Imai M, Hoshi Y, Okamoto H, Matsui T, Tsurimoto T, Matsubara K, Miyakawa Y, Mayumi M
Immunology Division, Jichi Medical School, Tochigi-Ken, Japan.
J Virol. 1987 Nov;61(11):3555-60. doi: 10.1128/JVI.61.11.3555-3560.1987.
Primary hepatocellular carcinoma cells (PLC/342) propagated in nude mice produce hepatitis B surface antigen of subtype adr, as well as core particles containing viral DNA and DNA polymerase. Free and integrated forms of hepatitis B virus (HBV) DNA in the tumor were isolated by molecular cloning, and their nucleotide sequences were determined. Both of the two representative clones of free HBV DNA had the same genomic length (3,158 base pairs) and had two stop codons as well as two deletions in the envelope gene. None of the seven distinct clones of integrated HBV DNA possessed the entire viral genome. The integrated clone sequences had deletions and rearrangements, and only two clones possessed the envelope gene including the promoter and enhancer sequences. The C gene, which codes for core protein, was preserved in the two free clones and one of the integrated clones. The P gene, which codes for DNA polymerase, had deletions at two positions of 21 and 36 base pairs in both free clones, but was carried in toto by one of the integrated clones. The nucleotide sequences of the S genes of two free and four integrated clones, as well as their two inverted repeats, were compared. All of the eight sequences of the S gene possessed two nucleotide substitutions in common that were not displayed by any of the reported HBV genomes. The sequences differed from one another by only 1.2%. They differed, however, from 11 reported HBV genomes of subtype adr by 2.4%, from an ayr genome by 1.9%, from 2 adw genomes by 6.9%, and from 2 ayw genomes by 5.9%. These results indicate that all free and integrated HBV DNA species in the PLC/342 tumor cell evolved from a common progenitor. The free HBV DNA underwent nucleotide substitutions during several integration events, resulting in integrated HBV DNA copies that were similar in sequence but distinct from the reported HBV genomes.
在裸鼠体内增殖的原发性肝癌细胞（PLC/342）产生adr亚型的乙型肝炎表面抗原，以及含有病毒DNA和DNA聚合酶的核心颗粒。通过分子克隆分离肿瘤中乙型肝炎病毒（HBV）DNA的游离形式和整合形式，并测定其核苷酸序列。游离HBV DNA的两个代表性克隆均具有相同的基因组长度（3158个碱基对），在包膜基因中有两个终止密码子以及两个缺失。整合HBV DNA的七个不同克隆均不具有完整的病毒基因组。整合克隆序列存在缺失和重排，只有两个克隆具有包括启动子和增强子序列的包膜基因。编码核心蛋白的C基因在两个游离克隆和一个整合克隆中得以保留。编码DNA聚合酶的P基因在两个游离克隆的21和36个碱基对的两个位置存在缺失，但在一个整合克隆中完整存在。比较了两个游离克隆和四个整合克隆的S基因及其两个反向重复序列的核苷酸序列。S基因的所有八个序列共有两个核苷酸替换，这在任何已报道的HBV基因组中均未出现。这些序列彼此之间仅相差1.2%。然而，它们与11个已报道的adr亚型HBV基因组相差2.4%，与一个ayr基因组相差1.9%，与两个adw基因组相差6.9%，与两个ayw基因组相差5.9%。这些结果表明，PLC/342肿瘤细胞中所有游离和整合的HBV DNA种类均源自一个共同的祖先。游离HBV DNA在几次整合事件中发生了核苷酸替换，导致整合的HBV DNA拷贝在序列上相似，但与已报道的HBV基因组不同。
