EV71和HBV病毒与宿主细胞相互作用的蛋白质组学分析

发布时间：2019-05-17 04:27

【摘要】：蛋白质组学是当今生命科学研究的重要工具之一。本论文综合运用稳定同位素标记和亚细胞结构分离等生化技术并结合基于新一代质谱的高通量蛋白质分析技术,对EV71和HBV这两种重要病毒感染所引起的宿主细胞蛋白变化进行了系统研究。EV71病毒能引起婴幼儿严重的中枢神经系统疾病,是我国乃至亚太地区公共健康安全的重大隐患之一。目前关于EV71病毒与宿主的相互作用组学研究报道较少,制约了我们对于病毒的了解。因此在本论文的第二章,我们系统地研究了病毒感染不同阶段的宿主细胞蛋白质组的动态变化。我们将不同的同位素标记的SILAC培养的细胞经过EV71感染,在8小时与20小时后提取宿主蛋白质,利用高分辨液质联用技术定量分析到了4114个宿主蛋白的感染前后的动态变化情况,发现其中有约17%的蛋白在不同的时间点发生了显著变化。通过对这些发生显著变化的蛋白进行分析,我们找到了受EV71感染后宿主细胞蛋白的一些变化规律,包括发现5个生物学进程和7个蛋白家族在感染后发生了显著的变化,尤其是histone蛋白家族在感染后持续下降。线粒体蛋白中有很多发生了明显的上下调,但是绝大部分只在某一个感染时间点发生变化。分层聚类分析结果也证实了这一点。此外,一些与病毒感染相关的蛋白家族,例如泛素化和SUMO化相关蛋白、膜泡运输蛋白和肿瘤坏死因子等蛋白,也发生了明显变化。通过进一步蛋白功能验证和筛选,我们发现宿主线粒体蛋白CHCH2对EV71的复制可能具有重要的影响,而这种影响可能是通过负调控宿主天然免疫信号通路来实现的。我国有超过9300万的乙肝患者,对于HBV病毒的研究有着重要的现实意义。在本论文的第三章中,我们采用一种源自Huh7细胞的HBV稳定表达细胞系Huh7.37,系统地研究了全细胞和亚细胞结构中蛋白质的表达变化。我们运用核质分离技术纯化了细胞核组分,运用蔗糖密度梯度超速离心技术富集了线粒体组分,并结合三重双甲基化标记和质谱分析技术,发现在全细胞组分、细胞核组分和线粒体组分中发生明显改变的信号通路和蛋白家族均不相同。我们在线粒体组分中发现了19个过氧化物酶体蛋白发生了明显的变化,这暗示了Huh7.37细胞中线粒体和过氧化物酶体之间的联系变得更加紧密。在亚细胞结构蛋白质组与全蛋白质组的比较过程当中,我们发现约有9.6%的核胞质共存蛋白在病毒复制过程中可能进入细胞核,而29.8%的线粒体胞质共存蛋白在这个过程中可能进入线粒体,这表明HBV的复制可能改变了这些蛋白在细胞中的定位。STRING分析显示,有30个蛋白在细胞核中的表达量明显下调,而它们在全细胞和线粒体中的变化不明显,甚至反而有所上调,因此我们推测这些蛋白可能在亚细胞结构间发生了穿梭。此外,我们初步探究了20个宿主蛋白对于HBV复制的影响,发现其中有10个蛋白能影响HBeAg的表达,但是仅有DDX1能同时影响HBsAg的表达。
[Abstract]:Proteomics is one of the most important tools in life science. In this paper, the changes of host cell protein caused by two important viral infections of EV71 and HBV were systematically studied by using biochemical techniques such as stable isotope labeling and subcellular structure separation, and combining with high-throughput protein analysis based on a new generation of mass spectrometry. The EV71 virus can cause severe central nervous system diseases in infants, and is one of the major hidden dangers of public health and safety in China and the Asia-Pacific region. The current study on the interaction between the EV71 virus and the host has reported less and restricted our understanding of the virus. Therefore, in the second chapter of this thesis, we systematically study the dynamic changes of host cell protein groups in different stages of viral infection. The cells of SIMILAC cultured with different isotopic labels were infected with EV71, and the host protein was extracted after 8 hours and 20 hours, and the dynamic changes of 4114 host proteins were quantitatively analyzed by high-resolution liquid chromatography. It was found that about 17% of the proteins had a significant change at different time points. By analyzing these proteins with significant changes, we found a number of changes in host cell proteins after EV71 infection, including the discovery of five biological processes and seven protein families with significant changes after infection, In particular the histone protein family continues to decline after infection. Many of the mitochondrial proteins have been significantly down-regulated, but most of them change only at a certain point of infection. The results of the hierarchical clustering analysis also confirmed this. In addition, some protein families associated with viral infections, such as ubiquitination and SUMO-related proteins, membrane vesicle transport proteins, and tumor necrosis factor proteins, have also changed significantly. By further protein function validation and screening, we have found that the host mitochondrial protein CHCH2 may have an important effect on the replication of the EV71, which may be achieved by negatively regulating the host's natural immune signal path. There are more than 93 million hepatitis B patients in China, and it is of great practical significance for the study of HBV. In the third chapter of this thesis, we used an HBV-stable expression cell line Huh7.37 derived from Huh7 cells, and systematically studied the expression of the protein in the whole cell and subcellular structure. The nuclear component was purified by the nuclear separation technique, and the mitochondrial fraction was enriched by the sucrose density gradient ultracentrifugation technique, and the whole cell fraction was found in combination with the triple double-methylation labeling and the mass-spectrum analysis technique. The signal pathway and the protein family that are significantly altered in the nuclear component and the mitochondrial component are not the same. We found a significant change in the mitochondrial fraction of the 19 peroxisome proteins, suggesting that the association between the mitochondria and the peroxidase in the Huh7.37 cell becomes more compact. During the comparison of the subcellular structural proteome and the whole proteome, we found that about 9.6% of the nuclear cytoplasmic co-existing proteins might enter the nucleus during the viral replication, while 29.8% of the mitochondrial cytoplasmic co-existing proteins may enter the mitochondria during this process, This suggests that the replication of HBV may alter the localization of these proteins in the cells. The STRING analysis showed that there were 30 proteins down-regulated in the cell nucleus, and their changes in the whole cell and the mitochondria were not significant, and even up-regulated, so we speculate that these proteins may be shuttling between the subcellular structures. In addition, we explored the effect of 20 host proteins on the replication of HBV, and found that 10 of them can affect the expression of HBeAg, but only DDX1 can affect the expression of HBsAg at the same time.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R373

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