Differentiated cells in prolonged hypoxia produce highly infectious native-like hepatitis C virus particles.

Standard Hepatitis C virus (HCV) cell culture models present an altered lipid metabolism and thus produce lipid-poor lipo-viral-particles (LVPs). These models are thereby weakly adapted to explore the complete natural viral life cycle. To overcome these limitations, we used an HCV cell culture model based on both cellular differentiation and sustained hypoxia to better mimic host cell environment. The long-term exposure of Huh7.5 cells to DMSO and hypoxia (1% O2 ) significantly enhanced the expression of major differentiation markers and the cellular hypoxia adaptive response by contrast with undifferentiated and normoxic (21% O2 ) standard conditions. Since hepatocyte-like differentiation and hypoxia are key regulators of intracellular lipid metabolism, we characterized the distribution of lipid droplets (LDs) and demonstrated that experimental cells significantly accumulate larger and more numerous LDs relative to standard cell culture conditions. An immunocapture and transmission electron microscopy method (IC-TEM) showed that differentiated and hypoxic Huh7.5 cells produced lipoproteins significantly larger than those produced by standard Huh7.5 cell cultures. The experimental cell culture model is permissive to HCV-JFH1 infection and produces very low buoyant density LVPs 6-fold more infectious than LVPs formed by standard JFH1-infected Huh7.5 cells. Finally, the IC-TEM approach and antibodies neutralization experiments revealed that LVPs were highly lipidated with a global ultrastructure and a E1E2-complex conformation close to the ones circulating in infected individuals. CONCLUSIONS: This relevant HCV cell culture model thus mimics the complete native intracellular HCV life cycle and, by extension, can be proposed as a model of choice for studies of other hepatotropic viruses.