【作者】 刘琳；

【导师】 于湘晖；

【作者基本信息】 吉林大学 ， 生物工程（专业学位）， 2017， 硕士

【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。更多还原

【Abstract】 Tetherin is a type II trans-membrane protein which possess a unique topology.Tetherin has an N-terminal cytoplasmic tail,followed by a trans-membrane helix,a coiled-coil ecto domain,and a C-terminal GPI anchor（or potentially second membrane anchor region）.Tetherin has been found to incorporate its two membrane embedding regions into host cell plasma membrane and nascent viruses membrane thus form a physical tether between the virions and the host cell so as to prevent the release of the virions.HIV-1 accessory protein Vpu is an 81 amino acids type I trans-membrane protein,with a single N-terminal transmembrane helix,and a C-terminal cytoplasmic domain containing two amphipathic helices.Early study indicated that Vpu has two major functions during the spread of HIV in vivo.The first is the down-regulation of CD4 receptors from the cell surface,through the interaction between their cytoplasmic domains and subsequently inducing CD4 degradation.The second is to enhance the release of progeny HIV-1 virionsby eliminating the tether effect of host restriction factor Tetherin.Similarly with its CD4 down-regulation activity,originally Vpu was proposed to eliminate the physical tether effect of host restriction factor Tetherin,by binding to Tetherin via trans-membrane helix interaction and leading to its surface down-regulation and degradation through ubiquitination and proteasome system.However,later research pointed out that Vpu can enhance the progeny virions release without degradation and cell surface down-regulation of Tetherin.Further study showed that Vpu can interfere with the recruitment of Tetherin to the virions budding site.However,some other report also indicated that BST2 still can be detected in the virions even in the presence of Vpu,which suggest that some other mechanisms are likely still waiting to be found.Vpu might form oligomers and possess weak selective monovalent cation channel activity.Vpu could interact with host cell background potassium channel TASΚ,which is widely expressed and responsible for maintaining the resting membrane potential.Previously,we had proposed and found that Vpu could induce a slight augmentation of intracellular calcium level.In this thesis,we tried to further confirm whether the rise of intracellular calcium concentration induced by Vpu is due to the triggering of voltage dependent calcium channels and how this triggering happen.And what kind of role of calcium-dependent downstream pathways could play to mediate HIV-1 Vpu antagonism of Tetherin,so as to provide new insight into the mechanism for Vpu antagonism of Tetherin.We first investigate the effect of Vpu on intracellular Ca²⁺ and the relationship with voltage dependent calcium channel in 293 T cells which stable expressing Vpu.The charge of intracellular calcium concentration was detected by inverted microscope,fluorescent-spectrophotometer and flow cytometry.The result shows that Vpu could up-regulate the level of Ca²⁺ in cells.We aslo detected the change of intracellular calcium concentration by VDCC antagonist and RNA silencingmethod,to determine the change of calcium concentration is mediated by voltage dependent calcium channel.Second,in this thesis,we further studied the effect of calcium ion mediated downstream pathway on Vpu antagonism of Tetherin.We used transmission electron microscope to observe whether Tetherin identify progeny virus budding site caused by its special topology,and interference of Vpu to this process and the effect of calcium ions on it.At the same time,we also used the Co-ip to verify whether Tetherin and Gag physical interaction,and whether Vpu has an impact on the process.The results showed that Vpu did not interfere with the localization of Tetherin in budding sites.In addition,we tried to explore the possible role of Vpu induced intracellular Ca²⁺ concentration on inhibiting the phosphorylation of Hem ITAM,and the further impact on the NF-κB signaling activity of Tetherin.In the end,we did some preliminary studies on the possible mechnism that Vpu might counteract Tetherin though activating certain cutting enzymes via calcium signaling.We explored the role of calcium ions on the activityand expression of matrix metalloproteinases and Furin attranscription and protein level.Although in contrast to our hypothesis,our result indicated that Furin does not cut Tetherin,we found some intersting phenomena that Tetherin might inhibit Furin.We found that SPP may also play a role in cutting the Tetherin through the prediction of the action site software,so we made a preliminary study on it.Through this thesis study,we achieved a more detailed understanding of the effect of Vpu on intracellular calcium concentration,and the possible downstream mechanism involved in Vpu’s counteraction of Tetherin,provided a new direction for the study of themechanism of Vpu antagonism to Tetherin.In the future,we will integrate the above preliminary exploration of the different pathways in to a more comprehensive understanding to reveal the Vpu antagonism to Tetherin mediated by calcium channel.更多还原