轮状病毒NSP1抑制天然免疫机制及其对宿主蛋白调控的研究

发布时间：2018-08-12 18:32

【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。
[Abstract]:Rotavirus (RV) is the main pathogen causing acute diarrhea in infants under 5 years old. It causes 600,000 deaths every year and seriously endangers human health.
The function of RV nonstructural protein 1 (NSP1) in antagonizing the innate immune response of RV host is not fully understood. Recent studies have shown that NSP1 can block the interferon (IFN) signaling pathway by mediating the degradation of interferon regulatory factor (IRF) family proteins and inhibiting the activation of nuclear transcription factor kappa B (NF-kappa B). However, there is evidence that the homology of RV NSP1 from different sources is relatively low, and there may be differences between the different NSP1 proteins and the way of innate immunity. For example, the IRF binding domain of NSP1 protein of pig RVOSU strain can not mediate the degradation of IRF protein. This suggests that NSP1 from different sources may antagonize host by many ways. In view of this, this study intends to further explore the target of interaction between NSP1 and IFN signaling pathway, explore the mechanism of NSP1 antagonizing host innate immunity, and analyze the regulation of NSP1 expression on host cell proteins by differential proteomics technology, in order to understand the function of NSP1.
Antagonistic effect of NSP1 on host's innate immunity
Firstly, we observed that NSP1, a virus strain known to inhibit IFN response by degrading IRF, could inhibit the activity of IFN-beta promoter even after the loss of IRF binding domain, suggesting that NSP1 had other targets for antagonizing IFN. Immunoprecipitation analysis showed that NSP1 could bind to RIG-I and decrease the expression of RIG-I protein. Moreover, RV NSP1 of different sources of human, monkey and pig can downregulate RIG-I. when RV infection. We found that with the increase of viral replication, RIG-I also showed changes in expression level, 4h RIG-I expression increased after infection, and RIG-I expression decreased gradually from 12h after infection, suggesting that RIG-I was indeed inhibited in the infection of RV, but the latter had a structure domain. Whether NSP1 has ubiquitin-ligase activity has been directly confirmed. We have confirmed that NSP1 is a ubiquitin-E3 ligase that enhances cell ubiquitination and self-ubiquitination, and that ubiquitin-ligase function is related to the N-terminal RING domain. However, the inhibition of NSP1 on RIG-I may not be through the ubiquitin-proteasome pathway. Bioinformatics predicts that RI The cleavage site of Caspase 1 is present in G-I. Whether RIG-I antagonizes the cleavage of RIG-I by activating Caspasel needs further study.
As a pattern recognition receptor, RIG-I plays an extremely important role in monitoring the invasion of RV and initiating innate immune response. This study found for the first time that NSP1 can reduce the expression of RIG-I protein to antagonize host IFN response. The results reveal that RIG-I is a new target for NSP1 and further enrich the innate immune mechanism for RV escaping host. Understanding of system.
Two, the effect of NSP1 on the proteome of host cells.
To further elucidate the mechanism of NSP1, we studied the effect of overexpression of NSP1 protein on the proteome of host cells by fluorescence differential gel electrophoresis (DIGE) and mass spectrometry. After overexpression of NSP1, 12 differentially expressed proteins were identified, of which 4 were up-regulated and 8 were down-regulated. Gene Ontology analysis showed that the differentially expressed proteins involved cytoskeleton, signal transduction, molecular chaperone, transcriptional regulation and other functions. Then we studied the interaction between up-regulated waveform protein (Vimentin) and NSP1. The localization of vimentin cells was rearranged after infection, suggesting that vimentin might be involved in viral replication and assembly. Laser confocal microscopy was used to observe the localization of vimentin cells after NSP1 transfection. These results suggest that the expression of NSP1 protein may be one of the causes of vimentin rearrangement after RV infection. NSP1 is involved in virus replication through cytoskeleton interaction. These results provide a new direction for further understanding the function of NSP1.
In conclusion, this study reveals for the first time that RV can antagonize RIG-I and inhibit innate immunity, and can regulate the expression and localization of vimentin in cells. These findings lay a foundation for further study of the mechanism of RV antagonizing innate immunity and RV replication.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R373

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