Dash S, Halim A B, Tsuji H, Hiramatsu N, Gerber M A
Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA.
Am J Pathol. 1997 Aug;151(2):363-73.
Hepatitis C virus (HCV) represents one of the major causes of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) around the world. Our knowledge of the life cycle of HCV, however, is limited. Current studies are hampered by the lack of a reproducible, high-level in vitro replication system of HCV. We sought to establish HCV replication in HepG2 cells by gene transfer of in vitro transcribed HCV RNA. In preliminary experiments, diethylaminoethyl-dextran led to more efficient gene transfer than cationic liposomes (lipofectin, lipofectamine, and DOTAP). Therefore, in subsequent experiments, HepG2 cells were transfected with full-length (9.6-kb) and near-full-length (9.4-kb) HCV RNA using diethylaminoethyl-dextran. Transfection with subgenomic HCV RNA and mock transfection were used as controls. Positive- and negative-strand HCV RNA sequences were detected by reverse transcription polymerase chain reaction (KT-PCR) for 60 days in the infectious HCV RNA transfected HepG2 cells. The presence of negative-strand HCV RNA, presumably representing replicative intermediates, was confirmed by ribonuclease protection assay. The intracellular levels of HCV RNA were measured by quantitative competitive RT-PCR from 10 to 50 days after transfection and were stable over this time period at moderately high levels (10(8) to 10(10) genomes per mg of total RNA). Expression of viral core and nonstructural proteins was detected in the cytoplasm of transfected cells by immunostaining. Virus-like particles measuring 50 to 60 nm in diameter were found by electron microscopy in cytoplasmic vesicles and conditioned media of the cells transfected with infectious HCV RNA but not in cells transfected with truncated HCV RNA. Culture supernatants of infectious HCV RNA transfected HepG2 cells were infectious for Daudi cells for three passages tested. The truncated HCV RNA lacking NS5 and 3' untranslated region (3' UTR) of HCV was replication incompetent. This is the first demonstration of HCV particles in HepG2 cells after transfection with infectious HCV RNA. We conclude that we have established a reproducible HCV replication system in HepG2 cells that can be used to study the life cycle of HCV and to test anti-HCV agents.
丙型肝炎病毒（HCV）是全球急性和慢性肝炎、肝硬化及肝细胞癌（HCC）的主要病因之一。然而，我们对HCV生命周期的了解有限。目前的研究因缺乏可重复的、高水平的HCV体外复制系统而受阻。我们试图通过体外转录的HCV RNA基因转移在HepG2细胞中建立HCV复制。在初步实验中，二乙氨基乙基葡聚糖比阳离子脂质体（lipofectin、lipofectamine和DOTAP）能更有效地进行基因转移。因此，在后续实验中，使用二乙氨基乙基葡聚糖将全长（9.6 kb）和近全长（9.4 kb）的HCV RNA转染到HepG2细胞中。用亚基因组HCV RNA转染和mock转染作为对照。在感染性HCV RNA转染的HepG2细胞中，通过逆转录聚合酶链反应（RT-PCR）检测正负链HCV RNA序列，持续60天。核糖核酸酶保护试验证实了负链HCV RNA的存在，推测其代表复制中间体。转染后10至50天，通过定量竞争性RT-PCR测量HCV RNA的细胞内水平，在此时间段内其水平稳定在中等高度（每毫克总RNA含10⁸至10¹⁰个基因组）。通过免疫染色在转染细胞的细胞质中检测到病毒核心蛋白和非结构蛋白的表达。通过电子显微镜在感染性HCV RNA转染细胞的细胞质囊泡和条件培养基中发现了直径为50至60 nm的病毒样颗粒，但在截短的HCV RNA转染细胞中未发现。感染性HCV RNA转染的HepG2细胞的培养上清液在测试的三代中对Daudi细胞具有感染性。缺乏HCV NS5和3'非翻译区（3'UTR）的截短HCV RNA无复制能力。这是在用感染性HCV RNA转染后首次在HepG2细胞中证明有HCV颗粒。我们得出结论，我们已在HepG2细胞中建立了可重复的HCV复制系统，可用于研究HCV的生命周期和测试抗HCV药物。
