Ikeda M, Sugiyama K, Mizutani T, Tanaka T, Tanaka K, Sekihara H, Shimotohno K, Kato N
Virology Division, National Cancer Center Research Institute, Tokyo, Japan.
Virus Res. 1998 Aug;56(2):157-67. doi: 10.1016/s0168-1702(98)00063-x.
We previously found that a human T-cell leukemia virus type I infected T-cell line, MT-2, was susceptible to hepatitis C virus (HCV) infection, and that cloned MT-2C cells could support HCV replication more persistently than the parental MT-2 cells. Recently we found that a non-neoplastic hepatocyte line, PH5CH, showed good susceptibility to HCV infection. In this study, we cloned PH5CH cells to obtain cells that supported more persistent HCV replication, and consequently three clones (PH5CH1, PH5CH7 and PH5CH8) in which intracellular HCV RNA could be detected at least 25 days postinoculation (p.i.) were obtained. Semi-quantitative analysis of HCV RNA indicated that HCV replicated in these cloned PH5CH cells was released into the culture medium. Semi-quantitative analysis of internalized HCV RNA after treatment of cloned PH5CH cells and parental PH5CH cells with proteinase K immediately after virus inoculation revealed that PH5CH1, PH5CH7 and PH5CH8 cells contained 10-fold higher levels of HCV RNA than low susceptible cloned PH5CH or parental PH5CH cells. Furthermore, we demonstrated that HCV replication was maintained for 70-100 days in these three clonal lines when the temperature of cell culture after virus inoculation was reduced from 37 to 32 degrees C. Moreover, we demonstrated that interferon alpha had antiviral effect on HCV-infected PH5CH8 cells. The three PH5CH clones obtained in this study will provide a useful tool for the study of HCV replication and proliferation, and for development of an assay system for antiviral agents.
我们之前发现，一株感染了I型人类T细胞白血病病毒的T细胞系MT-2对丙型肝炎病毒（HCV）感染敏感，并且克隆的MT-2C细胞比亲代MT-2细胞更能持久支持HCV复制。最近我们发现，一个非肿瘤性肝细胞系PH5CH对HCV感染表现出良好的敏感性。在本研究中，我们克隆了PH5CH细胞以获得能支持更持久HCV复制的细胞，结果得到了三个克隆株（PH5CH1、PH5CH7和PH5CH8），在接种后至少25天（p.i.）仍能检测到细胞内HCV RNA。对HCV RNA的半定量分析表明，在这些克隆的PH5CH细胞中复制的HCV被释放到了培养基中。在用蛋白酶K处理克隆的PH5CH细胞和亲代PH5CH细胞后，对病毒接种后立即内化的HCV RNA进行半定量分析发现，PH5CH1、PH5CH7和PH5CH8细胞中HCV RNA的水平比低敏感性克隆的PH5CH细胞或亲代PH5CH细胞高10倍。此外，我们证明，当病毒接种后细胞培养温度从37℃降至32℃时，这三个克隆株中的HCV复制可维持70 - 100天。而且，我们证明了α干扰素对HCV感染的PH5CH8细胞具有抗病毒作用。本研究中获得的三个PH5CH克隆株将为研究HCV复制和增殖以及开发抗病毒药物检测系统提供有用的工具。
