丙型肝炎病毒高水平复制细胞株的构建及其机制初步研究

旨在探讨丙型肝炎病毒(hepatitis C virus, HCV)cured细胞株的易感机制。本研究将体外转录的HCV RNA电转入肝癌细胞系Huh 7细胞，建立HCV复制子细胞株，用 γ-干扰素(interferon，IFN)处理复制子细胞株，获得HCV cured Huh 7A和Huh 7B细胞株。用插入报告基因的HCV毒株Jc1-G感染上述细胞株，分别进行荧光素酶活性测定、蛋白质印迹法和荧光定量聚合酶链反应(polymerase chain reaction，PCR)检测以验证其易感性。收集Huh 7、Huh 7.5、Huh 7A和Huh 7B细胞并利用IFN-α处理，之后用蛋白质印迹法及荧光定量PCR进行检测，验证细胞株中IFN诱生信号通路中关键因子内源性表达及抗病毒活性ISGs的激活水平。结果显示，在Huh 7A和Huh 7B细胞中检测不到病毒RNA，与Huh 7细胞一致。病毒感染实验中，与Huh 7细胞相比，Huh 7A和Huh 7B细胞株中荧光素酶活性增高百倍，病毒蛋白表达和RNA水平亦显著上调，与Huh 7.5细胞株中的表达水平接近。IFN信号通路实验中，与Huh 7细胞相比，Huh 7A和Huh 7B细胞株中RIG-I/MDA5/MAVS内源性蛋白表达和mRNA水平无明显差异；IFN-α处理细胞后IFN刺激基因isg56，mx1，mx2，oax1，oax2，viperin，cxcl10，ifitm1和ifitm3激活水平亦无显著变化。结果提示，本研究制备的Huh 7A和Huh 7B细胞株可支持HCV高水平复制，将为研究病毒复制机制提供有力的支持。

Generation of Huh 7 cured cell line with high permissiveness for hepatitis C virus replication

To establish cell line with high permissiveness for hepatitis C virus (HCV)infection, we established Huh 7 cell lines that harboring a HCV subgenomic replicon (sgJFH1) by blasticidin selection and then treated these replicon cells with interferon gamma to generate HCV cured cells. Two single clones were established (Huh 7A and Huh 7B, respectively). To determine whether these cured cells are more permissive for HCV, Huh7A and Huh7B cells were infected with the Jc1G strain of HCV and the activity of reporter gene Gaussia luciferase (Gluc), HCV protein and RNA level were determined. HCV has an intrinsic ability to trigger interferon production through RIG-I and MDA5 and its adaptors MAVS. To determine whether RIG-I or MDA5 or MAVS play a key role in permissive cell lines, we first analyzed the endogenous expression level of these proteins. Interferon-stimulated genes (ISGs) including ISG56, OAS1, OAS2, Viperin, CXCL10, IFITM1 and IFITM3 have been reported to control viral infection by directly interfering with the pathways and functions required during viral infection. To examine the capacity of the cured cell lines to induce ISGs by IFN, we determined the effect of interferon-α on the expression of ISGs associated with antiviral function in these permissive cell lines. Results showed that the HCV RNA could not be detected in cured cell lines Huh7A and Huh7B. In Huh7A and Huh7B cells infected with Jc1G, the Gluc activity was as high as that in Huh 7.5 cells and nearly 100 folds more than that in Huh 7 cells. Consistently, both HCV RNA and NS3 protein expression levels in Huh 7A, Huh 7B cells and Huh 7.5 cells increased significantly compared to that in Huh7 cells. Compared with parental Huh 7 cells, the expression of these proteins in Huh 7A and Huh 7B cells shows no obvious differences. The same results were observed in their mRNA level. Strikingly, in addition to RIG-I, MDA5 mRNA and protein levels in Huh 7.5 cells were also much less than that in Huh7 as well as Huh7A and Huh7B cells. The results showed that there was no significant difference in the stimulation of ISGs between Huh 7 cells and highly permissive cell lines, including MX1 that was reported to be responsible for the permissiveness for HCV replication in permissive cell lines. To further confirm the role of MX1 in our highly permissive cell lines, we treated cells with different concentration of interferon-α and found out that the expression levels of MX1 were still comparable among the four cell lines at indicated concentration. The data suggested that MX1 may not be associated with permissiveness of Huh 7.5 cells as well as Huh 7A and Huh 7B cells. These results showed that Huh 7A and Huh 7B cells were more permissive for HCV infection, which may provide a useful cell model to study HCV replication and identify novel targets for antiviral drugs.