VP2互作蛋白CK1α及CSGalNAcT2在鸡传染性法氏囊病病毒复制中的作用及分子机制研究

发布时间：2017-12-27 19:21

本文关键词：VP2互作蛋白CK1α及CSGalNAcT2在鸡传染性法氏囊病病毒复制中的作用及分子机制研究　出处：《中国农业科学院》2016年博士论文　论文类型：学位论文

【摘要】：传染性法氏囊病(Infectious Bursal Disease,IBD)是由传染性法氏囊病病毒(Infectious Bursal Disease Virus,IBDV)引起的急性、高度接触性传染病。该病主要侵害3-6周龄雏鸡的中枢免疫器官—法氏囊,有人形象地称该病为鸡的艾滋病‖。随着IBDV的不断进化变异,先后出现了经典毒株(classic IBDV,c IBDV)、变异毒株(variant IBDV,v IBDV)以及超强毒株(very virulent IBDV,vv IBDV),vv IBDV的高致死率给世界养禽业带了巨大的经济损失。近年来,在流行病学研究不断深入及反向遗传学技术日趋成熟的基础上,作为IBDV外层衣壳唯一组成成分的VP2蛋白,已被确认为是决定病毒毒力及细胞嗜性差异的重要分子基础。然而IBD发生与发展的实质是IBDV与宿主细胞相互博弈的过程。尽管在病毒层面已表明VP2蛋白对IBDV的细胞嗜性和毒力有重要影响,然而VP2蛋白在宿主细胞内的具体作用依然还不清楚。因此,深入了解VP2蛋白与宿主细胞的相互作用已成为目前揭开IBDV致病和免疫机制的研究重点。本研究从病毒与宿主相互作用的角度,采用不同的蛋白相互作用技术,以VP2蛋白为诱饵筛选出与其相互作用的宿主蛋白,并进一步探索它们在IBDV复制过程中的分子作用机制。首先,以免疫共沉淀技术与质谱技术相结合,在感染IBDV的易感细胞DF1中,利用VP2蛋白的特异性单克隆抗体,鉴定出了一个互作候选蛋白酪蛋白激酶CK1α。免疫共沉淀及激光共聚焦试验进一步证实,CK1α与VP2在IBDV感染过程中存在相互作用。与此同时,我们发现在IBDV复制过程中,CK1α呈下调表达。基因表达调控实验表明,CK1α下调表达能够抑制IBDV复制,反之则促进IBDV复制,另外CK1α激酶活性抑制剂D4476也会抑制IBDV复制,这表明CK1α对IBDV复制的影响与其激酶活性有关。综上,CK1α在IBDV复制过程中的下调表达可能是宿主抵抗IBDV的一种抗病毒应答。为深入揭示宿主通过下调CK1α来限制IBDV复制的分子机制,我们经过探索发现,IBDV感染能够诱导产生IFN-β,而CK1α过表达能够拮抗IFN-β的抗病毒效应,但这种拮抗作用不是源于对IFN-β本身表达量的负调控,而是源于对IFNAR1稳定性的负调控。CK1α负调控IFNAR1稳定性的具体机制是,CK1α能够泛素化降解IFNAR1。IFNAR1的C端胞内区存在一个保守的降解决定子序列,它与IFNAR1的泛素化密切相关,其磷酸化位点S545是CK1α发挥调控功能的关键位点。点突变实验证实,S545A的确能够削弱CK1α介导的IFNAR1泛素化。IBDV感染宿主细胞的过程中,在发现CK1α下调的同时,也发现了IFNAR1在细胞膜表面上的分布增加。因此我们认为,CK1α作为宿主的一种感应分子,在IBDV感染后被下调,进而通过负调控机制导致IFNAR1的上调,从而增强宿主限制IBDV复制的能力。在宿主的抗病毒压力下,病毒也进化出多种机制来利用宿主细胞的资源而利于自身复制。本研究还鉴定了另一个有利于IBDV复制的宿主细胞蛋白——高尔基体膜蛋白硫酸软骨素N-乙酰氨半乳糖胺基转移酶(CSGal NAc T2)。CSGal NAc T2能够与VP2蛋白在高尔基体上发生相互作用。结合基因芯片分析结果及荧光定量检测结果,我们证实,CSGal NAc T2在IBDV感染过程中表达上调,而CSGal NAc T2上调表达能够促进IBDV的复制,且此种促进作用依赖于高尔基体结构的完整性。高尔基体是IBDV“复制工厂”核糖核蛋白复合体(RNP)的所在地,VP2与CSGal NAc T2的相互作用进一步表明,IBDV能够利用宿主蛋白CSGal NAc T2招募VP2到高尔基体从而利于自身的组装和增殖。本研究首次发现了不同宿主蛋白与IBDV衣壳蛋白VP2互作所触发的宿主细胞对病毒复制不同的调控效应,揭示了在IBDV感染过程中CK1α触发的抗病毒机制,以及CSGal NAc T2介导的促进病毒复制的分子机理,对于深入认知IBDV的致病和免疫机制具有重要意义。
[Abstract]:Infectious Bursal Disease (IBD) is an acute and highly contagious infectious disease caused by infectious bursal disease virus (Infectious Bursal Disease Virus, IBDV). The disease mainly affects 3-6 week old chicks - central immune organs of IBD, people call the "chicken for AIDS disease. With the continuous evolution of IBDV, has appeared in the classical strains (classic IBDV, C, IBDV) (variant IBDV, V variant strain IBDV (very virulent) and vvIBDV IBDV, VV IBDV VV IBDV), the high mortality rate for the poultry industry in the world with huge economic losses. In recent years, based on the deepening of epidemiological research and the mature technology of reverse genetics, the VP2 protein as the sole component of outer capsid of IBDV has been identified as an important molecular basis for determining the virulence and cell tropism of viruses. However, the essence of the occurrence and development of IBD is the process of mutual game between IBDV and host cells. Although VP2 has been shown to play an important role in the cell tropism and virulence of IBDV, the specific role of VP2 protein in host cells is still unclear. Therefore, understanding the interaction of VP2 protein and host cell has become the focus of research on the pathogenesis and immune mechanism of IBDV. From the point of view of the interaction between virus and host, we used different protein interaction technologies to screen the host proteins interacting with VP2 protein, and further explored their molecular mechanism in the process of IBDV replication. First, combined immunoprecipitation and mass spectrometry, we identified an interaction protein casein kinase CK1 alpha in the susceptible cell DF1 infected with IBDV by using the specific monoclonal antibody of VP2 protein. Immunoprecipitation and laser confocal test further confirmed that CK1 alpha and VP2 interact in the process of IBDV infection. At the same time, we found that in the process of IBDV replication, the expression of CK1 alpha was downregulated. Gene expression regulation experiments showed that the down regulated expression of CK1 alpha could inhibit the replication of IBDV, whereas on the contrary, it promoted IBDV replication. Moreover, CK1 alpha kinase inhibitor D4476 also inhibited IBDV replication, indicating that the effect of CK1 on IBDV replication was related to its kinase activity. To sum up, the downregulation of CK1 alpha during IBDV replication may be an antiviral response of the host against IBDV. In order to further reveal the molecular mechanism of the host to limit IBDV replication through down-regulation of CK1 alpha, we have to explore found that IBDV infection could induce IFN- beta, CK1 alpha overexpression can antagonize the effect of antiviral IFN- beta, but this antagonism is not due to negatively regulate the expression amount of IFN- beta itself, but from the negative regulation on the stability of IFNAR1. The specific mechanism of CK1 alpha negatively regulates the stability of IFNAR1 is that CK1 alpha can degrade IFNAR1 by ubiquitination. There is a conserved degradation determining subsequence in the C terminal intracellular region of IFNAR1, which is closely related to ubiquitination of IFNAR1. Its phosphorylation site S545 is the key locus for CK1 alpha to exert regulatory function. The point mutation test confirmed that S545A could indeed weaken the CK1 - mediated IFNAR1 ubiquitination. In the process of IBDV infection of the host cells, the decrease of CK1 alpha was found, and the increase in the distribution of IFNAR1 on the surface of the cell membrane was also found. Therefore, we believe that CK1 alpha is an inducer of the host, which is down regulated after IBDV infection, and then leads to IFNAR1 up regulation by negative regulation mechanism, thereby enhancing the host's ability to limit IBDV replication. Under the anti-virus pressure of the host, the virus also evolved a variety of mechanisms to make use of the resources of the host cell to facilitate its own replication. Another N- cell membrane protein, chondroitin sulfate N-, acetylated Galacto transferase (CSGal NAc T2), which is favorable for IBDV replication, has been identified. CSGal NAc T2 can interact with the VP2 protein in the Golgi body. Combined with gene chip analysis and fluorescence quantitative detection results, we confirmed that CSGal NAc T2 expression was upregulated during IBDV infection, while CSGal NAc T2 up-regulated expression could promote IBDV replication, and this promotion effect was dependent on the integrity of Golgi body structure. The Golgi apparatus is the location of IBDV replication factory ribonuclein complex (RNP). The interaction between VP2 and CSGal NAc T2 further indicates that IBDV can recruit VP2 to Golgi body by CSGal NAc T2, enabling itself to assemble and proliferate. This is the first study found that different host proteins and IBDV capsid protein VP2 interaction triggered by the host cell replication regulation different effects on virus, reveals the antiviral mechanism of triggering CK1 alpha in the process of IBDV infection, and promote viral replication CSGal NAc T2 mediated molecular mechanism has important significance for in-depth understanding of IBDV the pathogenic and immune mechanism.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.65

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