Continuous de novo generation of spatially segregated hepatitis C virus replication organelles revealed by pulse-chase imaging.
Like all positive-sense RNA viruses, hepatitis C virus (HCV) induces host membrane alterations for its replication. In chronically infected cells, it is not known whether these viral replication organelles are being continually resupplied by newly synthesized viral proteins in situ, or whether they are generated de novo. Here we aimed to study temporal events in replication organelles formation and maturation.



Here we use pulse-chase labeling in combination with confocal microscopy, correlative light electron microscopy and biochemical methods to identify temporally distinct populations of replication organelles in living cells and study the formation, morphogenesis as well as compositional and functional changes of replication organelles over time.



We found that HCV replication organelles are continuously generated de novo at spatially distinct sites from preformed ones. This process is accompanied by accumulated intracellular membrane alteration, increased cholesterol delivery, NS5A phosphorylation, and positive-strand RNA content, and by eventual association with HCV core protein around lipid droplets. Generation of spatially segregated foci requires viral NS5A and the host factors phosphatidylinositol 4-kinase and oxysterol-binding protein, while association of foci with lipid droplets requires cholesterol.



Our results reveal that HCV replication organelles are not static structures, but instead are continuously generated and dynamically change in composition and possibly also in function.



Hepatitis C virus replication membrane structures are continuously generated at spatially distinct sites. New replication organelles are different in composition, and possibly also in function, compared to old replication organelles.
Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, United States.
Hongliang
H
Wang
Division of Gastroenterology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, United States; Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, United States; Medicine Service, Ann Arbor Veterans Administration Health System, Ann Arbor, MI, United States. Electronic address: andrewwt@med.umich.edu.
Andrew W
AW
Tai
D019870:1-Phosphatidylinositol 4-Kinase / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D016174:Hepacivirus / Q000235:genetics / Q000502:physiology; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D007425:Intracellular Membranes / Q000502:physiology; D015388:Organelles / Q000502:physiology*; D012367:RNA, Viral / Q000032:analysis; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D011987:Receptors, Steroid / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014773:Virology / Q000379:methods; D000098825:Oxysterol Binding Proteins
D012367:RNA, Viral; D011987:Receptors, Steroid; D017361:Viral Nonstructural Proteins; D000098825:Oxysterol Binding Proteins; D002784:Cholesterol; D019870:1-Phosphatidylinositol 4-Kinase; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
Cholesterol; Double membrane vesicles; Hepatitis C virus; Organelles; Pulse-chase imaging; SNAP; Viral replication