title: Development and application of hepatitis C reporter viruses with genotype 1 to 7 core-nonstructural protein 2 (NS2) expressing fluorescent proteins or luciferase in modified JFH1 NS5A.
abstract: To facilitate genotype-specific high-throughput studies of hepatitis C virus (HCV), we have developed reporter viruses using JFH1-based recombinants expressing core-nonstructural protein 2 (NS2) of genotype 1 to 7 prototype isolates. We introduced enhanced green fluorescent protein (EGFP) into NS5A domain III of the genotype 2a virus J6/JFH1 [2a(J6)]. During Huh7.5 cell culture adaptation, 2a(J6)-EGFP acquired a 40-amino-acid (aa) (Δ40) or 25-aa (Δ25) deletion in NS5A domain II, rescuing the impairment of viral assembly caused by the EGFP insertion. Δ40 conferred efficient growth characteristics to 2a(J6) tagged with EGFP, DsRed-Express2, mCherry, or Renilla luciferase (RLuc), yielding peak supernatant infectivity titers of 4 to 5 log(10) focus-forming units (FFU)/ml. 2a(J6) with Δ40 or Δ25 was fully viable in Huh7.5 cells. In human liver chimeric mice, 2a(J6)-EGFPΔ40 acquired various deletions in EGFP, while 2a(J6)Δ40 did not show an impaired viability. We further developed panels of JFH1-based genotype 1 to 7 core-NS2 recombinants expressing EGFP- or RLuc-NS5AΔ40 fusion proteins. In cell culture, the different EGFP recombinants showed growth characteristics comparable to those of the nontagged recombinants, with peak infectivity titers of 4 to 5 log(10) FFU/ml. RLuc recombinants showed slightly less efficient growth characteristics, with peak infectivity titers up to 10-fold lower. Overall, the EGFP and RLuc recombinants were genetically stable after one viral passage. The usefulness of these reporter viruses for high-throughput fluorescence- and luminescence-based studies of HCV-receptor interactions and serum-neutralizing antibodies was demonstrated. Finally, using RLuc viruses, we showed that the genotype-specific core-NS2 sequence did not influence the response to alfa-2b interferon (IFN-alfa-2b) and that genotype 1 to 7 viruses all responded to treatment with p7 ion channel inhibitors.
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Copenhagen University Hospital, Hvidovre, Kettegaard Alle 30, DK-2650 Hvidovre, Denmark.
authors.forename: Judith M
authors.initials: JM
authors.lastname: Gottwein
authors.forename: Tanja B
authors.initials: TB
authors.lastname: Jensen
authors.forename: Christian K
authors.initials: CK
authors.lastname: Mathiesen
authors.forename: Philip
authors.initials: P
authors.lastname: Meuleman
authors.forename: Stephanie B N
authors.initials: SB
authors.lastname: Serre
authors.forename: Jacob B
authors.initials: JB
authors.lastname: Lademann
authors.forename: Lubna
authors.initials: L
authors.lastname: Ghanem
authors.forename: Troels K H
authors.initials: TK
authors.lastname: Scheel
authors.forename: Geert
authors.initials: G
authors.lastname: Leroux-Roels
authors.forename: Jens
authors.initials: J
authors.lastname: Bukh
mesh_terms: D000818:Animals; D002460:Cell Line; D017930:Genes, Reporter; D042822:Genomic Instability; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D016174:Hepacivirus / Q000235:genetics / Q000254:growth & development*; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D050296:Microbial Viability; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014758:Viral Core Proteins / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D049452:Green Fluorescent Proteins; D008156:Luciferases; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; C097010:NS2 protein, Hepatitis C virus; C485184:enhanced green fluorescent protein; C069130:nucleocapsid protein, Hepatitis C virus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase