title: Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7.
abstract: Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (cLDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Current knowledge on assembly and release is primarily based on studies in genotype 2a cell culture systems; however, given the high genetic heterogeneity of HCV, variations might exist among genotypes. Here, we developed novel HCV strain JFH1-based recombinants expressing core-NS2 and NS5A from genotypes 1-7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core-NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core-NS2/NS5A recombinants produced infectivity titres of 10(2.5)-10(4.5) f.f.u. ml(-1). Co-localization analysis demonstrated that the core and NS5A proteins from all genotypes co-localized extensively, and there was no significant difference in protein co-localization among genotypes. In addition, we found that the core and NS5A proteins were highly associated with cLDs at 12 h post-infection but became mostly ER associated at later stages. Finally, we found that different genotypes showed varying levels of core/cLD co-localization, with a possible effect on viral assembly/release. In summary, we developed a panel of HCV genotype 1-7 core-NS2/NS5A recombinants producing infectious virus, and an immunostaining protocol detecting the core and NS5A proteins from seven different genotypes. These systems will allow, for the first time, investigation of core/NS5A interactions during assembly and release of HCV particles of all major genotypes.
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Andrea
authors.initials: A
authors.lastname: Galli
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Troels K H
authors.initials: TKH
authors.lastname: Scheel
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Jannick C
authors.initials: JC
authors.lastname: Prentoe
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Lotte S
authors.initials: LS
authors.lastname: Mikkelsen
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Judith M
authors.initials: JM
authors.lastname: Gottwein
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Jens
authors.initials: J
authors.lastname: Bukh
mesh_terms: D006528:Carcinoma, Hepatocellular; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly / Q000502:physiology; D014776:Virus Cultivation / Q000379:methods; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
chemical_list: D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase