CBFβ对Vif功能的调控以及进化压力下Vif功能的选择性研究
发布时间：2018-08-14 17:20
【摘要】：病毒感染性因子Vif(Viral infectivity factor)是HIV-1(Human immunodeficiency virus type 1)病毒的辅助蛋白之一,也是最早被发现具有拮抗宿主防御机制的HIV-1病毒功能性蛋白。并且在HIV-1的辅助蛋白中其拮抗宿主对病毒的防御机制也最先被揭示。Vif能够与宿主内的细胞因子Cul5,Elo B,Elo C,Rbx组装成Cullin-Ring E3泛素复合物,结合并诱导APOBEC3G、APOBEC3F等APOBEC3家族的蛋白泛素化降解。这一功能也完美的解释了在一些细胞系中,Vif能够促进HIV-1病毒的感染性的原因。随着CBFβ(core binding factorβ)作为Vif-Cullin-Ring E3复合物重要的辅助因子这一重要理论的提出,人们对该复合物的组装过程有了更加深入的了解。在CBFβ缺失的情况下,Vif只能够和Elo C/Elo B结合,不能够与Cul5结合,从而使E3复合物的组装有缺陷,不具有降解APOBEC3家族的蛋白的功能。即CBFβ能促进HIV-1 Vif蛋白降解靶蛋白。除此之外,研究还发现CBFβ也能够在原核细胞大肠杆菌E.coli中稳定Vif蛋白,提高Vif蛋白的可溶性,从而得到Vif蛋白的晶体结构。在天然情况下,CBFβ是调控宿主转录的关键性因子,它与RUNX(CBFα)结合,增强RUNX与DNA的亲和性,特别针对于细胞内一些关键的启动子和增强子,如巨噬细胞集落刺激因子MCSFR(macrophage colony-stimulating factor receptor)。CBFβ/RUNX形成异源二聚体能够影响人源细胞中很多基因的转录水平,从而调控细胞的分化和增殖。此外,Vif诱导细胞G2/M期阻滞以及Vif降解APOBEC3其他成员的选择性也成为了领域的研究热点。本论文围绕Vif、CBFβ、RUNX、APOBEC3H(A3H)之间相互联系进行了细致的研究和讨论,其中包括三个部分:我们在第一部分对于CBFβ参与Vif或RUNX功能的关键功能区域进行了细致的研究。通过检测CBFβ的N端和C端截短突变体,我们证明CBFβ15-126是帮助Vif降解A3G的最短功能区。CBFβLoop3的突变体对于RUNX1c介导的基因转录没有影响,但是Loop3前6个氨基酸的点突变抑制Vif对于A3G的降解。综上所述,CBFβLoop3是帮助Vif功能的重要区域,同时其突变不影响CBFβ在细胞内的正常功能。综上,Loop3可以作为设计抗HIV-1病毒药物的理想作用靶点。Vif与APOBEC3F/G之间的相互作用已经被广泛并且全面的研究,而关于Vif-A3H的结合相关信息鲜有报道。APOBEC3H在人类的细胞中有很多亚型,其中一些亚型,例如hap II,能够抑制HIV-1病毒的复制。在第二部分中,我们证明了不同于APOBEC3G/F,只有一些HIV-1亚型的Vif蛋白能够诱导A3H hap II的泛素化降解。位于HIV-1 Vif 39和49位的氨基酸,突变之后能够显著的降低Vif对A3H hap II降解的能力,但是依旧维持降解APOBEC3F/G的功能。这表明Vif与A3H hap II结合/降解的区域不同于Vif结构中A3F/G的功能区。Vif蛋白除了能够降解APOBEC3家族蛋白外,还有另外一个重要的功能,即作为细胞周期调控因子,诱导宿主细胞G2/M期阻滞。而这一功能经我们实验证明也需要CBFβ以及Vif组装的E3复合物。有趣的是,与Vif对于A3H hap II降解功能相似,只有部分Vif蛋白具有能够诱导细胞周期G2/M阻滞的能力。比如HIV-1 HXB2亚型的Vif蛋白不能够诱导细胞G2/M期阻滞,而HIV-1 NL4-3亚型的Vif蛋白则具有这个功能。在第三部分中,我们通过比较HXB2与NL4-3 Vif蛋白的氨基酸序列,发现位于31,33,36,47和50位氨基酸对于Vif诱导细胞G2/M期阻滞很重要,如果将HXB2 Vif上这5个位点的氨基酸突变成NL4-3 Vif位置上相对应的氨基酸,则赋予了其诱导细胞周期G2/M阻滞的能力。实验进一步证明,Vif蛋白对于A3H hap II降解或者诱导细胞G2//M期阻滞这两个显著的功能是具有选择性的,不能共存。而这一选择性也表明Vif蛋白仍具有一些潜在的新的机制还有待进一步挖掘。综上所述,本文对CBFβ蛋白结构上关键氨基酸进行扫描,通过功能实验发现了CBFβ影响Vif降解A3G或者参与RUNX功能的重要功能区;同时进一步探讨了CBFβ影响Vif调控细胞周期的分子机制;最后证明Vif在一定筛选压力下,通过选择性的拮抗A3H抗病毒因子或者诱导细胞G2/M期阻滞来增强病毒的生存能力。进一步完善了对于Vif蛋白本身的功能和行使功能的分子机制的研究。
[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.
【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
本文编号：2183565
[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.
【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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