Single-cell analysis of mature hepatocytes reveals an IRF1-driven restriction of HDV infection

Stem cell-derived hepatocyte-like cells (HLCs) are an in vitro 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 (p <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, p <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, p <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).