Comparative analysis of gene expression in HDV-infected dHepaRG cells upon treatment with IFN-a-2a or IFN-?1

Hepatitis D virus (HDV) causes the most severe form of chronic viral hepatitis, often leading to advanced liver disease and hepatocellular carcinoma. Viral cure is rarely achieved in infected patients. Interferon-alpha (IFN-a)-based therapies show suboptimal efficacy and low rates of sustained response, as reflected by their limited effect on HDV replication in vitro. Here, we show that HDV infection induces cellular resistance to IFN-a, marked by impaired STAT1 phosphorylation and reduced expression of interferon-stimulated genes (ISGs) in hepatocyte-like cells. This refractoriness depends on virus-induced innate immune activation, highlighting the role of HDV-induced ISG expression in regulating IFN signalling. We identify USP18 as a key mediator of IFN-a resistance in infected cells. Notably, ISG expression in response to type III IFN (IFN-?) remains intact, consistent with USP18's selective inhibition of IFN-a signalling. Collectively, these findings reveal the molecular mechanism of IFN-a resistance in HDV-infected hepatocytes and provide a rationale for developing novel therapies against this major public health threat. Overall design: For transcriptomic analyses, differentiated HepaRG (dHepaRG) cells were either inoculated with HDV virions at a multiplicity of infection (MOI) of approximately 100 viral genome equivalents (vge) per cell for 6 days or left non-infected. Cells were then treated for 8 h with either IFN-alpha-2a (500 IU/mL) or IFN-lambda-1 (100 ng/mL). As controls, non-infected dHepaRG cells were treated for 8 h with IFN-alpha-2a (500 IU/mL) or IL-29/IFN-lambda-1 (100 ng/mL). Additional control conditions included infection of dHepaRG cells for 8 h with Sendai virus (SeV, Cantell strain; AVS Bio) at 10 hemagglutination units (HAU)/mL or with encephalomyocarditis virus (EMCV, Sigean strain; kindly provided by Labib Bakkali-Kassimi, Anses, France) at an MOI of 0.70 vge/cell. Cells were lysed and total intracellular RNA was extracted using the Monarch Nucleic Acid Purification Kit (New England Biolabs).