Chang H K, Wang B Y, Yuh C H, Wei C L, Ting L P
Graduate Institute of Microbiology and Immunology, National Yang-Ming Medical College, Taipei, Taiwan, Republic of China.
Mol Cell Biol. 1989 Nov;9(11):5189-97. doi: 10.1128/mcb.9.11.5189-5197.1989.
The outer envelope of the 42-nm virion of the human hepatitis B virus (HBV) is composed of the large, the middle, and the major surface proteins. Whereas the middle and the major surface proteins are transcribed from the SPII promoter of the pre-S/S gene, the large surface protein is transcribed from the SPI promoter located upstream of SPII. We have previously shown that transcription of SPI (comprising nucleotides [nt] -380 to +17) occurs preferentially in differentiated hepatoma cell lines (H.K. Chang and L.P. Ting, Virology 170:176-183, 1989). In this report, we further demonstrated that a sequence of 95 base pairs in the upstream region of SPI (nt -95 to +17) was necessary and sufficient for such preferential expression in differentiated hepatoma cells. By analysis of the expression of the chloramphenicol acetyltransferase gene in a series of mutants with deletions at the 5' end of SPI, we identified a positive transcriptional cis-acting element mapping at nt -95 to -72 which appears to play a key role in the regulation of the expression of the large surface protein. This region shared a high degree of sequence homology with regulatory sequences of several liver-specific genes from human, mouse, and rat, with a consensus sequence (G/A)GTTA(A/C)TNNT(C/T)NNC(A/C). We further identified a nuclear factor present in the nuclear extracts of differentiated human hepatoma cell lines which interacted specifically with this element of the SPI promoter. This nuclear factor was similar to the rat liver-specific factor HNF-1, since an oligonucleotide containing the recognition sequence of HNF-1 could efficiently compete for the human factor in a footprinting assay. The sequence at nt -93 to -68 which was bound by this factor in SPI was termed the HNF-1-binding element. Activation of the SPI promoter by human differentiated hepatocyte nuclear factor 1, described in this report, probably explains, first, the formation of the 42-nm virion specifically in liver but not in several other tissues despite the synthesis of the middle and the major surface proteins in those tissues, and second, why only differentiated hepatoma cell lines are able to produce 42-nm-like virion particles on transfection by HBV DNA.
人类乙型肝炎病毒（HBV）42纳米病毒粒子的外膜由大、中、主表面蛋白组成。中表面蛋白和主表面蛋白由前S/S基因的SPII启动子转录而来，而大表面蛋白则由位于SPII上游的SPI启动子转录。我们之前已经表明，SPI（包含核苷酸[nt] -380至+17）的转录优先发生在分化的肝癌细胞系中（H.K. Chang和L.P. Ting，《病毒学》170:176 - 183，1989）。在本报告中，我们进一步证明，SPI上游区域（nt -95至+17）中的一段95个碱基对的序列对于在分化的肝癌细胞中的这种优先表达是必要且充分的。通过分析氯霉素乙酰转移酶基因在一系列SPI 5'端缺失突变体中的表达，我们鉴定出一个正向转录顺式作用元件，定位在nt -95至-72，它似乎在大表面蛋白表达的调控中起关键作用。该区域与来自人、小鼠和大鼠的几个肝脏特异性基因的调控序列具有高度的序列同源性，共有序列为（G/A）GTTA（A/C）TNNT（C/T）NNC（A/C）。我们进一步在分化的人肝癌细胞系的核提取物中鉴定出一种核因子，它与SPI启动子的这个元件特异性相互作用。这种核因子与大鼠肝脏特异性因子HNF - 1相似，因为在足迹分析中，包含HNF - 1识别序列的寡核苷酸能够有效地竞争人因子。在SPI中被该因子结合的nt -93至-68处的序列被称为HNF - 1结合元件。本报告中描述的人分化肝细胞核因子1对SPI启动子的激活，可能首先解释了尽管在其他几种组织中合成了中表面蛋白和主表面蛋白，但42纳米病毒粒子为何仅在肝脏中形成，而不在其他几种组织中形成；其次，解释了为什么只有分化的肝癌细胞系在转染HBV DNA后能够产生42纳米样病毒粒子。
