Hepatitis B virus compartmentalization and single-cell differentiation in hepatocellular carcinoma

Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) world-wide. The molecular mechanisms of viral hepatocarcinogenesis are still partially understood. Here, we applied two complementary single-cell RNA-sequencing protocols to investigate HBV–HCC host cell interactions at the single cell level of patient-derived HCC. Computational analyses revealed a marked HCC heterogeneity with a robust and significant correlation between HBV reads and cancer cell differentiation. Viral reads significantly correlated with the expression of HBV-dependency factors such asHLFin different tumor compartments. Analyses of virus-induced host responses identified previously undiscovered pathways mediating viral carcinogenesis, such as E2F- and MYC targets as well as adipogenesis. Mapping of fused HBV–host cell transcripts allowed the characterization of integration sites in individual cancer cells. Collectively, single-cell RNA-Seq unravels heterogeneity and compartmentalization of both, virus and cancer identifying new candidate pathways for viral hepatocarcinogenesis. The perturbation of pro-carcinogenic gene expression even at low HBV levels highlights the need of HBV cure to eliminate HCC risk.