State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071, China.
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, Shenzhen, 518035, China.
Virol Sin. 2017 Dec;32(6):465-475. doi: 10.1007/s12250-017-3983-x. Epub 2017 Sep 29.
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 (Huh7D) 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 (Huh7D) was used to construct a stable hNTCP-expressing cell line (Huh7D) using a recombinant lentivirus system. Then, the morphological characteristics and differentiation molecular markers of Huh7D cells with or without DMSO treatment were characterized. Finally, the susceptibility of Huh7D cells to HBV infection was assessed. Our results showed that Huh7D cells were resistant to 2.5% DMSO / 4% PEG8000, whereas the others were not. Huh7D cells were established to express a high level of hNTCP compared to liver extracts, and Huh7D cells rapidly transformed into a non-dividing, well-differentiated polarized phenotype under DMSO treatment. Huh7D 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.
需要能够有效感染乙型肝炎病毒 (HBV) 的细胞模型来研究该病毒的完整生命周期，包括病毒进入的早期步骤。抵抗二甲基亚砜/聚乙二醇 (DMSO/PEG)、人钠离子牛磺胆酸共转运蛋白 (hNTCP) 的表达和分化状态是成功建立 HBV 感染模型的限制因素。在本研究中，我们使用肝癌细胞系 (Huh7D) 来克服这些限制因素，使其对 HBV 感染具有极好的易感性。为了实现这一目标，我们用 2.5% DMSO/4% PEG8000 测试了不同的肝癌细胞系，选择一株具有抗性的细胞系 (Huh7D)，使用重组慢病毒系统构建稳定表达 hNTCP 的细胞系 (Huh7D)。然后，我们对未经 DMSO 处理和经 DMSO 处理的 Huh7D 细胞的形态特征和分化分子标记进行了分析。最后，我们评估了 Huh7D 细胞对 HBV 感染的敏感性。结果显示， Huh7D 细胞对 2.5% DMSO/4% PEG8000 具有抗性，而其他细胞系则没有。与肝提取物相比， Huh7D 细胞能够高表达 hNTCP，并且在 DMSO 处理下， Huh7D 细胞迅速转化为非分裂、高度分化的极化表型。 Huh7D 细胞完全支持 HBV 感染的整个生命周期。这种细胞培养系统将有助于分析宿主-病毒相互作用，这应该有助于发现抗病毒药物和疫苗。
