title: Differential sensitivity of 5'UTR-NS5A recombinants of hepatitis C virus genotypes 1-6 to protease and NS5A inhibitors.
abstract: OBJECTIVE
  Hepatitis C virus (HCV) therapy will benefit from the preclinical evaluation of direct-acting antiviral (DAA) agents in infectious culture systems that test the effects on different virus genotypes. We developed HCV recombinants comprising the 5' untranslated region-NS5A (5-5A) from genotypes 1-6 and 2a(JFH1) NS5B-3' untranslated region, and tested the effects of NS3 protease and NS5A inhibitors on these recombinants.
  
  
  METHODS
  The HCV 5-5A recombinants with previously identified mutations in the NS3-helicase (F1464L), NS4A (A1672S), and NS5B (D2979G) were adapted and improved, by incorporating additional recovered mutations that increased their propagation in Huh7.5 cells. Concentration-response profiles were determined for each DAA agent in replicate infected Huh7.5 cells.
  
  
  RESULTS
  Developed efficient 1a(H77), 1a(TN), 3a(S52), 4a(ED43), 5a(SA13), and 6a(HK6a) 5-5A recombinants did not require mutations after viral passage in the NS3 protease or NS5A domain-I regions targeted by the drugs. They were inhibited in a concentration-dependent manner by the NS3 protease inhibitors telaprevir, boceprevir, asunaprevir, simeprevir, vaniprevir, faldaprevir, and MK-5172 and by the NS5A inhibitor daclatasvir. The 1a(TN) 5-5A and JFH1-independent full-length viruses had similar levels of sensitivity to the DAA agents, validating the 5-5A recombinants as surrogates for full-length viruses in DAA testing. Compared with the 1a(TN) full-length virus, the 3a(S52) 5-5A recombinant was highly resistant to all protease inhibitors, and the 4a(ED43) recombinant was highly resistant to telaprevir and boceprevir, but most sensitive to other protease inhibitors. Compared with other protease inhibitors, MK-5172 had exceptional potency against all HCV genotypes. The NS5A inhibitor daclatasvir had the highest potency observed, but with genotype-dependent activity.
  
  
  CONCLUSIONS
  The mutations F1464L, A1672S, and D2979G permitted the development of efficient HCV recombinants comprising genotype-specific 5' untranslated region-NS5A (5-5A), which include the natural NS3 protease and NS5A domain-I drug targets. The robust replication of adapted 5-5A recombinants allowed for direct comparison of NS3 protease and NS5A inhibitors against HCV strains of genotypes 1-6.
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Li
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Santseharay
authors.initials: S
authors.lastname: Ramirez
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Daryl
authors.initials: D
authors.lastname: Humes
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Sanne B
authors.initials: SB
authors.lastname: Jensen
authors.affiliation: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital 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, Hvidovre Hospital and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Electronic address: jbukh@sund.ku.dk.
authors.forename: Jens
authors.initials: J
authors.lastname: Bukh
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002219:Carbamates; D006528:Carcinoma, Hepatocellular / Q000473:pathology / Q000821:virology; D002352:Carrier Proteins / Q000235:genetics; D045744:Cell Line, Tumor; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008099:Liver / Q000187:drug effects / Q000473:pathology / Q000821:virology; D008113:Liver Neoplasms / Q000473:pathology / Q000821:virology; D009154:Mutation / Q000235:genetics; D009842:Oligopeptides / Q000494:pharmacology; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology*; D011759:Pyrrolidines; D014633:Valine / Q000031:analogs & derivatives; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
chemical_list: D000998:Antiviral Agents; D002219:Carbamates; D002352:Carrier Proteins; D007093:Imidazoles; D047908:Intracellular Signaling Peptides and Proteins; D009842:Oligopeptides; D011392:Proline; D011480:Protease Inhibitors; D011759:Pyrrolidines; D014633:Valine; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus; C486464:telaprevir; C512204:N-(3-amino-1-(cyclobutylmethyl)-2,3-dioxopropyl)-3-(2-((((1,1-dimethylethyl)amino)carbonyl)amino)-3,3-dimethyl-1-oxobutyl)-6,6-dimethyl-3-azabicyclo(3.1.0)hexan-2-carboxamide; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C549273:daclatasvir
keywords: Cultures; Drug Resistance; PI; Vaccine