蓝舌病病毒感染诱导细胞自噬的作用和机制

发布时间：2017-12-27 04:08

本文关键词：蓝舌病病毒感染诱导细胞自噬的作用和机制　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】：蓝舌病(Bluetongue,BT)是由蓝舌病病毒(Bluetongue virus,BTV)引起的一种重要虫媒传染病,可引起反刍动物特别是绵羊较高的发病率和致死率,对畜牧业的发展和畜产品的经济贸易造成潜在的巨大威胁。BTV属于呼肠孤病毒科环状病毒属,是一种复杂的双链分节段RNA病毒,拥有双层衣壳,无囊膜。作为环状病毒属的一个典型代表,BTV在分子结构、生化特征、流行病学和诊断方法等分子生物学领域已取得了重大进展,然而,BTV感染与宿主细胞应答之间的相互作用并没有得到充分的关注,因此从宿主的角度去探索BTV的致病机制显得尤为重要。病毒作为一种专性细胞内寄生物,其在感染过程中必定会与宿主的胞内应答发生相互作用。细胞自噬(Autophagy)作为一种保守的胞内适应性应答,在维持细胞稳态,促进新陈代谢方面起着举足轻重的作用。它可将细胞内的衰老细胞器、外源微生物等成分包裹在一个称为自噬体的双层膜结构中,运送至溶酶体进行降解回收。自噬是对抗病原体的天然防御机制,但最近越来越多的证据表明许多病毒已进化出多种策略抵抗、逃逸、甚至利用细胞自噬促进自身增殖,如流感病毒、丙型肝炎病毒、登革热病毒、轮状病毒等。对于BTV而言,其复制感染与细胞自噬之间的相互作用及其启动机制便是本研究中要探索的问题。首先,采用三种经典方法对BTV1感染BSR细胞后是否诱导自噬进行了检测,包括透射电镜观察自噬体的形成、激光共聚焦检测荧光聚点的变化以及Western blot分析LC3的转化。结果表明,与阴性对照相比,BTV1感染后诱导BSR细胞产生了自噬体样的双层膜结构,GFP-LC3荧光聚点显著累积,而且LC3发生了明显的类别转化。此外,在BTV的自然宿主靶细胞-原代羊舌上皮细胞上进行了分析,得到了类似的结果。由此证实BTV1感染可以诱导细胞自噬的发生。其次,进行了基于自噬性降解的自噬潮分析。自噬潮,即自噬的整个动态连续的过程,可通过p62的降解、LC3-II的周转、自噬体和溶酶体共定位分析等综合评估。研究结果显示,BTV1感染明显促进了自噬底物p62/SQSTM1的降解,而且溶酶体抑制剂CQ的配合使用导致了LC3-II和p62的蓄积,BTV1感染后自噬体标记GFP-LC3和溶酶体标记LysoTracker也出现明显的共定位,表明BTV1诱导了自噬的起始和自噬性降解过程,即BTV1感染后触发了完整的自噬潮反应。再次,判定BTV1诱导自噬是否严格依赖于病毒的有效复制。灭活病毒UV-BTV1不能诱导相同的自噬现象证实了该推论。进一步应用自噬早期阻断药物3-MA和晚期阻断药物CQ处理细胞或者干扰自噬关键基因Beclin 1,可见自噬活性明显降低,病毒复制也受到了显著的抑制;相反,自噬诱导剂Rapamycin处理细胞后激活了自噬反应,促进了BTV1的复制。上述结果证明了BTV1的有效复制是诱导自噬的关键因素,并可以利用自噬促进自身增殖。最后,为了深入探索BTV1感染激活自噬的机制,对可能涉及的多条信号级联反应进行了系统分析。结果显示,BTV1感染后明显抑制了自噬的中央调控分子mTOR的磷酸化及其下游底物p70S6K的活性,从而启动了自噬。而后探索mTOR上游可能的调控信号,首先检测发现BTV1感染激发细胞自噬不依赖于ERK1/2信号通路。而药物处理和突变质粒转染显示mTOR活性降低部分取决于PI3K/Akt的失活,相反,激活Akt后p-mTOR有所恢复,自噬也相应被抑制,说明Akt信号通路参与了BTV1诱导的自噬的启动过程。最为明显的是AMPK途径的影响,通过药物处理和RNA干扰实验等联合使用,证实病毒感染显著提高了胞浆内Ca2+的浓度,并介导了Ca MKKβ的激活,作用于下游的AMPK,从而导致mTOR的抑制和自噬的激活。试验进一步证实TSC2是Akt/AMPK及mTOR之间信号传导的关键中介分子。结果表明BTV1可通过抑制Akt和活化Ca2+介导的AMPK来共同作用于下游的TSC2-mTOR途径,从而诱导自噬的激活,促进病毒自身的复制。综上所述,本研究首次阐明了BTV1感染诱导的细胞自噬在BTV1的复制中发挥着重要作用,而且系统分析了调控两者之间相互作用的信号途径。这些发现推动了BTV与宿主相互作用领域的研究,成为探索BTV发病机制的新视角,为抗病毒药物研发奠定了基础。
[Abstract]:Bluetongue (Bluetongue, BT) by bluetongue virus (Bluetongue virus BTV) is an important infectious disease caused by insect, especially sheep ruminant animal can cause high morbidity and mortality, causing huge potential threat to the development of animal husbandry and animal products trade. BTV belongs to the Reoviridae like virus, is a kind of complex double stranded segments of RNA virus, has a double capsid, no envelope. As a typical representative of the orbivirus genus, BTV in the molecular structure, biochemical characteristics, epidemiology and diagnostic methods of molecular biology have made significant progress, however, the interaction between BTV infection and host cell responses have not been fully concerned, because the host from the point of view to explore the pathogenic mechanism of BTV is is particularly important. The virus, as a specific intracellular parasite, is bound to interact with the intracellular response of the host in the process of infection. Cellular autophagy (Autophagy), as a conserved intracellular adaptive response, plays an important role in maintaining cell homeostasis and promoting metabolism. It can encapsulate cell senescent organelles and exogenous microorganisms in a bilayer membrane structure called autophagosome, transported to lysosomes, and can be degraded and recovered. Autophagy is a natural defense mechanism against pathogens, but recently more and more evidence that many viruses have evolved a variety of strategies of resistance, escape, and even the use of autophagy promote their proliferation, such as influenza virus, hepatitis C virus, dengue virus, rotavirus. For BTV, the interaction between the replicative infection and the autophagy and the mechanism of its initiation are the problems to be explored in this study. First of all, using three classical methods to induce autophagy in BTV1 infected BSR cells were detected, including conversion change formation, transmission electron microscope observation of autophagosomes detected by laser confocal fluorescence technique and Western blot analysis LC3. The results show that, compared with the negative control, the double membrane autophagosomes like BSR cells induced by BTV1 infection, GFP-LC3 fluorescence and LC3 accumulation significantly accumulated obviously category transformation. In addition, the target cells of the natural host of BTV, the primary goat tongue epithelial cells, were analyzed, and similar results were obtained. It is confirmed that BTV1 infection can induce autophagy. Secondly, autophagy analysis based on autophagy was carried out. Autophagy, the whole process of autophagy, can be evaluated through p62 degradation, turnover of LC3-II, CO localization of autophagosomes and lysosomes. The results showed that BTV1 infection could promote the degradation of autophagy substrate p62/SQSTM1, and with the lysosomal inhibitor CQ use has led to the accumulation of LC3-II and p62, BTV1 after infection of autophagosome marker GFP-LC3 and lysosomal marker LysoTracker also appeared significant co localization, suggesting initiation and autophagic degradation of BTV1 induced autophagy, BTV1 infection after the complete reaction of tidal triggering autophagy. Again, it is determined whether the BTV1 induced autophagy is strictly dependent on the effective replication of the virus. Inactivated virus UV-BTV1 can not induce the same autophagy, which confirms this inference. Further application of autophagy early blocking drugs and late 3-MA cells treated with CQ blocking drugs or interfere with the key autophagy gene Beclin 1, visible autophagy activity decreased significantly, virus replication was significantly inhibited; on the contrary, the agent Rapamycin cells after activation of autophagy reaction induced autophagy, promote the replication of BTV1. The above results show that the effective replication of BTV1 is the key factor in inducing autophagy and can use autophagy to promote the proliferation of autophagy. Finally, in order to explore the mechanism of activation of autophagy by BTV1 infection, a systematic analysis of the possible multi signal cascade reaction was carried out. The results showed that BTV1 infection significantly inhibited the phosphorylation of the autophagy, the central regulatory molecule mTOR, and the activity of the downstream substrate p70S6K, thus initiating autophagy. Then we explored the possible regulatory signals in the upstream of mTOR. First, it was found that the autophagy of BTV1 infected cells was not dependent on the ERK1/2 signaling pathway. Drug treatment and mutation plasmid transfection showed that the decrease of mTOR activity was partly determined by the inactivation of PI3K/Akt. On the contrary, after activation of Akt, p-mTOR recovered and autophagy was also inhibited, indicating that the Akt signaling pathway was involved in the initiation process of BTV1 induced autophagy. The most obvious is the influence of AMPK pathway. The combination of drug treatment and RNA interference test confirmed that virus infection significantly increased the concentration of Ca2+ in the cytoplasm, and mediated the activation of Ca MKK beta, which played a role in the downstream AMPK, resulting in the inhibition of mTOR and the activation of autophagy. The test further confirmed that TSC2 is the key mediator of signal transduction between Akt/AMPK and mTOR. The results showed that BTV1 could co activate downstream TSC2-mTOR pathway by inhibiting Akt and activated Ca2+ mediated AMPK, thereby inducing autophagy and promoting the replication of virus itself. To sum up, this study first elucidates that BTV1 induced autophagy plays an important role in the replication of BTV1, and systematically analyzes the signaling pathways regulating the interaction between them. These findings promote the research on the interaction between BTV and host, and become a new perspective to explore the pathogenesis of BTV and lay a foundation for the research and development of antiviral drugs.
【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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