Persistent Hepatocyte Plasticity and Lipid Metabolic Reprogramming After Hepatitis C Virus Cure

Chronic hepatitis C virus (HCV) infection disrupts hepatocyte polarity and promotes epithelial-mesenchymal transition (EMT), contributing to liver disease progression. Although direct-acting antivirals (DAAs) eradicate HCV infection in most patients, the risk of fibrosis progression and hepatocellular carcinoma persists after viral clearance. Whether antiviral cure fully restores hepatocyte differentiation, polarity, and epithelial identity programs remains unclear. We investigated whether HCV-induced EMT and lipid metabolism alterations are fully reversible following DAA treatment.

Huh7 hepatoma cells and Huh7 cells stably expressing a full-length HCV replicon were cultured in three-dimensional Matrigel to assess morphogenesis, polarity-associated features, and EMT. Spheroid morphology, canalicular-like organization, EMT markers, and transcriptomic signatures were analyzed using microscopy, immunofluorescence, RT-qPCR, and microarray analysis. The effects of DAA treatment (sofosbuvir with or without velpatasvir) were evaluated in vitro. Publicly available transcriptomic data from liver biopsies collected before therapy and within five days after its completion, were analyzed.

HCV replicon expression altered canalicular-like organization and induced actin-rich protrusions in 90% of Huh7-R spheroids. DAA treatment efficiently suppressed HCV replicon expression and partially restored epithelial morphology, reducing protrusions to 25%, without normalizing them to the control level (2.5%). EMT-associated changes, including reduced E-cadherin and increased N-cadherin expression, were only partially corrected by DAA treatment. Transcriptomic EMT scoring showed persistent mesenchymal features in DAA-treated cells and in patient biopsies collected at the end of treatment. In parallel, genes involved in lipid metabolism and hepatocyte differentiation remained dysregulated.

These findings show incomplete restoration of epithelial organization after suppression of HCV replicon expression in vitro and incomplete normalization of selected transcriptional signatures at the end of DAA treatment in patient liver samples. They suggest that cellular plasticity and metabolic reprogramming may persist after treatment and contribute to long-term liver disease risk.