Sugata F, Chen H S, Kaneko S, Purcell R H, Miller R H
Hepatitis Virus Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Virology. 1994 Nov 15;205(1):314-20. doi: 10.1006/viro.1994.1647.
During the course of woodchuck hepatitis virus (WHV) replication three virus-specific mRNA transcripts that encode four essential proteins are produced. The transcripts are 3.6, 2.3, and 0.7 kb in size. The 3.6-kb transcript serves as the replicative intermediate as well as the template for translation of the nucleocapsid and polymerase proteins. The 2.3-kb mRNA serves as the template for translation of the virus envelope proteins. Both the 3.6- and 2.3-kb transcripts are polyadenylated and are readily found in the cytoplasm of infected hepatocytes. However, the 0.7-kb transcript, specific for the X gene, accumulates in the nucleus of infected cells and is polyadenylated poorly in hepatocytes. Thus, while it is likely that the 0.7-kb transcript is the template for translation of the X protein, it is possible that it also has a function at the RNA level to regulate virus replication or gene expression. In order to characterize the WHV X promoter we cloned the region of the WHV8 genome encompassing the viral enhancer through the amino terminus of the X gene into the vector pSV0CAT. We transfected Huh-7 and WLC-3 cells with the WHV X promoter construct, along with a plasmid encoding human growth hormone to control for transfection efficiency, and assayed for the presence of chloramphenicol acetyl transferase activity. We found that the WHV X promoter was about one-half as active as the well-studied simian virus 40 early or Rous sarcoma virus promoters. Next, we made a series of 5' and 3' deletion mutants and mapped the WHV X promoter to a 21-nucleotide domain (1482-GGGGAAGCTGACGTCCTTTCC-1502) which is approximately 100 bp downstream of the corresponding promoter in hepatitis B virus. Further analysis, using oligonucleotide-directed mutagenesis, demonstrated that the essential nucleotides comprising the WHV X promoter are located in a 10-nucleotide domain near the initiation codon of the X gene. Mutation of either nucleotide T at position 1490 or G at position 1491 within this domain was sufficient to reduce the level of promoter activity by 100-fold. Thus, we have defined the important nucleotides within the promoter of the WHV X transcript which is a first step in understanding the role of this transcript in WHV replication and gene expression.
在土拨鼠肝炎病毒（WHV）复制过程中，会产生三种编码四种必需蛋白质的病毒特异性mRNA转录本。这些转录本的大小分别为3.6、2.3和0.7 kb。3.6 kb的转录本既是复制中间体，也是核衣壳蛋白和聚合酶蛋白翻译的模板。2.3 kb的mRNA是病毒包膜蛋白翻译的模板。3.6 kb和2.3 kb的转录本都进行了多聚腺苷酸化，并且很容易在受感染肝细胞的细胞质中找到。然而，X基因特异性的0.7 kb转录本在受感染细胞的细胞核中积累，并且在肝细胞中多聚腺苷酸化程度很低。因此，虽然0.7 kb转录本可能是X蛋白翻译的模板，但它也有可能在RNA水平上具有调节病毒复制或基因表达的功能。为了表征WHV X启动子，我们将WHV8基因组中包含病毒增强子直至X基因氨基末端的区域克隆到载体pSV0CAT中。我们用WHV X启动子构建体转染Huh-7和WLC-3细胞，同时转染一个编码人生长激素的质粒以控制转染效率，并检测氯霉素乙酰转移酶活性的存在。我们发现WHV X启动子的活性约为研究充分的猿猴病毒40早期启动子或劳氏肉瘤病毒启动子的一半。接下来，我们制作了一系列5'和3'缺失突变体，并将WHV X启动子定位到一个21个核苷酸的区域（1482 - GGGGAAGCTGACGTCCTTTCC - 1502），该区域位于乙型肝炎病毒相应启动子下游约100 bp处。使用寡核苷酸定向诱变的进一步分析表明，构成WHV X启动子的必需核苷酸位于X基因起始密码子附近的一个10个核苷酸的区域内。该区域内第1490位的核苷酸T或第1491位的核苷酸G发生突变足以使启动子活性水平降低100倍。因此，我们已经确定了WHV X转录本启动子内的重要核苷酸，这是了解该转录本在WHV复制和基因表达中作用的第一步。
