Yaginuma K, Nakamura I, Takada S, Koike K
Department of Gene Research, Cancer Institute, JFCR, Tokyo, Japan.
J Virol. 1993 May;67(5):2559-65. doi: 10.1128/JVI.67.5.2559-2565.1993.
Recent studies have demonstrated the transacting function of the X gene product of hepatitis B virus. However, little information is available on the regulation of X gene expression. In this report, we first investigate a cellular factor regulating X gene transcription by DNA transfection, using the human hepatoma cell line HuH-7, which is permissive for HBV replication as well as X mRNA transcription. A sequence-specific cellular factor was found to bind to the promoter region upstream of the first ATG (nucleotide [nt] 1248) of the X open reading frame. DNase I footprinting analysis showed the binding sequence of this factor to be situated between nt 1097 and 1119, where an 8-bp palindrome structure resides. S1 nuclease analysis of X gene transcripts demonstrated the binding site to be adjacent to two major start sites (nt 1117 and 1125) of X mRNA. Second, we demonstrate that introduction of a mutation into the binding site gives rise to a loss of the binding with a concomitant shift of the transcription start site of X mRNA beyond the 8-bp palindrome structure, causing it to become more heterogeneous. Thus, the promoter-binding protein appears to be involved in directing the transcription initiation site of the X gene toward the downstream region of the X promoter when X protein is produced from X mRNA.
最近的研究已经证实了乙型肝炎病毒X基因产物的反式作用功能。然而，关于X基因表达调控的信息却很少。在本报告中，我们首先利用人肝癌细胞系HuH-7研究一种通过DNA转染调节X基因转录的细胞因子，该细胞系允许HBV复制以及X mRNA转录。发现一种序列特异性细胞因子可与X开放阅读框第一个ATG（核苷酸[nt]1248）上游的启动子区域结合。DNase I足迹分析表明该因子的结合序列位于nt 1097和1119之间，此处存在一个8碱基对的回文结构。对X基因转录本的S1核酸酶分析表明结合位点与X mRNA的两个主要起始位点（nt 1117和1125）相邻。其次，我们证明在结合位点引入突变会导致结合丧失，同时X mRNA的转录起始位点会移至8碱基对回文结构之外，使其变得更加不均一。因此，当从X mRNA产生X蛋白时，启动子结合蛋白似乎参与将X基因的转录起始位点导向X启动子的下游区域。
