Lange Frauke, Garn Jonathan, Bruhn Matthias, Pietschmann Thomas, Carpentier Arnaud
Institute for Experimental Virology, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between Hannover Medical School (MHH) and Helmholtz Centre for Infection Research (HZI), Hannover, Germany.
Institute for Experimental Infection Research, TWINCORE Centre for Experimental and Clinical Infection Research, a joint venture between Hannover Medical School (MHH) and Helmholtz Centre for Infection Research (HZI), Hannover, Germany.
JHEP Rep. 2025 Apr 22;7(8):101429. doi: 10.1016/j.jhepr.2025.101429. eCollection 2025 Aug.
BACKGROUND & AIMS: Stem cell-derived hepatocyte-like cells (HLCs) are an model of hepatocytes reproducing mature hepatic functions. However, heterogeneous or imperfect differentiation may limit their biological relevance. HLCs are susceptible to all primary hepatitis viruses, including HDV. Importantly, HLCs support limited HDV replication, at a significantly lower level than hepatoma cell lines.
We used single-cell RNA sequencing (scRNAseq) to analyse control and HDV-infected HLCs. We assessed maturation and heterogeneity of the HLCs population. We visualised viral genomic and antigenomic sequences abundance and innate immune response at the single-cell level. Further functional characterisation was performed in HLCs and hepatoma cell lines.
HLCs form a population of hepatocytes exhibiting various levels of maturation, with a minor hybrid population of myofibroblast/hepatocyte cells associated with immature phenotype. Upon HDV inoculation, Interferon-Stimulated Genes expression was induced in infected cells, but not in bystander HLCs. Moreover, Interferon Regulatory Factor 1 (IRF1) was enriched in infected HLCs with undetectable levels of viral genome replication, suggesting it may restrict viral infection. Decreasing IRF1 expression in HLCs by 50% improved susceptibility to HDV infection by 10-fold ( <0.01). Moreover, IRF1 overexpression in hepatoma cell lines restored physiological basal level of IRF1 effector antiviral genes and inhibited HDV infection (∼50% reduction after 7 days, <0.01). Importantly, in IRF1 expressing cells, cell division-mediated spread was inhibited and infection was significantly decreased after 2 weeks of culture (>1.5 log decrease, <0.001).
scRNAseq of HLCs identified IRF1 as a potent restriction factor of HDV infection, through an antiviral mechanism blocking HDV infection at an early step of the viral cycle.
We performed single-cell RNA sequencing of control and HDV-inoculated stem cell-derived hepatocytes, and characterised them in terms of hepatic differentiation, viral abundance and response to infection. We identified IRF1 as a constitutive cellular factor restricting HDV infection in mature hepatocytes, particularly targeting HDV in the cytoplasm. This work improves our understanding of mature cell culture models for HDV, needed to decipher its host-pathogens interactions. Identification of antiviral effector genes opens the way to the development of new host targeted antiviral strategies, particularly for targeting cell division-mediated spread that is not inhibited by currently used entry inhibitors (Hepcludex).
干细胞来源的肝样细胞（HLCs）是再现成熟肝功能的肝细胞模型。然而，异质性或不完全分化可能会限制其生物学相关性。HLCs对所有原发性肝炎病毒均易感，包括丁型肝炎病毒（HDV）。重要的是，HLCs支持有限的HDV复制，其水平显著低于肝癌细胞系。
我们使用单细胞RNA测序（scRNAseq）分析对照和HDV感染的HLCs。我们评估了HLCs群体的成熟度和异质性。我们在单细胞水平上可视化了病毒基因组和反基因组序列的丰度以及先天免疫反应。在HLCs和肝癌细胞系中进行了进一步的功能表征。
HLCs形成了一群表现出不同成熟水平的肝细胞群体，还有一小部分与未成熟表型相关的肌成纤维细胞/肝细胞杂交群体。接种HDV后，感染细胞中诱导了干扰素刺激基因表达，但旁观者HLCs中未诱导。此外，干扰素调节因子1（IRF1）在病毒基因组复制水平不可检测的感染HLCs中富集，表明它可能限制病毒感染。将HLCs中IRF1的表达降低50%可使对HDV感染的易感性提高10倍（P<0.01）。此外，在肝癌细胞系中过表达IRF1可恢复IRF1效应抗病毒基因的生理基础水平并抑制HDV感染（7天后降低约50%，P<0.01）。重要的是，在表达IRF1的细胞中，细胞分裂介导的传播受到抑制，培养2周后感染显著减少（>1.5个对数级下降，P<0.001）。
HLCs的scRNAseq确定IRF1是HDV感染的有效限制因子，通过一种抗病毒机制在病毒周期的早期阶段阻断HDV感染。
我们对对照和接种HDV的干细胞来源的肝细胞进行了单细胞RNA测序，并在肝分化、病毒丰度和感染反应方面对其进行了表征。我们确定IRF1是一种在成熟肝细胞中限制HDV感染的组成性细胞因子，特别是在细胞质中靶向HDV。这项工作增进了我们对HDV成熟细胞培养模型的理解，这对于解读其宿主-病原体相互作用是必要的。抗病毒效应基因的鉴定为开发新的宿主靶向抗病毒策略开辟了道路，特别是针对目前使用的进入抑制剂（Hepcludex）未抑制的细胞分裂介导的传播。
