【作者】 乔文涛；

【导师】 耿运琪；

【作者基本信息】 南开大学 ， 微生物学， 2004， 博士

【摘要】 泡沫病毒基因组成分、生活周期及基因调控方式与传统的反转录病毒区别较大。它的基因转录至少需要两个启动子协同完成，两类启动子的活性都依赖于反式激活因子Tas(transactivator of spmavirus，Tas)，同时Tas与其它病毒蛋白共同与宿主细胞因子作用，协调病毒与宿主细胞间的相互关系，决定病毒的感染方式。其作用方式和机制尚不清楚。 本文以牛泡沫病毒中国毒株(Bovine foamy virus 3026，BFV3026)为材料，研究其Tas蛋白(即Borf1)的特征，探索其转录调节的分子机理。本文的工作内容包括以下4部分：(1)通过生物信息学分析，获得有关Borf1蛋白分子进化、生化性质及翻译后修饰等有关数据；(2)对BFV borf1基因转录本进行研究，通过对其不同感染时间转录本数量、启动子使用及剪接方式的追踪，探讨病毒基因表达和复制的调节机制；(3)完成对BFV Borf1蛋白的可溶性表达、精细纯化，为抗体制备提供优质抗原，获得高滴度抗血清，完成目的蛋白的定位及其定位相关序列的界定。同时也为利用结构生物学直观解析其作用机制打下基础；(4)利用哺乳动物细胞双杂交系统，界定BFV Borf1蛋白激活域，加深对Borf1蛋白的认识，为利用酵母双杂交系统筛选Borf1功能相关因子奠定基础。开展Borf1与宿主细胞因子组蛋白乙酰化酶间的相互作用关系研究。 本文通过5′RACE试验首次直接观察到泡沫病毒时序调节中启动子的转换。并发现在BFV 3026的内部启动子近上游存在“神秘”转录起始点，该起始点在泡沫病毒的自然感染进程中发挥作用。在不同感染时间，borf1转录本拼接方式明显不同，暗示存在转录后调控。在原核细胞中可溶性表达Borf-1重组蛋白，并纯化至电泳纯。得到优质抗血清。发现Borf-1蛋白主要定位于细胞核，其入核不是通过简单的被动扩散，而是需要细胞蛋白的辅助，定位相关序列位于羧基端167-249位氨基酸之间。Borf1激活作用存在以下特点：Borf-1激活病毒自身启动子不需要宿主细胞种属特异性细胞因子。其激活结构域为羧基端198-249位氨基酸，其中198-217位氨基酸为最小激活域。Borf1的激活作用机制与细胞组蛋白乙酰化酶相关。更多还原

【Abstract】 The genomic nature, replication strategy and the gene expression of foamy viruses(FV) diverge in many aspects from what is commonly accepted as the rules of retrovirus. The transcription of FV genes is dependent on two distinct promoters. A transactivator, termed Tas, is required for transcription from the two viral promoters by interacting with Tas response elements (TREs) in both promoters. Tas also interacts with host cell factors. Nevertheless, little is known about the mechanism of the Tas transactivation.To characterize and define molecular mechanism of Tas transcriptional regulation, the property and functional feature of bovine foamy virus transactivator, known as Borf1, were studied. The unrooted tree of Tas from foamy virus genomes, basic biochemical property and posttranslational modify of Borfl were predicted through bioinformatics analysis. The borfl mRNA pattern at different time point was observed by 5 primer rapid amplification of cDNA ends (5’RACE). After the protein was expressed in BL21 strain of E. coli in a soluble form and purified by 3 kinds of chromatography, its high qualitative antiserum from mouse was prepared and used to analyze its cellular localization. To further explore the mechanism that Borfl regulates gene expression by transcriptional transactivation, its activation domain was mapped by mammalian two-hybrid system and the interaction between Borfl and p300/CBP was detected.It is the first report observed directly that the promoters changed during foamy virus infection. And a cryptic start site of transcription was found, which was located upstream of the internal promoter TATA box and used in the natural infection. Combined with the different splicing patterns of borfl transcript in the different time point, it suggested that there was a posttranscription regulation in the BFV replication. To fulfill its transactivation function, this protein was predominately located in the nuclear and related sequence was between amino acids (aa) 167 to 249. Detected by Gal4 binding domain fusion protein analysis in mammalian cells, the transcriptional activation domain of Borfl was defined to an acidic C-terminal region from amino acids 198 to 249. Importantly, the data from transfection, co-localization and co-immunoprecipitation demonstrated that p300/CBP was involved in the process of Borfl transactivation. Further study is required to complete our understanding on the network mechanism of Borfl gene regulation.更多还原