本文關(guān)鍵詞：HIV-1與病毒限制因子相互作用的新機(jī)制研究　出處：《中國科學(xué)院研究生院(武漢病毒研究所)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： HIV-1 Vpr APOBEC3G(A3G) Tetherin 復(fù)制

【摘要】：HIV-1輔助蛋白與宿主病毒限制因子的相互作用是HIV-1/AIDS研究領(lǐng)域的重要組成部分,明晰兩者間功能的相互拮抗對于更加深入地理解HIV-1致病機(jī)制和研發(fā)行之有效的HIV-1/AIDS治療策略至關(guān)重要。APOBEC3G(A3G)是細(xì)胞內(nèi)重要的天然免疫抗HIV-1因子,但其抑制病毒復(fù)制的功能可被HIV-1 Vif蛋白所拮抗。通過研究我們發(fā)現(xiàn),HIV-1的另一種輔助蛋白Vpr與A3G也可能存在密切的功能聯(lián)系,為此我們開展研究深入探討兩者相互作用的細(xì)節(jié)。首先我們通過應(yīng)用基于計算機(jī)的結(jié)構(gòu)匹配蛋白相互作用(protein interactions by structural matching,PRISM)算法預(yù)測出Vpr與A3G存在直接的相互作用,繼而通過實驗的方法驗證了兩者可在體內(nèi)直接結(jié)合形成復(fù)合體。其次我們研究了此相互作用的潛在功能意義,發(fā)現(xiàn)Vpr可顯著下調(diào)A3G包裝入新出芽HIV-1病毒粒子中的能力,此功能限制了A3G抗病毒功能的發(fā)揮,回復(fù)了A3G所介導(dǎo)的對HIV-1復(fù)制的抑制。進(jìn)一步的實驗證明了Vpr可顯著降低A3G的表達(dá),且此下調(diào)體現(xiàn)在蛋白水平上,與A3G的轉(zhuǎn)錄無關(guān),深入研究后我們發(fā)現(xiàn)Vpr誘導(dǎo)的A3G表達(dá)下調(diào)是通過依賴于Vpr BP的泛素-蛋白酶體降解途徑實現(xiàn)的。最后,通過突變體實驗我們證實了Vpr抑制A3G病毒粒子包裝歸因于上述Vpr誘導(dǎo)的A3G蛋白翻譯后降解。本研究揭示了Vpr與A3G的功能拮抗關(guān)系,這豐富了Vpr蛋白的功能多樣性,此外A3G可被不同的E3泛素連接酶復(fù)合體所誘導(dǎo)降解這一現(xiàn)象的發(fā)現(xiàn)表明HIV-1與細(xì)胞泛素連接酶復(fù)合體系統(tǒng)間復(fù)雜串?dāng)_(crosstalk)互作的存在,這提示今后為更加準(zhǔn)確地揭示HIV-1輔助蛋白與細(xì)胞限制因子間博弈的細(xì)節(jié),應(yīng)采取系統(tǒng)化的研究而不單單是只針對兩種蛋白的相互作用孤立地進(jìn)行實驗。Tetherin蛋白是定位于細(xì)胞膜系統(tǒng)上的另一種重要的HIV-1限制因子,以往圍繞Tetherin與HIV-1關(guān)系的研究主要集中在Tetherin滯留HIV-1病毒粒子于質(zhì)膜表面從而限制了病毒釋放和傳播這一功能上。我們前期在針對Vpu和Tetherin相互關(guān)系的課題研究中發(fā)現(xiàn)一個可重復(fù)的實驗現(xiàn)象,即Tetherin對胞內(nèi)HIV-1的復(fù)制可能存在前人未報道過的調(diào)控過程,為此我們展開進(jìn)一步的實驗以探索Tetherin對HIV-1復(fù)制的調(diào)控及其機(jī)制。首先我們發(fā)現(xiàn)了外源超量表達(dá)的Tetherin對HIV-1假病毒的單輪復(fù)制存在明顯的上調(diào)效應(yīng),且此上調(diào)存在一個有趣的現(xiàn)象,即低劑量Tetherin上調(diào)病毒復(fù)制的能力明顯,而劑量高時此上調(diào)效應(yīng)反而下降。然后我們證明了低劑量Tetherin對HIV-1基因組的轉(zhuǎn)錄也有顯著激活。隨后的實驗結(jié)果表明內(nèi)源組成型表達(dá)的Tetherin對HIV-1基因組的復(fù)制也存在顯著調(diào)控,且結(jié)果與外源轉(zhuǎn)染實驗相一致。緊接著,我們發(fā)現(xiàn)Tetherin具有顯著激活HIV-1 LTR啟動子的功能,且后續(xù)的實驗證明此激活效應(yīng)依賴于LTR上的NF-κB轉(zhuǎn)錄因子結(jié)合位點。本研究表明Tetherin蛋白可能通過NF-κB信號通路激活HIV-1 LTR啟動子的活性,進(jìn)而促進(jìn)了HIV-1基因組的復(fù)制,這可能是Tetherin除了限制HIV-1毒粒釋放之外的另一個潛在的重要功能。由于Tetherin可極低量表達(dá)于多種HIV-1靶標(biāo)細(xì)胞中,且是一種I型干擾素誘導(dǎo)蛋白,所以我們推測Tetherin增強(qiáng)HIV-1基因組復(fù)制的功能可體現(xiàn)在活化HIV-1潛伏感染上,通過激活HIV-1潛伏感染使得機(jī)體免疫系統(tǒng)啟動識別并清除病毒的過程。從此角度分析,Tetherin上調(diào)HIV-1基因組復(fù)制的能力是其發(fā)揮抗病毒活性的另一種體現(xiàn)。
[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.

【學(xué)位授予單位】：中國科學(xué)院研究生院(武漢病毒研究所)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R512.91

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