【摘要】：病毒感染性因子Vif(Viral infectivity factor)是HIV-1(Human immunodeficiency virus type 1)病毒的輔助蛋白之一,也是最早被發(fā)現(xiàn)具有拮抗宿主防御機(jī)制的HIV-1病毒功能性蛋白。并且在HIV-1的輔助蛋白中其拮抗宿主對(duì)病毒的防御機(jī)制也最先被揭示。Vif能夠與宿主內(nèi)的細(xì)胞因子Cul5,Elo B,Elo C,Rbx組裝成Cullin-Ring E3泛素復(fù)合物,結(jié)合并誘導(dǎo)APOBEC3G、APOBEC3F等APOBEC3家族的蛋白泛素化降解。這一功能也完美的解釋了在一些細(xì)胞系中,Vif能夠促進(jìn)HIV-1病毒的感染性的原因。隨著CBFβ(core binding factorβ)作為Vif-Cullin-Ring E3復(fù)合物重要的輔助因子這一重要理論的提出,人們對(duì)該復(fù)合物的組裝過(guò)程有了更加深入的了解。在CBFβ缺失的情況下,Vif只能夠和Elo C/Elo B結(jié)合,不能夠與Cul5結(jié)合,從而使E3復(fù)合物的組裝有缺陷,不具有降解APOBEC3家族的蛋白的功能。即CBFβ能促進(jìn)HIV-1 Vif蛋白降解靶蛋白。除此之外,研究還發(fā)現(xiàn)CBFβ也能夠在原核細(xì)胞大腸桿菌E.coli中穩(wěn)定Vif蛋白,提高Vif蛋白的可溶性,從而得到Vif蛋白的晶體結(jié)構(gòu)。在天然情況下,CBFβ是調(diào)控宿主轉(zhuǎn)錄的關(guān)鍵性因子,它與RUNX(CBFα)結(jié)合,增強(qiáng)RUNX與DNA的親和性,特別針對(duì)于細(xì)胞內(nèi)一些關(guān)鍵的啟動(dòng)子和增強(qiáng)子,如巨噬細(xì)胞集落刺激因子MCSFR(macrophage colony-stimulating factor receptor)。CBFβ/RUNX形成異源二聚體能夠影響人源細(xì)胞中很多基因的轉(zhuǎn)錄水平,從而調(diào)控細(xì)胞的分化和增殖。此外,Vif誘導(dǎo)細(xì)胞G2/M期阻滯以及Vif降解APOBEC3其他成員的選擇性也成為了領(lǐng)域的研究熱點(diǎn)。本論文圍繞Vif、CBFβ、RUNX、APOBEC3H(A3H)之間相互聯(lián)系進(jìn)行了細(xì)致的研究和討論,其中包括三個(gè)部分:我們?cè)诘谝徊糠謱?duì)于CBFβ參與Vif或RUNX功能的關(guān)鍵功能區(qū)域進(jìn)行了細(xì)致的研究。通過(guò)檢測(cè)CBFβ的N端和C端截短突變體,我們證明CBFβ15-126是幫助Vif降解A3G的最短功能區(qū)。CBFβLoop3的突變體對(duì)于RUNX1c介導(dǎo)的基因轉(zhuǎn)錄沒(méi)有影響,但是Loop3前6個(gè)氨基酸的點(diǎn)突變抑制Vif對(duì)于A3G的降解。綜上所述,CBFβLoop3是幫助Vif功能的重要區(qū)域,同時(shí)其突變不影響CBFβ在細(xì)胞內(nèi)的正常功能。綜上,Loop3可以作為設(shè)計(jì)抗HIV-1病毒藥物的理想作用靶點(diǎn)。Vif與APOBEC3F/G之間的相互作用已經(jīng)被廣泛并且全面的研究,而關(guān)于Vif-A3H的結(jié)合相關(guān)信息鮮有報(bào)道。APOBEC3H在人類(lèi)的細(xì)胞中有很多亞型,其中一些亞型,例如hap II,能夠抑制HIV-1病毒的復(fù)制。在第二部分中,我們證明了不同于APOBEC3G/F,只有一些HIV-1亞型的Vif蛋白能夠誘導(dǎo)A3H hap II的泛素化降解。位于HIV-1 Vif 39和49位的氨基酸,突變之后能夠顯著的降低Vif對(duì)A3H hap II降解的能力,但是依舊維持降解APOBEC3F/G的功能。這表明Vif與A3H hap II結(jié)合/降解的區(qū)域不同于Vif結(jié)構(gòu)中A3F/G的功能區(qū)。Vif蛋白除了能夠降解APOBEC3家族蛋白外,還有另外一個(gè)重要的功能,即作為細(xì)胞周期調(diào)控因子,誘導(dǎo)宿主細(xì)胞G2/M期阻滯。而這一功能經(jīng)我們實(shí)驗(yàn)證明也需要CBFβ以及Vif組裝的E3復(fù)合物。有趣的是,與Vif對(duì)于A3H hap II降解功能相似,只有部分Vif蛋白具有能夠誘導(dǎo)細(xì)胞周期G2/M阻滯的能力。比如HIV-1 HXB2亞型的Vif蛋白不能夠誘導(dǎo)細(xì)胞G2/M期阻滯,而HIV-1 NL4-3亞型的Vif蛋白則具有這個(gè)功能。在第三部分中,我們通過(guò)比較HXB2與NL4-3 Vif蛋白的氨基酸序列,發(fā)現(xiàn)位于31,33,36,47和50位氨基酸對(duì)于Vif誘導(dǎo)細(xì)胞G2/M期阻滯很重要,如果將HXB2 Vif上這5個(gè)位點(diǎn)的氨基酸突變成NL4-3 Vif位置上相對(duì)應(yīng)的氨基酸,則賦予了其誘導(dǎo)細(xì)胞周期G2/M阻滯的能力。實(shí)驗(yàn)進(jìn)一步證明,Vif蛋白對(duì)于A3H hap II降解或者誘導(dǎo)細(xì)胞G2//M期阻滯這兩個(gè)顯著的功能是具有選擇性的,不能共存。而這一選擇性也表明Vif蛋白仍具有一些潛在的新的機(jī)制還有待進(jìn)一步挖掘。綜上所述,本文對(duì)CBFβ蛋白結(jié)構(gòu)上關(guān)鍵氨基酸進(jìn)行掃描,通過(guò)功能實(shí)驗(yàn)發(fā)現(xiàn)了CBFβ影響Vif降解A3G或者參與RUNX功能的重要功能區(qū);同時(shí)進(jìn)一步探討了CBFβ影響Vif調(diào)控細(xì)胞周期的分子機(jī)制;最后證明Vif在一定篩選壓力下,通過(guò)選擇性的拮抗A3H抗病毒因子或者誘導(dǎo)細(xì)胞G2/M期阻滯來(lái)增強(qiáng)病毒的生存能力。進(jìn)一步完善了對(duì)于Vif蛋白本身的功能和行使功能的分子機(jī)制的研究。
[Abstract]:Viral infectivity factor (Vif) is one of the auxiliary proteins of HIV-1 virus, and it is also the first functional protein of HIV-1 virus that has been found to antagonize host defense mechanism. The ability to assemble Cullin-Ring E3 ubiquitin complexes with cytokines Cul5, Elo B, Elo C, and Rbx in host cells to bind to and induce ubiquitination of APOBEC3 family proteins such as APOBEC3G, APOBEC3F. This function also perfectly explains why Vif can promote HIV-1 infection in some cell lines. With CBFbeta (core binding factor binding factor) As an important auxiliary factor of Vif-Cullin-Ring E3 complex, the assembling process of Vif-Cullin-Ring E3 complex has been deeply understood. In the absence of CBF-beta, Vif can only bind to Elo C/Elo B, but can not bind to Cu5, which makes the assembling of E3 complex defective and does not degrade APOBEC3 family. In addition, it was also found that CBFbeta could stabilize the Vif protein in E. coli and enhance the solubility of Vif protein, thus obtaining the crystal structure of Vif protein. X (CBFalpha) binding enhances the affinity of RUNX to DNA, especially for some key promoters and enhancers in cells, such as macrophage colony-stimulating factor receptor (MCSFR). The formation of heterodimers of CBFbeta/RUNX can affect the transcription level of many genes in human cells, thereby regulating the cell's transcription. In addition, the G2/M phase arrest induced by Vif and the selectivity of other members of Vif-degrading APOBEC3 have also become the focus of research in this field. This paper focuses on the relationship among Vif, CBF-beta, RUNX, APOBEC3H (A3H), including three parts: in the first part, we discuss the involvement of CBF-beta in Vif or RUNX. CBF beta 15-126 was proved to be the shortest functional region to help Vif degrade A3G by detecting N-terminal and C-terminal truncated mutants of CBF beta. The mutant of CBF beta Loop3 had no effect on RUNX1c-mediated gene transcription, but point mutation of the first six amino acids of Loop3 inhibited Vif's degradation of A3G. In summary, CBF-beta Loop3 is an important region that helps Vif function, and its mutation does not affect the normal function of CBF-beta in cells. In summary, Loop3 can be an ideal target for designing anti-HIV-1 drugs. The interaction between Vif and APOBEC3F/G has been extensively and comprehensively studied, and information about the binding of Vif-A3H has been obtained. There are few reports. APOBEC3H has many subtypes in human cells, some of which, such as hap II, can inhibit the replication of HIV-1 virus. In the second part, we demonstrated that unlike APOBEC 3G/F, only some of the HIV-1 subtypes of Vif proteins can induce ubiquitination degradation of A3H hap II. Amino acids at sites 39 and 49 of HIV-1 Vif, mutations. Vif can significantly reduce the ability of Vif to degrade A3H hap II, but still maintain the function of degrading APOBEC3F/G. This indicates that the binding/degrading region of Vif to A3H hap II is different from that of A3F/G in Vif structure. Interestingly, similar to Vif's ability to degrade A3H hap II, only some Vif proteins have the ability to induce G2/M arrest of cell cycle. For example, the Vif protein of HIV-1 HXB2 subtype cannot induce fineness. In the third part, by comparing the amino acid sequences of HXB2 and NL4-3 Vif proteins, we found that amino acids at positions 31, 33, 36, 47 and 50 were important for Vif-induced G2/M phase arrest, if the amino acids at these five sites on HXB2 Vif were mutated into NL4-3 Vif sites. The presence of corresponding amino acids endowed Vif with the ability to induce G2/M arrest of cell cycle. It was further demonstrated that Vif protein was selective and could not coexist with A3H hap II degradation or G2/M phase arrest. In summary, the key amino acids in the structure of CBF-beta protein were scanned, and the important functional regions of CBF-beta affecting the degradation of A3G or participating in RUNX function were found by functional experiments. The molecular mechanism of CBF-beta affecting the regulation of cell cycle by Vif was further explored. Vif proteins can selectively antagonize A3H antiviral factors or induce G2/M phase arrest to enhance the viability of the virus.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R373

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