Shimoda A, Sugata F, Chen H S, Miller R H, Purcell R H
Hepatitis Viruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA.
Virus Res. 1998 Jul;56(1):25-39. doi: 10.1016/s0168-1702(98)00050-1.
The genetic organization of hepadnaviruses is unusual in that all cis-acting regulatory sequences are located within genes. Thus, in the mammalian hepadnavirus genome, the presurface, surface, and X transcript promoters reside within the polymerase gene while the pregenome transcript promoter is located within the X gene. In this study we have identified two additional promoters within the woodchuck hepatitis virus (WHV) X gene that stimulate production of transcripts in vitro. First, we cloned regions of the WHV X gene into a promoterless expression vector (pGL2) to examine their ability to promote expression of firefly luciferase and mapped a previously unidentified promoter to positions 1475-1625 of the WHV8 genome. Deletion analysis revealed that the essential domain of this promoter, termed the ORF5/deltaX transcript promoter, mapped to nucleotides 1525-1625. Analysis revealed that this transcript initiated at nucleotide 1572 in both human (HuH-7) and woodchuck (WLC-3) hepatoma cell lines. Consistent with this finding, DNA footprinting analysis revealed protection of nucleotides 1567-1578 on the positive strand of the WHV8 genome. The function of this transcript in vivo is unclear, however, it may be used to produce a truncated form of the X protein that initiates at an AUG codon at position 1743-1745 on the WHV8 genome. Next, a second promoter was identified at positions 1625-1975 that was responsible for production of an antisense transcript. The activity of this promoter was comparable to that of the previously characterized surface transcript promoter of WHV in the absence of an enhancer. The antisense transcript promoter resides immediately upstream of open reading frame (ORF) 6, a previously identified ORF on the strand opposite of the known WHV protein-encoding sequences, that is thought to represent a vestigial gene. Analysis indicates that the antisense transcript had multiple start sites: nucleotides 1683 and 1762 on the WHV8 genome when assayed in HuH-7 cells, and nucleotide 1786 when assayed in WLC-3 cells. These data are consistent with footprinting analysis of supercoiled WHV DNA that revealed that the regions encompassing nucleotides 1696-1685, 1781-1766, and 1801-1787 on the negative sense DNA strand were protected from nuclease degradation. It is possible that such a transcript was once used in protein expression in an ancestral virus and may now be used for genetic control of WHV replication and/or gene expression. Overall, these data are consistent with the presence of a bidirectional promoter complex within the WHV X gene.
嗜肝DNA病毒的基因组织方式不同寻常，因为所有顺式作用调控序列都位于基因内部。因此，在哺乳动物嗜肝DNA病毒基因组中，前S、S和X转录启动子位于聚合酶基因内，而前基因组转录启动子位于X基因内。在本研究中，我们在土拨鼠肝炎病毒（WHV）X基因内鉴定出另外两个在体外可刺激转录物产生的启动子。首先，我们将WHV X基因的区域克隆到无启动子表达载体（pGL2）中，以检测它们促进萤火虫荧光素酶表达的能力，并将一个先前未鉴定的启动子定位到WHV8基因组的1475 - 1625位。缺失分析表明，这个启动子的核心区域，称为ORF5/δX转录启动子，定位到核苷酸1525 - 1625。分析显示，该转录物在人（HuH - 7）和土拨鼠（WLC - 3）肝癌细胞系中均起始于核苷酸1572。与此发现一致，DNA足迹分析显示WHV8基因组正链上的核苷酸1567 - 1578受到保护。然而，该转录物在体内的功能尚不清楚，它可能用于产生一种截短形式的X蛋白，该蛋白在WHV8基因组上1743 - 1745位的AUG密码子处起始。接下来，在1625 - 1975位鉴定出第二个启动子，它负责产生反义转录物。在没有增强子的情况下，这个启动子的活性与先前鉴定的WHV表面转录启动子相当。反义转录启动子紧邻开放阅读框（ORF）6的上游，ORF6是在与已知WHV蛋白质编码序列相反的链上先前鉴定出的一个开放阅读框，被认为代表一个退化基因。分析表明反义转录物有多个起始位点：在HuH - 7细胞中检测时，WHV8基因组上的核苷酸1683和1762；在WLC - 3细胞中检测时，核苷酸1786。这些数据与超螺旋WHV DNA的足迹分析一致，足迹分析显示负义DNA链上包含核苷酸1696 - 1685、1781 - 1766和1801 - 1787的区域免受核酸酶降解。这种转录物有可能曾经在一种原始病毒的蛋白质表达中被使用，现在可能用于WHV复制和/或基因表达的遗传控制。总体而言，这些数据与WHV X基因内存在双向启动子复合体一致。
