The conserved amino-terminal region (amino acids 1-20) of the hepatitis B virus X protein shows a transrepression function
The conserved amino-terminal region (amino acids 1-20) of the hepatitis B virus X protein shows a transrepression function
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DOI：
10.1016/j.virusres.2004.05.006
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发表时间：
2004-10-01
期刊：
影响因子：
5
通讯作者：
Kumar, V
中科院分区：
文献类型：
--
作者：
Misra, KP;Mukherji, A;Kumar, V
B型肝炎病毒的X蛋白或HBx是一种多功能调节蛋白，具有混杂反式激活因子的名声。尽管HBx的N-末端“A”区（氨基酸1-20）是哺乳动物嗜肝DNA病毒基因组中最保守的区域，但已发现其不具有反式激活功能[Proc.Natl. Acad. Sci. U.S.A.93，1996，647]。为了阐明其生物学作用，通过聚合酶链反应扩增对应于X基因A区的DNA序列，并克隆为72个碱基对的HBx突变体X17。为了增强表达蛋白的细胞内生化稳定性，将单体X17多聚化，并将A区的2-10个单位长的串联重复序列（X17-n）克隆到哺乳动物表达载体中。X17构建体的表达通过体外转录和翻译以及在肝癌细胞中转染后通过RT-PCR证实。使用病毒（RSV-LTR、HIV 1-LTR和HBx）和细胞基因启动子（c-Jun和表皮生长受体）的氯霉素乙酰转移酶报告基因构建体研究X17的功能。X17多聚体不仅抑制HBx介导的所有报告基因的反式激活，而且抑制它们的基础活性。抑制作用取决于细胞中转染的X17质粒的量以及X17表达载体中存在的重复单元的数量。此外，X17相关的反式激活抑制不是细胞毒性效应。因此，我们的数据表明，HBx的N-末端“A”结构域具有负调控功能。(C)2004 Elsevier B. V.保留所有权利。
The X protein of hepatitis B virus or HBx is a multifunctional regulatory protein that carries the fame of a promiscuous transactivator. Although, the N-terminal 'A' region of HBx (amino acids 1-20) is the most conserved region among mammalian hepadnavirus genomes, it has been found to be dispensable for transactivation function [Proc. Natl. Acad. Sci. U.S.A. 93, 1996, 647]. To elucidate its biological role, DNA sequence corresponding to the A region of X gene was amplified by polymerase chain reaction and cloned as a 72 base pair HBx mutant X17. In order to augment the intracellular biochemical stability of the expressed protein, the monomeric X17 was multimerized and 2-10 units long tandem repeats of the A region (X17-n) were cloned in a mammalian expression vector. Expression of the X17 constructs was confirmed by in vitro transcription and translation, as well as by RT-PCR after transfection in hepatoma cells. The function of X17 was investigated using the chloramphenicol acetyl transferase reporter constructs of viral (RSV-LTR, HIV1-LTR and HBx) and cellular gene promoters (c-Jun and epidermal growth receptor). Not only did the X17 multimers inhibit the HBx-mediated transactivation of all the reporter genes, but also their basal activities. The inhibition was dependent on the amount of X17 plasmid transfected in cells as well as on the number of repeat units present in the X17 expression vectors. Further, the X17-related inhibition of transactivation was not a cytotoxic effect. Thus, our data suggests that the N-terminal 'A' domain of HBx has a negative regulatory function. (C) 2004 Elsevier B.V. All rights reserved.