组的影响为了进一步阐明NSP1的作用机制,我们利用荧光差异凝胶电泳(DIGE)技术结合质谱鉴定研究了过表达NSP1蛋白对宿主细胞蛋白质组的影响。过表达NSP1后,初步鉴定到12个差异表达蛋白质,其中4个表达上调,8个表达下调。通过Western blot分析,我们对表达下调差异蛋白质Prohibitin、ERP29进行了验证,结果与DIGE结果一致。Gene Ontology分析显示差异表达蛋白涉及细胞骨架、信号转导、分子伴侣、转录调控等功能。随后我们研究了表达上调的波形蛋白(Vimentin)和NSP1相互作用。免疫荧光显示RV感染后,波形蛋白细胞定位发生重新排列,提示波形蛋白可能与病毒复制和装配有关。进而我们利用激光共聚焦方法观察了转染NSP1后波形蛋白细胞定位变化,结果显示,不表达NSP1蛋白的细胞中,波形蛋白成网状纤维分布,而表达NSP1的细胞中,波形蛋白形成聚集体,并与NSP1蛋白共定位,提示NSP1蛋白表达可能是导致RV感染后波形蛋白发生重新排列的原因之一,NSP1通过与细胞骨架相互作用参与病毒复制。这些结果为进一步了解NSP1功能提供研究方向。综上所述,本研究首次揭示了RV可拮抗RIG-I而抑制天然免疫,且可调控细胞波形蛋白的表达和定位,这些发现为深入研究RV拮抗天然免疫的机制和RV复制机制打下了基础。更多还原

【Abstract】 Rotavirus (RV) is the major cause of acute diarrhea in children less than5years of age, leading to approximately600,000deaths annually. Better understanding of the mechanisms of rotavirus pathogenesis, especially the viral strategies to subvert and evade host’s antiviral responses, will help us to identify novel strategies for developing antiviral reagents and vaccines.The function of RV nonstructural protein1(NSPl) has not been elucidated. It has been shown that the interaction between NSP1and host is essential for subverting innate immune response. NSP1represses the innate immunity by at least2mechanisms. First, NSP1can induce proteasome-dependent degradation of the interferon transcription factors (IRF3, IRF7, and IRF5) to inhibite the IFN response. Second, NSP1can inhibit nuclear factor-KB (NF-κB) activation by inducing proteasome-dependent degradation of β-TrCP. However, it is reported NSP1is the least conserved protein encoded by the rotavirus genome in different strains. Furthermore, current evidences indicated that the NSP1effect to subvert innate immunity is rotavirus strain-specific. For examples, the NSP1from OSU strain can not induce IRF3degradation.Taken together, we question if NSP1from different rotavirus strains may target a range of cellular substrates of antiviral signaling pathways. In this study, we indentified the new targets of NSP1to better understand the strategies for RV to antagonize innate immunity. We also investigated the host cell proteins modulated by NSP1overexpression using a quantitative proteomics technology to add understandings to the functions of NSP1.1. Rotavirus NSPl antagonizes innate immunity by interacting with RIG-I Previous studies have shown that RV SA11strain NSP1inhibits IFN response by binding and degradating IRFs through its IRF3binding domain. However, we found that the truncated NSP1of SA11strain lacking IRF3binding domain reserve its ability to inhibite RIG-I-induced IFN-β promoter activity, indicating that there may exist other targets by which NSP1inhibit IFN response. Immunoprecipitation assay showed that NSP1binds and degrades RIG-I, a crucial pathogen recognition receptor to sensor pathogen invasion and induce IFN response. Similar results were obtained in RVs strains from other species, human (WA) and porcine (OSU) in addation to simian RV. We also found that during RV infection, RIG-I expression decreased with rotavirus replication. It was predicted that there is a RING domain in NSP1. We proved that the NSP1is a viral E3ubiquitin ligase but the degradation of RIG-I is not through the ubiquitin-proteasome pathway. Bioinformatics analysis showed that there is a caspase1cleavage site in RIG-I. Whether NSP1might cleave RIG-I by active caspase1needs to be further investigated.2. Influence on host cell proteome by rotavirus NSP1In order to further explore the function of NSP1, by using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS) techniques, we analyze the proteomes changes of host cells by over expressing NSP1.A total of12quantitatively altered proteins were identified. Among them, expression of8proteins decresed, while expression of4proteins increased.GO analysis showed that these proteins involved in components of the cytoskeleton, signal transduction, molecular chaperone and transcription. We verified the down-regulate expression of proteins, prohibitin and ERP29by western blot, which is in agreement with DIGE analysis. Then we studied the interaction between Vimentin, an expression increase protein belongs to cytoskeleton, and NSP1. RV infection leads to rearrangement of vimentin which serves virus replication and assemble. We found that expression of NSP1also lead to the rearrangement of vimentin and can co-localized with RV VP6, which indicates vimentin may associate with RV replication and assemble. NSP1maybe is one of the reasons responsible for vimentin rearrangement during RV infection.In summary, our study reveal that rotavirus NSP1can inhibit innate immunity by antagonizing RIG-I, and can modulate the expression and rearrangement of vimentin for the first time. These findings add insights into RV replication and the mechanism by which RV subvert host innate immunity.更多还原