Yamamoto M, Hayashi N, Takehara T, Ueda K, Mita E, Tatsumi T, Sasaki Y, Kasahara A, Hori M
Department of Internal Medicine and Therapeutics, Osaka University School of Medicine, Suita, Osaka, Japan.
Hepatology. 1999 Jul;30(1):300-7. doi: 10.1002/hep.510300105.
Hepatitis B virus (HBV) is one of the major causes of chronic liver diseases and hepatocellular carcinoma. In this study, we used a single chain antibody (sFv), which is a man-made antibody with a strong affinity of immunoglobulin, to inhibit HBV replication. Because HBV replication can only take place in the viral nucleocapsid made of HBV core protein (HBc), we generated anti-HBc sFv and examined whether intracellular anti-HBc sFv could inhibit viral replication in the human hepatoblastoma-derived cell line that produces HBV (HB611). With respect to HBV replication intermediates, both single-stranded and partially double-stranded DNA intermediates were markedly suppressed in the cells expressing anti-HBc sFv, although HBV RNA intermediates were not affected. This suggested that intracellular anti-HBc sFv inhibited HBV DNA replication by inhibiting reverse transcription from HBV pregenome RNA to single-stranded DNA. Because the sFv-HBc complex was detected in the cells expressing anti-HBc sFv by immunoprecipitation analysis but the quantity of intracellular HBc was not affected, the anti-HBc sFv was suggested to inhibit HBV DNA replication by interfering with the function of HBc. These results indicate that intracellular sFv against HBc might be effective as a novel active molecule for gene therapy of hepatitis B.
乙型肝炎病毒（HBV）是慢性肝病和肝细胞癌的主要病因之一。在本研究中，我们使用了一种单链抗体（sFv），它是一种具有强免疫球蛋白亲和力的人工合成抗体，来抑制HBV复制。由于HBV复制只能在由HBV核心蛋白（HBc）构成的病毒核衣壳中进行，我们制备了抗-HBc sFv，并检测细胞内抗-HBc sFv是否能在产生HBV的人肝母细胞瘤衍生细胞系（HB611）中抑制病毒复制。关于HBV复制中间体，在表达抗-HBc sFv的细胞中，单链和部分双链DNA中间体均受到显著抑制，尽管HBV RNA中间体未受影响。这表明细胞内抗-HBc sFv通过抑制从HBV前基因组RNA到单链DNA的逆转录来抑制HBV DNA复制。由于通过免疫沉淀分析在表达抗-HBc sFv的细胞中检测到了sFv-HBc复合物，但细胞内HBc的量未受影响，因此提示抗-HBc sFv通过干扰HBc的功能来抑制HBV DNA复制。这些结果表明，细胞内抗-HBc sFv可能作为一种新型活性分子，对乙型肝炎的基因治疗有效。
