Takada S, Koike K
Department of Gene Research, Cancer Institute, Tokyo, Japan.
Proc Natl Acad Sci U S A. 1990 Aug;87(15):5628-32. doi: 10.1073/pnas.87.15.5628.
To investigate the expression and transactivation function of the X gene in integrated hepatitis B virus (HBV) DNA from chronic hepatitis tissues, a series of transfectants containing cloned integrated HBV DNAs was made and analyzed for X mRNA expression and trans-activation activity by using a chloramphenicol acetyltransferase assay. Most of the integrated HBV DNAs expressed X mRNA and encoded a product with trans-activation activity in spite of the loss of the 3' end region of the X gene due to integration. From cDNA cloning and sequence analysis of X mRNA transcribed from native or integrated HBV DNA, the X protein was found to be translated from the X open reading frame without splicing. For integrated HBV DNA, transcription was extended to a cellular flanking DNA and an X gene-cell fusion transcript was terminated by using a cellular poly(A) signal. The amino acid sequence deduced from an X-cell fusion transcript indicated truncation of the carboxyl-terminal five amino acids, but the upstream region of seven amino acids conserved among hepadnaviruses was retained in the integrated HBV DNA, suggesting that this conserved region is essential for the transactivation function of the X protein. These findings support the following explanation for hepatocarcinogenesis by HBV DNA integration: the expression of a cellular oncogene(s) is transactivated at the time of chronic infection by the increasing amounts of the integrated HBV gene product(s), such as the X-cell fusion product.
为研究慢性肝炎组织中整合型乙型肝炎病毒（HBV）DNA的X基因的表达及反式激活功能，构建了一系列含有克隆整合型HBV DNA的转染子，并通过氯霉素乙酰转移酶分析检测X mRNA表达及反式激活活性。尽管由于整合导致X基因3'端区域缺失，但大多数整合型HBV DNA仍表达X mRNA并编码具有反式激活活性的产物。通过对天然或整合型HBV DNA转录的X mRNA进行cDNA克隆和序列分析，发现X蛋白由X开放阅读框翻译而来，无需剪接。对于整合型HBV DNA，转录延伸至细胞侧翼DNA，且X基因-细胞融合转录本通过细胞聚腺苷酸化信号终止。从X-细胞融合转录本推导的氨基酸序列表明羧基末端的五个氨基酸被截短，但嗜肝DNA病毒中七个保守氨基酸的上游区域保留在整合型HBV DNA中，提示该保守区域对X蛋白的反式激活功能至关重要。这些发现支持了关于HBV DNA整合导致肝癌发生的以下解释：在慢性感染时，整合型HBV基因产物（如X-细胞融合产物）数量增加，从而反式激活细胞癌基因的表达。
