【摘要】 牛泡沫病毒 (BFV)具有复杂的基因组结构 ,其基因组除编码反转录病毒共有的 gag、pol、env 三个结构基因之外 ,在其env和 3′LTR之间有两个重叠的读码框 (ORF - 1和ORF - 2 ) ,编码Borf- 1、Borf- 2、Bet等多种调节蛋白 ,其中Borf- 1(2 49aa)为BFV反式激活因子 (Tas)。为研究Borf - 1的结构与功能 ,Borf - 1在LTR上的应答元件及作用机制 ,Borf- 2、Bet等调节蛋白在BFV基因表达调控中的作用 ,本研究以本室分离鉴定的BFV30 2 6中国毒株为材料 ,克隆Borf- 1等基因片段构建系列质粒 ,与带有luc报告基因的LTR系列缺失质粒共转染 ,作瞬时表达分析。结果表明 ,Borf- 137aa～ 114aa、C端 2 18aa～ 2 49aa为其行使激活功能所必需区域 ;Tas在BFVLTR上的应答元件 (TRE)位于 - 983/ - 6 6 8(TREI)、- 470 / - 140 (TREII)和RU5区 ,其中TREI、TREII均位于U3区 ,为正调控元件 ,RU5区为负调控元件。进一步研究证明 ,TREII能在异源启动子 (BIVLTR)上行使功能 ,且与其自身的方向无关 ,类似于增强子元件。此外还发现 ,RU5区具有抑制其下游基因表达的功能 ,该区在异源启动子 (BIVLTR)之后使其下游基因表达量降低。计算机分析结果表明 ,BFVRU5区的负调控作用可能是因为该区mRNA具有稳定的二级结构 ,从而抑制其下游基因的表达更多还原

【Abstract】 Bovine foamy virus (BFV), a member of retrovirus family, possesses a complex genome structure. In addition to structural genes gag, pol, and env, the genome contains two open reading frames (ORF-1 and ORF-2) located between the env gene and 3′LTR. ORF-1 and ORF-2 encode several different proteins via alternative splicing, including Borf-1, Borf-2, and Bet. In order to analyze the structure of the Borf-1, which is a transcriptional transactivator of gene expression directed by the BFV LTR, as well as the Tas responsive elements (TRE) in LTR and the requirements of other proteins for the BFV replication in vitro, we constructed serial Borf-1 mutants, Borf-2 and Bet expression plasmids by cloning ORF genes from the genome of fetal bovine lung (FBL) cells infected with the China strain BFV3026. The results of transient expression assays in FBL cells showed that the residues 37 to 113 and 218 to 249 (carboxyl-terminal residues) are essential for the Borf-1 activity. TREII showed the properties of a transcriptional enhancer by functioning in a heterologous promoter BIV LTR and independently of its orientation. The RU5 region of BFV LTR down-regulated the Borf-1 transactivation by 14-folds. Computer analysis of this region revealed a stable secondary structure, which may result in the RU5 inhibition on LTR-directed gene expression. Our results also showed that Borf-2 was dispensable for BFV LTR-directed gene expression in vitro. In addition, the BFV Bet protein as well as the Borf-1 mutants had a trans-dominant inhibitory effect on wild type Borf-1-mediated transactivating ability. These results showed that the mechanism of transactivation in foamy viruses is significantly different from that in lentiviruses and oncoviruses.更多还原