钙离子参与下的相关通路在HIV-1 Vpu拮抗Tetherin过程中的作用

发布时间：2018-11-05 12:10

【摘要】：Tetherin(BST-2,CD317)是一种具有特殊拓扑结构的II型膜蛋白,它的结构包括N端的胞质尾端,单次穿膜区,胞外的螺旋区以及C端的磷脂酰肌醇锚定区(潜在的二次穿膜区)。目前发现Tetherin能够嵌入宿主细胞质膜和新生的病毒膜,以抑制病毒的释放。HIV-1附属蛋白Vpu是一种编码81个氨基酸的I型膜蛋白,早期的研究表明,Vpu在HIV-1传播过程中有两个主要的功能,其一是Vpu可以下调细胞表面的CD4受体,阻止病毒感染细胞中gp160/CD4复合体形成,从而使宿主细胞被不同病毒多重感染;另外Vpu可以解除掉Tetherin(BST-2,CD317)系留病毒的作用,促进子代病毒的释放。Vpu拮抗Tetherin作用的分子机制目前仍未完全明确。起初,有报道猜测Vpu拮抗Tetherin与降解CD4受体的机制相似,即通过穿膜螺旋区与Tetherin相互结合,通过泛素化酶和蛋白酶体途径对Tetherin本身起到降解作用,使之不能够完成病毒的物理系留作用。然而后来的研究证实Vpu在不降解,也不下调Tetherin在细胞表面表达量的前提下也能够促进病毒释放,说明上述理论并不完备。也有报道间接证明Tetherin能够特异性识别病毒出芽位点,因而猜测Vpu拮抗Tetherin可能通过破坏其识别作用达到促进病毒释放的作用。还有一些其他的报道显示在有Vpu存在的情况下,Tethern仍然可以被包装进子代病毒但却无法发挥系留作用,综上说明Vpu拮抗Tetherin的关键分子机制仍有待进一步阐明。Vpu自身的同源寡聚体能形成弱选择性的钾离子通道,同时Vpu也能够干扰TASK导致宿主细胞膜的去极化,我们猜测这种去极化现象能够进而触发电压依赖型钙离子通道,引起细胞内的钙离子浓度上升,在之前的研究中已经初步证实了这种现象的发生。本篇论文就是在此基础上,进一步确认Vpu引起细胞内钙离子浓度上升是由于触发哪种电压依赖型钙离子通道导致,这种作用与其自身离子通道活性的相关性,以及钙离子参与下的相关下游通路在HIV-1 Vpu拮抗Tetherin过程中的作用,从而为Vpu拮抗Tetherin提供一个新思路。在本论文中,我们首先在稳定表达Vpu的293T细胞中探究Vpu对细胞内钙离子浓度的影响以及与电压依赖型钙离子通道的关系。我们在稳定表达Vpu的细胞中利用绿色荧光钙指示剂Fluo-4 AM标记细胞内的钙离子,利用倒置显微镜、分光光度仪以及流式细胞仪检测细胞内钙离子的浓度变化,检测到Vpu对细胞内的钙离子有上调作用;同时我们又通过VDCC拮抗剂以及RNA沉默VDCC的方法检测细胞内的钙离子浓度变化,来确定钙浓度变化的确是通过介导电压依赖型钙离子通道。其次,在本篇论文里,我们又进一步研究了钙离子作为媒介的下游通路对Vpu拮抗Tetherin的影响。我们利用了透射电子显微镜观察Tetherin是否通过其特殊的拓扑结构识别子代病毒出芽造成的细胞膜膜曲率变化,以及Vpu对这一过程的干扰和钙离子对其的影响;同时,我们还利用了Co-IP验证是否Tetherin与Gag物理相互结合,以及Vpu是否对此过程有影响,结果表明Vpu不干扰Tetherin定位于出芽位点的过程。除此之外,我们还尝试探究出Vpu可能通过提升细胞内钙离子浓度,抑制Tetherin的Hem ITAM磷酸化,进而影响其NF-κB信号传导活性,以及一系列通路中钙离子通道起到作用。最后,我们又对Vpu通过激活水解酶从而切割Tetherin的这种可能的机制进行了进一步研究。从基因水平和蛋白水平验证钙离子在Tetherin影响基质金属酶和Furin酶活性上的作用以及Tetherin潜在的Furin切割位点突变后对病毒释放的影响,结果表明Furin并不切割Tetherin,但有趣的是Tetherin却可能抑制Furin自身的活性。通过作用位点软件预测,我们发现信号肽酶对于Tetherin也可能有潜在切割作用,所以我们对此也做了初步探究。通过上述实验,我们更精细的探究了Vpu对于细胞内的钙离子浓度的影响通过何种方式介导,以及初步明确钙离子通道与拮抗Tetherin活性相关的下游分子机制,为Vpu拮抗Tetherin的机制的进一步阐明提供了崭新的方向。在未来,我们会对以上初步探究的各项通路综合起来,更全面的揭示出Vpu依赖钙离子拮抗Tetherin的途径。
[Abstract]:Terethin (BST-2, CD317) is a type II membrane protein with a special topological structure. Its structure includes the cytoplasmic tail end of N terminal, single pass membrane region, spiral region outside the cell, and phospholipid and inositol anchoring region of C terminal (potential secondary puncture zone). Tethin is now found to be able to embed the host cytoplasmic membrane and the nascent viral membrane to inhibit the release of the virus. HIV-1 accessory protein Vpu is an I-type membrane protein encoding 81 amino acids. Early studies have shown that Vpu has two major functions in the transmission of HIV-1, one of which is that Vpu can down-regulate the CD4 receptor on the cell surface and prevent the formation of gp160/ CD4 complex in virus infected cells. so that the host cell is infected with different viruses; and the Vpu can release the effect of the teethin (BST-2, CD317) captive virus, and promote the release of the progeny virus. The molecular mechanism of Vpu antagonizing the role of Tethin is still not completely clear at present. At first, it was reported that Vpu antagonized the mechanism of Tethin and the degradation of CD4 receptor, that is, through the combination of membrane helix region and Tethin, Tethin itself was degraded by ubiquitination enzyme and proteome pathway, so that it could not complete the physical system retention of the virus. However, subsequent studies confirmed that Vpu could also promote viral release without degrading, or reducing the amount of Tethin's expression on the surface of cells, suggesting that the theory was not complete. It was also reported that Tethin was able to specifically identify the viral replicon site, thus guessing that Vpu antagonized the role of Tethin to promote viral release by disrupting its recognition. There are some other reports showing that, in the presence of Vpu, Tethn can still be packaged into the progeny virus, but it is unable to play a mooring function, and the key molecular mechanism of Vpu antagonizing Tethin remains to be further elucidated. Vpu's own homologous oligomer forms a weakly selective potassium ion channel while Vpu can also interfere with TASK leading to depolarization of the host cell membrane, and we suspect that this depolarization phenomenon can trigger a voltage-dependent calcium ion channel, causing a rise in calcium ion concentration in the cell, This phenomenon has been preliminarily confirmed in previous studies. On the basis of this, we further confirm that Vpu causes intracellular calcium ion concentration to rise as a result of which voltage-dependent calcium ion channels trigger the correlation between this effect and its own ion channel activity. and the action of the relevant downstream pathway under the participation of calcium ions in the process of antagonizing Tethin by HIV-1Vpu, thereby providing a new idea for Vpu to antagonize Tethin. In this paper, we first explored the influence of Vpu on intracellular calcium ion concentration and the relationship with voltage-dependent calcium ion channels in 293T cells stably expressing Vpu. The calcium ions in cells were labeled with green fluorescent calcium indicator Fluo-4AM in the cells stably expressing Vpu, and the concentration of calcium ions in cells was detected by an inverted microscope, a spectrophotometer and a flow cytometer, and the up-regulation effect of Vpu on calcium ions in the cells was detected. At the same time, we detected the change of calcium ion concentration in the cells by means of VDCC antagonist and RNA silencing VDCC to determine that the change of calcium concentration is indeed by mediating the voltage-dependent calcium ion channel. Secondly, in this paper, we further study the influence of calcium ion as the downstream pathway of the medium on Vpu antagonist Tethin. We use the transmission electron microscope to observe whether Tethin has identified the change of membrane curvature of membrane membrane caused by sub-generation of virus by its special topological structure, and the interference of Vpu on the process and the effect of calcium ion on it; meanwhile, We also use Co-IP to verify whether Tethin and Gag are physically bonded to each other, and whether Vpu is affected by this process, and the results show that Vpu does not interfere with the process of Tethin to be located at the site. In addition, we tried to show that Vpu could inhibit the phosphorylation of Hem ITAM by increasing the intracellular calcium ion concentration, thus affecting the signal transduction activity of NF-Sepharose B and the role of calcium ion channels in a series of pathways. Finally, we further studied the possible mechanism of Vpu by activating the hydrolase to cut Tethin. The effect of calcium ions on the activity of matrix metalloenzymes and Furin enzymes and the effects of Tethin potential Furin cleavage site mutations on viral release were verified from gene levels and protein levels, and the results indicated that Furin did not cut Tethin, but it was interesting that Tethin could inhibit Furin's own activity. We have also made a preliminary inquiry about the potential cleavage of Tethin through the prediction of active site software. Through the above experiments, we examine the influence of Vpu on the concentration of calcium ions in the cells and the mechanism of downstream molecular mechanism related to the activity of Tethin, and provide a new direction for the further elucidation of the mechanism of Vpu antagonizing Tethin. In the future, we can comprehensively reveal the ways of Vpu-dependent calcium ion antagonizing Tethin.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.91

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