Generation of infectious hepatitis C virus in immortalized human hepatocytes
Generation of infectious hepatitis C virus in immortalized human hepatocytes
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DOI：
10.1128/jvi.80.9.4633-4639.2006
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发表时间：
2006-05-01
影响因子：
5.4
通讯作者：
Ray, RB
中科院分区：
文献类型：
--
作者：
Kanda, T;Basu, A;Ray, RB
由于缺乏有效的用于病毒生长的细胞培养系统，了解丙型肝炎病毒（HCV）生物学的进展仍然是一个挑战。在这项研究中，我们检测了HCV核心蛋白介导的永生化人肝细胞（IHH）对HCV生长的影响。通过电穿孔将HCV基因型la（克隆H77）的体外转录全长RNA导入IHH。从HCV基因组转染的IHH中分离的细胞RNA的逆转录pcr表明，病毒RNA发生了复制。用HCV全长基因组转染的IHH也通过间接免疫荧光显示病毒蛋白表达。相反，转染聚合酶缺陷型HCV (H77/GND) RNA作为阴性对照的细胞没有表现出病毒基因组的表达。免疫金标记证实了El蛋白在转染rna的IHH粗内质网中的定位。细胞质中可见50 nm左右的病毒样颗粒。用转染HCV全长基因组的细胞培养基接种后，幼稚IHH以稀释依赖的方式表达NS5a蛋白，但事先用HCV感染患者血清孵育培养基抑制NS5a的表达。转染全长H77 RNA的IHH细胞培养基的ns5a阳性免疫荧光产生4.5 × 10(4) ~ 1 × 10(5)个病灶形成单位/ml。将HCV基因型2a （JFH1）的RNA转染到IHH中，观察到类似水平的病毒生长。综上所述，我们的结果表明IHH支持HCV基因组复制和病毒组装。
Progress in understanding hepatitis C virus (HCV) biology has remained a challenge due to the lack of an efficient cell culture system for virus growth. In this study, we examined HCV core protein-mediated immortalized human hepatocytes (IHH) for growth of HCV. In vitro-transcribed full-length RNA from HCV genotype la (clone H77) was introduced into IHH by electroporation. Reverse transcription-PCR of cellular RNA isolated from HCV genome-transfected IHH suggested that viral RNA replication occurred. IHH transfected with the full-length HCV genome also displayed viral protein expression by indirect immunofluorescence. In contrast, cells transfected with polymerase-defective HCV (H77/GND) RNA as a negative control did not exhibit expression of the viral genome. Immunogold labeling demonstrated localization of El protein in the rough endoplasmic reticulum of RNA-transfected IHH. Virus-like particles of similar to 50 nm were observed in the cytoplasm. After being inoculated with culture media of cells transfected with the full-length HCV genome, naive IHH displayed NS5a protein expression in a dilution-dependent manner, but expression of NS5a was inhibited by prior incubation of culture medium with HCV-infected patient sera. NS5a-positive immunofluorescence of cell culture media of IHH transfected with full-length H77 RNA yielded similar to 4.5 X 10(4) to 1 X 10(5) focus-forming units/ml. A similar level of virus growth was observed upon transfection of RNA from HCV genotype 2a (JFH1) into IHH. Taken together, our results suggest that IHH support HCV genome replication and virus assembly.