本文關(guān)鍵詞：不同種屬慢病毒Vif蛋白影響HIV-1增殖和Vif蛋白與HIV-1 Gag蛋白相互作用分子機制的研究　出處：《吉林大學》2017年博士論文　論文類型：學位論文

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【摘要】：病毒感染性因子(Viral infectivity factor,Vif),作為一種慢病毒輔助因子,在不同種屬慢病毒中是一類保守的蛋白。研究表明,Vif蛋白通過抵抗宿主限制因子人載脂蛋白B m RNA編輯催化多肽(Apolipoprotein B m RNA-editing catalytic polypeptide-like protein,APOBEC)家族來協(xié)助病毒在非允許性細胞中的復制。宿主的APOBEC蛋白家族是一類活化誘導的胞嘧啶脫氨基酶,可以介導病毒基因組發(fā)生致死性突變,具有極為有效的抗病毒功能;而Vif蛋白可以在宿主細胞內(nèi)利用宿主的泛素蛋白酶復合體誘導APOBEC家族蛋白的泛素化降解,抵抗宿主對病毒的防御。由于不同種屬宿主具有不盡相同的APOBEC家族蛋白,對病毒造成的生存選擇壓力不同,這些慢病毒的Vif蛋白也進化出了不同的策略來克服宿主的限制,因此,長期的進化使得不同種屬病毒的Vif蛋白的功能存在相互隔離:雖然都是Vif拮抗APOBEC蛋白,但是牛免疫缺陷病毒(Bovine immunodeficiency virus,BIV)的Vif不能抵抗人和猴的APOBEC蛋白,而人免疫缺陷病毒(Human immunodeficiency virus type 1,HIV-1)和猴免疫缺陷病毒(Simian immunodeficiency virus,SIV)的Vif也不能夠拮抗牛的APOBEC家族蛋白。雖然如此,不同種屬的慢病毒Vif蛋白在進化過程中是否也保留了某些共性?既往研究中還未見報道。本研究采用細胞生物學、分子生物學、生物化學和病毒學等相關(guān)技術(shù),首次發(fā)現(xiàn):即使在沒有抗病毒因子APOBEC蛋白存在的情況下,BIV Vif可以顯著地抑制HIV-1的產(chǎn)生、感染性和增殖。這一發(fā)現(xiàn)具有重要的意義:一個來源于不同種屬慢病毒的輔助因子可以抵抗HIV-1,并且這種來源不同的輔助因子BIV Vif與HIV-1 Vif在功能上并沒有關(guān)聯(lián)性,是相互獨立的,這無疑為抗HIV-1提供了新的思路。一方面,BIV Vif本身可能成為一種潛在的抗病毒因子;另一方面,BIV Vif又是如何抵抗HIV-1的?深入研究其抗病毒作用機制也將為宿主抵抗病毒的研究提供更多線索。于是我們進一步的對其中的分子機制進行了探究,對于受到抑制的病毒進行了詳細的分析,研究結(jié)果表明:首先,BIV Vif能夠在一定程度上抑制病毒的產(chǎn)量;但是通過對病毒產(chǎn)量和病毒感染能力的詳細分析,這并不是BIV Vif抵抗HIV-1的最主要原因;第二,更重要的是,BIV Vif還能夠直接抑制新產(chǎn)生病毒的感染性;第三,更加深入的研究發(fā)現(xiàn),BIV Vif可以抑制出芽病毒中組織特異性抗原(或稱衣殼前體蛋白,Group specific antigen,Gag/Pr55Gag)的剪切,從而干擾了病毒復制周期中至關(guān)重要的一個環(huán)節(jié)——病毒的成熟。病毒成熟的核心過程即為出芽病毒中的Pr55Gag按照嚴格順序和位點被病毒蛋白酶剪切為組成病毒顆粒的各個層面的衣殼蛋白,包括:基質(zhì)(Matrix,MA/p17)、衣殼(Capsid,CA/p24)、核衣殼(Nucleocapsid,NC/p7)和p6,另外還有兩個位于CA和NC、NC和p6中間的間隔肽1(Spacer peptide,SP1/p2)和間隔肽2(Spacer peptide,SP2/p1)。如上各個部分不僅對于病毒的結(jié)構(gòu)組成缺一不可,并且需要按照嚴格順序進行剪切產(chǎn)生。而BIV Vif可以抑制Pr55Gag剪切的第一步,NC/p2間的剪切。因此,BIV Vif能夠顯著地抑制HIV-1的感染性。這一部分研究初步闡明了BIV Vif抑制HIV-1的內(nèi)在原因。接下來,一個重要的問題是為什么只有BIV Vif可以抑制HIV-1,而HIV-1或是SIV的Vif不能?根據(jù)我們的研究結(jié)果顯示:相比于等量表達的HIV-1 Vif或是SIV Vif,BIV Vif能夠更強地結(jié)合Pr55Gag。同時,不能結(jié)合Pr55Gag的BIV Vif突變體失去了抑制HIV-1的能力。這就解釋了雖然HIV-1、SIV、BIV Vif都可以與HIV-1 Pr55Gag相互作用,但是因為BIV Vif與Pr55Gag的結(jié)合能力最強,所以BIV Vif可以顯著地抑制HIV-1,而HIV-1或是SIV的Vif因與Pr55Gag的結(jié)合能力弱而不能抑制HIV-1。這部分研究進一步闡明了BIV Vif抑制HIV-1的分子機制。同時,不同種屬的慢病毒Vif蛋白,包括HIV-1、SIV、BIV Vif都保留了與HIV-1 Pr55Gag相互作用的能力。而這種結(jié)合的保守性提示了對于病毒來說Vif與Pr55Gag的結(jié)合非常重要,可能對病毒的復制周期具有重要功能。Vif與HIV-1 Pr55Gag的相互作用無疑是BIV Vif拮抗病毒的核心環(huán)節(jié),并且,這個結(jié)合在不同種屬Vif間的保守性也暗示了其對病毒可能具有重要功能。因此,接下來我們利用分子克隆技術(shù)結(jié)合免疫共沉淀技術(shù)探索了二者結(jié)合的結(jié)合決定域;實驗結(jié)果表明:對于HIV-1的Pr55Gag來說,Pr55Gag中對于病毒組裝和成熟起重要作用的CA碳末端和p2區(qū)域?qū)r55Gag與Vif的結(jié)合必不可少;對于HIV Vif和BIV Vif,它們的氮末端,尤其是第30位酪氨酸(Tyrosine,Y)和第33位精氨酸(Arginine,R)或者組氨酸(Histidine,H),對于Vif-Pr55Gag相互結(jié)合作用至關(guān)重要。Vif與HIV-1 Pr55Gag相互作用的保守決定域的鑒定對于后續(xù)BIV Vif拮抗病毒的應(yīng)用研究以及后續(xù)研究Vif與HIV-1 Pr55Gag相互作用的生物學功能具有重要的意義。BIV Vif在上述人胚腎細胞系HEK 293T體系中,有效地抵抗了HIV-1的復制和增殖。那么更重要的是,這種抗病毒作用在HIV的靶細胞CD4陽性T細胞中是否仍然如此有效呢?接下來,我們在CD4陽性T細胞系中穩(wěn)定表達BIV Vif,實驗證明,在BIV Vif的表達不影響細胞周期和增殖的前提下,BIV Vif在CD4陽性T細胞中能夠有效抑制HIV-1的增殖。綜上所述,本研究利用現(xiàn)代生物學技術(shù),首次發(fā)現(xiàn)牛艾滋病病毒輔助蛋白Vif可以作為一種外源的抗病毒因子,能夠顯著抑制HIV-1的產(chǎn)生、感染性和增殖;不僅如此,我們還對BIV Vif拮抗病毒的原因和分子機制進行了深入的研究和探討,BIV Vif通過在細胞內(nèi)有效地結(jié)合HIV-1 Pr55Gag,抑制了病毒出芽后的成熟過程,從而顯著地抑制了HIV-1感染性、復制和增殖;同時我們首次發(fā)現(xiàn)HIV-1 Vif、SIV Vif和BIV Vif都可以與HIV-1 Pr55Gag相互結(jié)合,這是首次在真核細胞中鑒定出二者的相互結(jié)合;更進一步,我們還鑒定出了二者相互結(jié)合的決定域。這些發(fā)現(xiàn)無論是為后續(xù)相關(guān)研究的進行還是抗病毒治療新策略的開發(fā)都提供了重要的理論基礎(chǔ)和潛在靶點。
[Abstract]:Viral infectivity factor (Vif), a kind of lentivirus auxiliary factor, is a kind of conservative protein in lentiviruses of different species. Studies have shown that Vif protein is able to assist virus replication in non permissive cells by resisting the host limiting factor, human apolipoprotein B m RNA editing catalytic polypeptide (Apolipoprotein B m RNA-editing catalytic polypeptide-like protein). The host of the APOBEC family is a class of activation induced cytosine deaminase, mediated by viral genome occurred lethal mutation, has extremely effective antiviral function; ubiquitination and degradation of Vif protein by ubiquitin proteasome can induce the host family of APOBEC proteins in host cells, resistance of host defense against virus. Because of the different species have different host APOBEC protein family, choice of survival pressure caused by viruses, these slow virus Vif protein has also evolved different strategies to overcome host restriction, therefore, the evolution of the different species of virus Vif protein function are isolated from each other though they are: the Vif antagonist APOBEC protein, but the bovine immunodeficiency virus (Bovine immunodeficiency, virus, BIV) Vif can not resist the monkey and human APOBEC protein, and human immunodeficiency virus (Human immunodeficiency virus type 1, HIV-1) and simian immunodeficiency virus (Simian immunodeficiency, virus, SIV) Vif is not able to antagonize bovine APOBEC protein family. However, does the lentivirus Vif protein in different species have some similarities in the evolution process and have not been reported in previous studies. In this study, cell biology, molecular biology, biochemistry and virology were used for the first time. It was first discovered that BIV Vif can significantly inhibit HIV-1 production, infection and proliferation even without the presence of antiviral factor APOBEC protein. This finding has important implications: a cofactor derived from different species of lentivirus can resist HIV-1, and the different sources of BIV Vif and HIV-1 Vif cofactor in function and no relevance is independent of each other, which undoubtedly provides a new way for anti HIV-1. On the one hand, BIV Vif itself may become a potential antiviral factor. On the other hand, how BIV Vif resists HIV-1? Further study of its antiviral mechanism will provide more clues for host's research on virus resistance. So we made further exploration of the molecular mechanism for the inhibition of virus, are analyzed in detail. The results show that: firstly, BIV Vif can inhibit the virus to a certain extent, the yield; but through a detailed analysis of the yield and virus infection ability, the main reason for this is not BIV Vif resistance HIV-1; second, more importantly, BIV Vif also can inhibit the infection of new virus directly; third, further research found that BIV Vif can inhibit the virus budding tissue specific antigen (or capsid precursor protein, Group specific antigen, Gag/Pr55Gag) of the shear, which interfere with a - the virus link in the virus replication cycle is mature. Virus core maturation process is budding virus in Pr55Gag according to the strict order and sites of virus protease cleavage for each level consists of the virion capsid protein, including: matrix (Matrix, MA/p17), (Capsid, CA/p24), capsid Nucleocapsid (Nucleocapsid, NC/p7) and P6, another two in spacer peptide CA and NC, NC and P6 in the middle of 1 (Spacer peptide, SP1/p2 (Spacer) and spacer peptide 2 peptide, SP2/p1). The above parts are not only dispensable for the structure of the virus, but also need to be cut in strict order. And BIV Vif can inhibit the first step of Pr55Gag shearing, the shear between NC/p2. Therefore, BIV Vif can significantly inhibit the infection of HIV-1. This part of the study preliminarily elucidated the intrinsic reasons for the inhibition of HIV-1 by BIV Vif. Next, an important question is why only BIV Vif can inhibit HIV-1, while HIV-1 or Vif of SIV can not. According to our research results, compared with the HIV-1 Vif or SIV Vif with equal expression, BIV can be combined more strongly. At the same time, the BIV Vif mutant was not combined with Pr55Gag to lose the ability to inhibit HIV-1. This explains that although HIV-1, SIV and BIV Vif can interact with HIV-1 Pr55Gag, but BIV BIV can significantly inhibit the Pr55Gag because BIV Vif has the strongest binding capacity with Pr55Gag. This part of the study further elucidated the molecular mechanism of BIV Vif inhibition of HIV-1. At the same time, the lentivirus Vif proteins of different species, including HIV-1, SIV, and BIV Vif, retain their ability to interact with HIV-1 Pr55Gag. The conservatism of this combination suggests that the combination of Vif and Pr55Gag is very important for the virus, and may have an important function for the replication cycle of the virus. The interaction between Vif and HIV-1 Pr55Gag is undoubtedly the core part of BIV Vif antagonistic virus. Moreover, the conservatism of this binding among different Vif species also implies that it may play an important role in virus. Therefore, we use molecular cloning technique combined with immune technology explores the combination of decision domain with two co precipitation; experimental results show that: for HIV-1 Pr55Gag, Pr55Gag for virus assembly and maturation indispensable important role of CA and P2 C-terminal region of the Pr55Gag and Vif combination of Vif and BIV Vif for HIV; N, the end of their, especially tyrosine thirtieth (Tyrosine, Y) and thirty-third arginine (Arginine, R) or histidine (Histidine, H), the Vif-Pr55Gag combination is crucial. Identification of the conservative domain of the interaction of Vif and HIV-1 Pr55Gag for subsequent BIV Vi
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R373

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1 鄭雯雯;不同種屬慢病毒Vif蛋白影響HIV-1增殖和Vif蛋白與HIV-1 Gag蛋白相互作用分子機制的研究[D];吉林大學;2017年

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