H5N1与人蛋白质相互作用网络构建及PB2与RNF31相互作用研究

发布时间：2018-03-11 05:36

本文选题：H5N1禽流感病毒　切入点：相互作用网络　出处：《天津大学》2014年硕士论文　论文类型：学位论文

【摘要】：甲型H5N1高致病性禽流感出现的人畜共患现象已引起社会恐慌。H5N1病毒侵染机体，与宿主蛋白相互作用以完成病毒复制和转录，为了确定宿主蛋白在H5N1病毒致病过程中发挥的作用，，本实验室前期通过酵母双杂交技术，以病毒蛋白为诱饵，对人脾文库进行筛选，筛选出与H5N1禽流感病毒的10个病毒蛋白相互作用的179个宿主蛋白。本研究通过生物信息学方法构建了病毒蛋白与人蛋白质相互作用网络，发现这些相互作用多集中在病毒聚合酶复合体，参与调解包括细胞凋亡、免疫应答、蛋白转运、RNA加工等多个生物学过程；与随机网络蛋白相比发现，H5N1相互作用的蛋白在网络中的连接度和介度均高于其他蛋白，且更倾向于连接在一起；同时对宿主蛋白和宿主蛋白邻近蛋白进行基因富集分析，发现这些蛋白主要集中在NF-κB、WNT、MAPK、p53及凋亡通路；此外还将H5N1与其他禽流感毒株以及其他病原体的人相互作用蛋白质进行基因功能分析，为更好的解释不同流感毒株或不同病原体侵染宿主产生不同机体反应提供思路。基于相互作用网络分析，我们选择病毒聚合酶PB2与人RNF31这对相互作用进行深入研究。PB2是流感病毒聚合酶的一个重要亚基，它与病毒毒力相关并决定病毒的宿主范围，PB2多个氨基酸残基的改变都可以引起宿主范围的改变；RNF31是E3泛素连接酶的一种，其对NF-κB转录活性的调节具有重要作用。本研究发现，RNF31下调时抑制对TNF-α介导的NF-κB转录活性，下调NF-κB下游与炎症相关因子表达，抑制TNF-α介导的IκBα的磷酸化，同时细胞凋亡增多。另外本研究通过间接免疫荧光、核质分离发现PB2与RNF31共定位于胞核，通过Co-IP证实PB2与RNF31相互作用发生在胞核内，相互作用结构域分别位于PB2N端1-471肽段和RNF31C端910-1062肽段。同时，PB2抑制NF-κB转录活性和NF-κB下游与凋亡相关的靶基因表达；干涉RNF31，PB2对NF-κB转录活性的抑制作用消失；流式细胞分析发现PB2促进细胞凋亡，与下调RNF31促凋亡结果相一致，提示PB2是通过RNF31影响NF-κB转录活性和其介导的生物学功能。此外，稳定性实验和胞核Co-IP发现，RNF31不仅能微弱影响PB2的稳定性，也能增强PB2与聚合酶另一个亚基PB1相互作用。推测RNF31可能在影响流感聚合酶的形成过程中发挥重要作用，为分析流感病毒致病机制和寻找药物靶点提供新的思路。
[Abstract]:The emergence of highly pathogenic avian influenza A (H5N1) has caused social panic. H5N1 viruses infect the body and interact with host proteins to complete viral replication and transcription, in order to determine the role of host proteins in the pathogenesis of H5N1. In our laboratory, the human spleen library was screened by yeast two-hybrid technique and viral protein was used as bait. 179 host proteins interacting with 10 viral proteins of H5N1 avian influenza virus were screened. In this study, a network of interactions between viral proteins and human proteins was constructed by bioinformatics. It was found that these interactions were mainly concentrated in virus polymerase complexes and involved in many biological processes, including apoptosis, immune response, protein transporter RNA processing and so on. Compared with random network proteins, it was found that proteins interacting with H5N1 had higher connectivity and intermedium in the network than other proteins, and were more likely to be linked together, and the host proteins and host protein adjacent proteins were also analyzed for gene enrichment. It was found that these proteins were mainly concentrated in NF- 魏 B, WNTG MAPKN, p53 and apoptotic pathways. In addition, the gene function analysis of human interaction proteins between H5N1 and other avian influenza strains and other pathogens was carried out. In order to better explain the different influenza strains or different pathogens infected host to produce different body reaction ideas. Based on the interaction network analysis, we select virus polymerase PB2 and human RNF31 to study the interaction. PB2 is an important subunit of influenza virus polymerase. It is associated with virulence of the virus and determines the host range of the virus. Changes in the amino acid residues of PB2 can cause changes in the host range. RNF31 is one of the E3 ubiquitin ligases. It plays an important role in regulating the transcriptional activity of NF- 魏 B. in this study, we found that the down-regulation of RNF31 inhibits the transcriptional activity of NF- 魏 B mediated by TNF- 伪, down-regulates the expression of down-stream NF- 魏 B and inflammatory related factors, inhibits the phosphorylation of I 魏 B 伪 mediated by TNF- 伪, and increases apoptosis. In addition, PB2 and RNF31 were colocated in the nucleus by indirect immunofluorescence, and the interaction between PB2 and RNF31 was confirmed by Co-IP in the nucleus. The interaction domain was located at the PB2N terminal 1-471 peptide and the RNF31C terminal 910-1062, respectively. At the same time, PB2 inhibited NF- 魏 B transcription activity and NF- 魏 B downstream apoptosis-related target gene expression, and interfered with the inhibition of NF- 魏 B transcriptional activity by RNF31tPB2. Flow cytometry showed that PB2 promoted apoptosis, which was consistent with down-regulation of RNF31, suggesting that PB2 affected NF- 魏 B transcriptional activity and its mediated biological function through RNF31. Stability test and nuclear Co-IP showed that RNF31 not only weakly affected the stability of PB2, but also enhanced the interaction of PB2 with another subunit of polymerase, PB1. It is suggested that RNF31 may play an important role in the formation of influenza polymerase. It provides a new idea for analyzing the pathogenic mechanism of influenza virus and searching for drug target.
【学位授予单位】：天津大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R511.7

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