Generation of Huh 7 cured cell line with high permissiveness for hepatitis C virus replication
SONG Wuhui, YI Zhigang
Key Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), and Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Shanghai Medical College, Fudan University, Shanghai 200032, China
Abstract：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.
宋武慧，易志刚. 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[J]. 微生物与感染, 2020, 15(5): 285-292.
SONG Wuhui, YI Zhigang. Generation of Huh 7 cured cell line with high permissiveness for hepatitis C virus replication. JOURNAL OF MICROBES AND INFECTIONS, 2020, 15(5): 285-292.