The human liver is an essential multifunctional organ, and liver diseases are rising with limited treatment options. However, the cellular composition of the liver remains poorly understood. Here, we performed single-cell RNA-sequencing of ~10,000 cells from normal liver tissue of 9 human donors to construct a human liver cell atlas. Our analysis revealed previously unknown sub-types among endothelial cells, Kupffer cells, and hepatocytes with transcriptome-wide zonation of some of these populations. We reveal heterogeneity of the EPCAM + population, which comprises hepatocyte-biased and cholangiocyte populations as well as a TROP2 int progenitor population with strong potential to form bipotent liver organoids. As proof-of-principle, we utilized our atlas to unravel phenotypic changes in hepatocellular carcinoma cells and in human hepatocytes and liver endothelial cells engrafted into a mouse liver. Our human liver cell atlas provides a powerful resource enabling the discovery of previously unknown cell types in the normal and diseased liver.

Hepatitis C virus (HCV) entry is dependent on coreceptor complex formation between the tetraspanin superfamily member CD81 and the tight junction protein claudin-1 (CLDN1) on the host cell membrane. The receptor tyrosine kinase EGFR acts as a cofactor for HCV entry by promoting CD81-CLDN1 complex formation via unknown mechanisms. We identify the GTPase HRas, activated downstream of EGFR signaling, as a key host signal transducer for EGFR-mediated HCV entry. Proteomic analysis revealed that HRas associates with tetraspanin CD81, CLDN1, and the previously unrecognized HCV entry cofactors integrin β1 and Ras-related protein Rap2B in hepatocyte membranes. HRas signaling is required for lateral membrane diffusion of CD81, which enables tetraspanin receptor complex assembly. HRas was also found to be relevant for entry of other viruses, including influenza. Our data demonstrate that viruses exploit HRas signaling for cellular entry by compartmentalization of entry factors and receptor trafficking.