Productive HBV infection of well-differentiated, hNTCP-expressing human hepatoma-derived(Huh7) cells  

作　　者：Ming Zhou Kaitao Zhao Yongxuan Yao Yifei Yuan Rongjuan Pei Yun Wang Jizheng Chen Xue Hu Yuan Zhou Xinwen Chen Chunchen Wu 

机构地区：[1]State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences [2]Shenzhen Xenotransplantation Research and Development Center, State and Local Joint Cancer Genome Clinical Application of Key Technology Laboratory, Shenzhen Second People's Hospital, First Affiliated Hospital of Shenzhen University [3]Institute of Immunology, Zhongshan School of Medicine, Guangdong Provincial Key Laboratory of Organ Donation and Transplant Immunology, Sun Yat-sen University

出　　处：《Virologica Sinica》2017年第6期465-475,共11页中国病毒学（英文版）

基　　金：supported by the National Natural Science Foundation of China (Grant number: 81601760, 31621061 and 81461130019);General Financial Grant from the China Postdoctoral Science Foundation (Grant number: 2016M602587);the Shenzhen Foundation of Science and Technology (Grant number: JCYJ20160425 104534335);supported by the Youth Innovation Promotion Association CAS (No.201603)

摘　　要：Feasible and effective cell models for hepatitis B virus(HBV) infection are required for investigating the complete lifecycle of this virus, including the early steps of viral entry. Resistance to dimethyl sulfoxide/polyethylene glycol(DMSO/PEG), h NTCP expression, and a differentiated state are the limiting factors for successful HBV infection models. In the present study, we used a hepatoma cell line(Hu7^(hDNTCPh)) to overcome these limiting factors so that it exhibits excellent susceptibility to HBV infection. To achieve this goal, different hepatoma cell lines were tested with 2.5% DMSO/4%PEG8000, and one resistant cell line(Huh7 D) was used to construct a stable h NTCP-expressing cell line(Hu7^(hDNTCPh)) using a recombinant lentivirus system. Then, the morphological characteristics and differentiation molecular markers of Hu7^(hDNTCPh) cells with or without DMSO treatment were characterized. Finally, the susceptibility of Hu7^(hDNTCPh) cells to HBV infection was assessed. Our results showed that Huh7 D cells were resistant to 2.5% DMSO/4% PEG8000, whereas the others were not. Hu7^(hDNTCPh) cells were established to express a high level of h NTCP compared to liver extracts, and Hu7^(hDNTCPh) cells rapidly transformed into a non-dividing, well-differentiated polarized phenotype under DMSO treatment. Hu7^(hDNTCPh) cells fully supported the entire lifecycle of HBV infection. This cell culture system will be useful for the analysis of host-virus interactions, which should facilitate the discovery of antiviral drugs and vaccines.Feasible and effective cell models for hepatitis B virus （HBV） infection are required for investigating the complete lifecycle of this virus, including the early steps of viral entry. Resistance to dimethyl sulfoxide/polyethylene glycol （DMSO/PEG）, hNTCP expression, and a differentiated state are the limiting factors for successful HBV infection models. In the present study, we used a hepatoma cell line （Huh7DhNTcP） to overcome these limiting factors so that it exhibits excellent susceptibility to HBV infection. To achieve this goal, different hepatoma cell lines were tested with 2.5% DMSO 1 4% PEG8000, and one resistant cell line （Huh7D） was used to construct a stable hNTCP-expressing cell line （Huh7DhNTcp） using a recombinant lentivirus system. Then, the morphological characteristics and differentiation molecular markers of Huh7Dh^cw cells with or without DMSO treatment were characterized. Finally, the susceptibility of Huh7DhNTcP cells to HBV infection was assessed. Our results showed that Huh7D cells were resistant to 2.5% DMSO ! 4% PEGS000, whereas the others were not. Huh7DhNTcP cells were established to express a high level of hNTCP compared to liver extracts, and Huh7Dh^TcP cells rapidly transformed into a non.dividing, well-differentiated polarized phenotype under DMSO treatment. Huh7DhNTcP cells fully supported the entire lifecycle of HBV infection. This cell culture system will be useful for the analysis of host-virus interactions, which should facilitate the discovery of antiviral drugs and vaccines.

关 键 词：Hepatitis B virus(HBV) Na+/taurocholate cotransporting polypeptide(NTCP) HUH7 dimethyl sulfoxide(DMSO) polyethylene glycol(PEG) susceptibility 

分 类 号：R512.62[医药卫生—内科学]