不同种属慢病毒Vif蛋白影响HIV-1增殖和Vif蛋白与HIV-1 Gag蛋白相互作用分子机制的研究

发布时间：2017-12-27 10:17

本文关键词：不同种属慢病毒Vif蛋白影响HIV-1增殖和Vif蛋白与HIV-1 Gag蛋白相互作用分子机制的研究　出处：《吉林大学》2017年博士论文　论文类型：学位论文

【摘要】：病毒感染性因子(Viral infectivity factor,Vif),作为一种慢病毒辅助因子,在不同种属慢病毒中是一类保守的蛋白。研究表明,Vif蛋白通过抵抗宿主限制因子人载脂蛋白B m RNA编辑催化多肽(Apolipoprotein B m RNA-editing catalytic polypeptide-like protein,APOBEC)家族来协助病毒在非允许性细胞中的复制。宿主的APOBEC蛋白家族是一类活化诱导的胞嘧啶脱氨基酶,可以介导病毒基因组发生致死性突变,具有极为有效的抗病毒功能;而Vif蛋白可以在宿主细胞内利用宿主的泛素蛋白酶复合体诱导APOBEC家族蛋白的泛素化降解,抵抗宿主对病毒的防御。由于不同种属宿主具有不尽相同的APOBEC家族蛋白,对病毒造成的生存选择压力不同,这些慢病毒的Vif蛋白也进化出了不同的策略来克服宿主的限制,因此,长期的进化使得不同种属病毒的Vif蛋白的功能存在相互隔离:虽然都是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也不能够拮抗牛的APOBEC家族蛋白。虽然如此,不同种属的慢病毒Vif蛋白在进化过程中是否也保留了某些共性?既往研究中还未见报道。本研究采用细胞生物学、分子生物学、生物化学和病毒学等相关技术,首次发现:即使在没有抗病毒因子APOBEC蛋白存在的情况下,BIV Vif可以显著地抑制HIV-1的产生、感染性和增殖。这一发现具有重要的意义:一个来源于不同种属慢病毒的辅助因子可以抵抗HIV-1,并且这种来源不同的辅助因子BIV Vif与HIV-1 Vif在功能上并没有关联性,是相互独立的,这无疑为抗HIV-1提供了新的思路。一方面,BIV Vif本身可能成为一种潜在的抗病毒因子;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机制也将为宿主抵抗病毒的研究提供更多线索。于是我们进一步的对其中的分子机制进行了探究,对于受到抑制的病毒进行了详细的分析,研究结果表明:首先,BIV Vif能够在一定程度上抑制病毒的产量;但是通过对病毒产量和病毒感染能力的详细分析,这并不是BIV Vif抵抗HIV-1的最主要原因;第二,更重要的是,BIV Vif还能够直接抑制新产生病毒的感染性;第三,更加深入的研究发现,BIV Vif可以抑制出芽病毒中组织特异性抗原(或称衣壳前体蛋白,Group specific antigen,Gag/Pr55Gag)的剪切,从而干扰了病毒复制周期中至关重要的一个环节——病毒的成熟。病毒成熟的核心过程即为出芽病毒中的Pr55Gag按照严格顺序和位点被病毒蛋白酶剪切为组成病毒颗粒的各个层面的衣壳蛋白,包括:基质(Matrix,MA/p17)、衣壳(Capsid,CA/p24)、核衣壳(Nucleocapsid,NC/p7)和p6,另外还有两个位于CA和NC、NC和p6中间的间隔肽1(Spacer peptide,SP1/p2)和间隔肽2(Spacer peptide,SP2/p1)。如上各个部分不仅对于病毒的结构组成缺一不可,并且需要按照严格顺序进行剪切产生。而BIV Vif可以抑制Pr55Gag剪切的第一步,NC/p2间的剪切。因此,BIV Vif能够显著地抑制HIV-1的感染性。这一部分研究初步阐明了BIV Vif抑制HIV-1的内在原因。接下来,一个重要的问题是为什么只有BIV Vif可以抑制HIV-1,而HIV-1或是SIV的Vif不能?根据我们的研究结果显示:相比于等量表达的HIV-1 Vif或是SIV Vif,BIV Vif能够更强地结合Pr55Gag。同时,不能结合Pr55Gag的BIV Vif突变体失去了抑制HIV-1的能力。这就解释了虽然HIV-1、SIV、BIV Vif都可以与HIV-1 Pr55Gag相互作用,但是因为BIV Vif与Pr55Gag的结合能力最强,所以BIV Vif可以显著地抑制HIV-1,而HIV-1或是SIV的Vif因与Pr55Gag的结合能力弱而不能抑制HIV-1。这部分研究进一步阐明了BIV Vif抑制HIV-1的分子机制。同时,不同种属的慢病毒Vif蛋白,包括HIV-1、SIV、BIV Vif都保留了与HIV-1 Pr55Gag相互作用的能力。而这种结合的保守性提示了对于病毒来说Vif与Pr55Gag的结合非常重要,可能对病毒的复制周期具有重要功能。Vif与HIV-1 Pr55Gag的相互作用无疑是BIV Vif拮抗病毒的核心环节,并且,这个结合在不同种属Vif间的保守性也暗示了其对病毒可能具有重要功能。因此,接下来我们利用分子克隆技术结合免疫共沉淀技术探索了二者结合的结合决定域;实验结果表明:对于HIV-1的Pr55Gag来说,Pr55Gag中对于病毒组装和成熟起重要作用的CA碳末端和p2区域对Pr55Gag与Vif的结合必不可少;对于HIV Vif和BIV Vif,它们的氮末端,尤其是第30位酪氨酸(Tyrosine,Y)和第33位精氨酸(Arginine,R)或者组氨酸(Histidine,H),对于Vif-Pr55Gag相互结合作用至关重要。Vif与HIV-1 Pr55Gag相互作用的保守决定域的鉴定对于后续BIV Vif拮抗病毒的应用研究以及后续研究Vif与HIV-1 Pr55Gag相互作用的生物学功能具有重要的意义。BIV Vif在上述人胚肾细胞系HEK 293T体系中,有效地抵抗了HIV-1的复制和增殖。那么更重要的是,这种抗病毒作用在HIV的靶细胞CD4阳性T细胞中是否仍然如此有效呢?接下来,我们在CD4阳性T细胞系中稳定表达BIV Vif,实验证明,在BIV Vif的表达不影响细胞周期和增殖的前提下,BIV Vif在CD4阳性T细胞中能够有效抑制HIV-1的增殖。综上所述,本研究利用现代生物学技术,首次发现牛艾滋病病毒辅助蛋白Vif可以作为一种外源的抗病毒因子,能够显著抑制HIV-1的产生、感染性和增殖;不仅如此,我们还对BIV Vif拮抗病毒的原因和分子机制进行了深入的研究和探讨,BIV Vif通过在细胞内有效地结合HIV-1 Pr55Gag,抑制了病毒出芽后的成熟过程,从而显著地抑制了HIV-1感染性、复制和增殖;同时我们首次发现HIV-1 Vif、SIV Vif和BIV Vif都可以与HIV-1 Pr55Gag相互结合,这是首次在真核细胞中鉴定出二者的相互结合;更进一步,我们还鉴定出了二者相互结合的决定域。这些发现无论是为后续相关研究的进行还是抗病毒治疗新策略的开发都提供了重要的理论基础和潜在靶点。
[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
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R373

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