Differentiated Cells in Prolonged Hypoxia Produce Highly Infectious Native‐Like Hepatitis C Virus Particles

Background and Aims Standard hepatitis C virus (HCV) cell‐culture models present an altered lipid metabolism and thus produce lipid‐poor lipoviral particles (LVPs). These models are thereby weakly adapted to explore the complete natural viral life cycle. Approach and Results To overcome these limitations, we used an HCV cell‐culture model based on both cellular differentiation and sustained hypoxia to better mimic the host‐cell environment. The long‐term exposure of Huh7.5 cells to DMSO and hypoxia (1% O 2 ) significantly enhanced the expression of major differentiation markers and the cellular hypoxia adaptive response by contrast with undifferentiated and normoxic (21% O 2 ) standard conditions. Because 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 (IC) and transmission electron microscopy (TEM) method 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–Japanese fulminant hepatitis (JFH1) infection and produces very‐low‐buoyant‐density LVPs that are 6‐fold more infectious than LVPs formed by standard JFH1‐infected Huh7.5 cells. Finally, the IC–TEM approach and antibody‐neutralization experiments revealed that LVPs were highly lipidated, had a global ultrastructure and a conformation of the envelope glycoprotein complex E1E2 close to that of 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.