HIV-1与病毒限制因子相互作用的新机制研究

发布时间：2018-01-02 06:21

本文关键词：HIV-1与病毒限制因子相互作用的新机制研究　出处：《中国科学院研究生院(武汉病毒研究所)》2016年博士论文　论文类型：学位论文

更多相关文章： HIV-1 Vpr APOBEC3G(A3G) Tetherin 复制

【摘要】：HIV-1辅助蛋白与宿主病毒限制因子的相互作用是HIV-1/AIDS研究领域的重要组成部分,明晰两者间功能的相互拮抗对于更加深入地理解HIV-1致病机制和研发行之有效的HIV-1/AIDS治疗策略至关重要。APOBEC3G(A3G)是细胞内重要的天然免疫抗HIV-1因子,但其抑制病毒复制的功能可被HIV-1 Vif蛋白所拮抗。通过研究我们发现,HIV-1的另一种辅助蛋白Vpr与A3G也可能存在密切的功能联系,为此我们开展研究深入探讨两者相互作用的细节。首先我们通过应用基于计算机的结构匹配蛋白相互作用(protein interactions by structural matching,PRISM)算法预测出Vpr与A3G存在直接的相互作用,继而通过实验的方法验证了两者可在体内直接结合形成复合体。其次我们研究了此相互作用的潜在功能意义,发现Vpr可显著下调A3G包装入新出芽HIV-1病毒粒子中的能力,此功能限制了A3G抗病毒功能的发挥,回复了A3G所介导的对HIV-1复制的抑制。进一步的实验证明了Vpr可显著降低A3G的表达,且此下调体现在蛋白水平上,与A3G的转录无关,深入研究后我们发现Vpr诱导的A3G表达下调是通过依赖于Vpr BP的泛素-蛋白酶体降解途径实现的。最后,通过突变体实验我们证实了Vpr抑制A3G病毒粒子包装归因于上述Vpr诱导的A3G蛋白翻译后降解。本研究揭示了Vpr与A3G的功能拮抗关系,这丰富了Vpr蛋白的功能多样性,此外A3G可被不同的E3泛素连接酶复合体所诱导降解这一现象的发现表明HIV-1与细胞泛素连接酶复合体系统间复杂串扰(crosstalk)互作的存在,这提示今后为更加准确地揭示HIV-1辅助蛋白与细胞限制因子间博弈的细节,应采取系统化的研究而不单单是只针对两种蛋白的相互作用孤立地进行实验。Tetherin蛋白是定位于细胞膜系统上的另一种重要的HIV-1限制因子,以往围绕Tetherin与HIV-1关系的研究主要集中在Tetherin滞留HIV-1病毒粒子于质膜表面从而限制了病毒释放和传播这一功能上。我们前期在针对Vpu和Tetherin相互关系的课题研究中发现一个可重复的实验现象,即Tetherin对胞内HIV-1的复制可能存在前人未报道过的调控过程,为此我们展开进一步的实验以探索Tetherin对HIV-1复制的调控及其机制。首先我们发现了外源超量表达的Tetherin对HIV-1假病毒的单轮复制存在明显的上调效应,且此上调存在一个有趣的现象,即低剂量Tetherin上调病毒复制的能力明显,而剂量高时此上调效应反而下降。然后我们证明了低剂量Tetherin对HIV-1基因组的转录也有显著激活。随后的实验结果表明内源组成型表达的Tetherin对HIV-1基因组的复制也存在显著调控,且结果与外源转染实验相一致。紧接着,我们发现Tetherin具有显著激活HIV-1 LTR启动子的功能,且后续的实验证明此激活效应依赖于LTR上的NF-κB转录因子结合位点。本研究表明Tetherin蛋白可能通过NF-κB信号通路激活HIV-1 LTR启动子的活性,进而促进了HIV-1基因组的复制,这可能是Tetherin除了限制HIV-1毒粒释放之外的另一个潜在的重要功能。由于Tetherin可极低量表达于多种HIV-1靶标细胞中,且是一种I型干扰素诱导蛋白,所以我们推测Tetherin增强HIV-1基因组复制的功能可体现在活化HIV-1潜伏感染上,通过激活HIV-1潜伏感染使得机体免疫系统启动识别并清除病毒的过程。从此角度分析,Tetherin上调HIV-1基因组复制的能力是其发挥抗病毒活性的另一种体现。
[Abstract]:The interaction between HIV-1 protein and host auxiliary virus restriction factor is an important part of the research field of HIV-1/AIDS antagonism between the two clear function for more in-depth understanding of.APOBEC3G is essential for HIV-1 pathogenesis and treatment strategies of HIV-1/AIDS (A3G) is the development of effective natural immune cells important anti HIV-1 factor, but the inhibition of viral replication the function of HIV-1 Vif protein can be antagonized. Through research we found that HIV-1 is another auxiliary protein of Vpr and A3G may be closely related to function, we carry out in-depth research on the interaction between the two details. First we through the application of protein interaction, based on the structure of the computer (protein interactions by structural matching. PRISM) algorithm to predict the direct interaction between Vpr and A3G, and then verified by experimental methods two Can be directly combined to form a complex in vivo. Secondly, we study the potential functional significance of this interaction, we found that Vpr significantly decreased A3G packaged into virus particles budding new HIV-1, this function limits the A3G antiviral function, responded to the A3G mediated inhibition of HIV-1 replication in further experiments. It proves that Vpr significantly reduced the expression of A3G, and this is reflected in the down-regulation of the protein level, has nothing to do with the transcription of A3G, after further study we found the expression of Vpr induced down-regulation of A3G is dependent on Vpr BP through the ubiquitin proteasome pathway implementation. Finally, through experiments we confirmed the inhibition of Vpr mutant A3G viral particle package was ascribed to the Vpr induced A3G protein post-translational degradation. This study reveals the antagonistic relationship between Vpr and A3G function, which enriches the function of Vpr protein diversity, in addition to A3G by different E3 Discovery of ubiquitin ligase complex induced degradation of this phenomenon indicates that HIV-1 and ubiquitin ligase complex cells system complex crosstalk (crosstalk) interactions exist, suggesting that in the future to more accurately reveal the HIV-1 accessory protein and cell factor limiting game details, should take a systematic study and not merely the interaction for the two kinds of protein isolation experiments of.Tetherin protein is another important factor limiting HIV-1 located on the cell membrane system, previous research on Tetherin's relationship with HIV-1 mainly in Tetherin stranded HIV-1 virus particles on the plasma membrane surface so as to limit the spread of the virus and release function. We found a pre repeatable experimental phenomena in the research for the relationship between Vpu and Tetherin, Tetherin on the replication of intracellular HIV-1 may have not reported previous The regulation process of the road, so we carried out further experiments to explore the regulation of Tetherin on HIV-1 and its mechanism of replication. First we found overexpression of Tetherin on exogenous HIV-1 pseudovirion single round replication has obvious effect of raising, and this has raised an interesting phenomenon, namely the ability of low dose Tetherin increased virus replication obviously, the effect of raising and high doses decreased. Then we prove that the transcription of low dose Tetherin on the HIV-1 genome was also activated. Then the experimental results show that replication of endogenous constitutively expressed Tetherin of HIV-1 genome was obvious regulation, and the results are consistent with the exogenous transfection experiments. Then, we found that Tetherin has a significant activation of HIV-1 LTR promoter function, and prove the following experiment NF- kappa B transcription factor activation of this effect was dependent on the LTR binding site Point. This study suggests that Tetherin protein may activate HIV-1 promoter activity of LTR via NF- B pathway, and then promote the replication of the HIV-1 genome, which may be Tetherin in addition to another potentially important function beyond the limit of HIV-1 virion release. Because Tetherin can be a very low level of expression in a variety of HIV-1 target cells. And is a kind of type I interferon induced protein, so we speculate that Tetherin enhanced HIV-1 genome replication function can be reflected in the activation of latent infection of HIV-1, through the activation of HIV-1 latent infection the immune system starts to recognize and eliminate the virus. From the angle of analysis, Tetherin upregulation of HIV-1 genome replication ability is another manifestation of the play antiviral activity.

【学位授予单位】：中国科学院研究生院(武汉病毒研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R512.91

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