title: Generation of a chimeric hepatitis C replicon encoding a genotype-6a NS3 protease and assessment of boceprevir (SCH503034) sensitivity and drug-associated mutations.
abstract: BACKGROUND
  Genotype (gt)6 HCV is common amongst HCV-positive populations of the Asia-Pacific region but cell culture models for this gt have only recently been developed. Boceprevir (SCH503034) is a clinically available inhibitor of the HCV NS3 protein. We investigated the efficacy of boceprevir for inhibiting replication of a chimeric gt1b replicon encoding a gt6a NS3 protease and defined the development of mutations in the protease when boceprevir treatment was applied.
  
  
  METHODS
  We constructed a chimeric gt1b subgenomic replicon encoding a gt6 NS3 protease (NS3p) sequence (gt6NS3p_gt1b). The boceprevir EC50 value against replication of this replicon was determined using quantitative reverse transcriptase PCR. Next-generation sequencing was used to identify nucleotide changes associated with boceprevir resistance. The replication capacities of chimeric replicons containing mutations associated with boceprevir resistance were determined by colony formation efficiency assays.
  
  
  RESULTS
  The boceprevir EC50 value for the gt6NS3p_gt1b replicon was 535 ±79 nM. Boceprevir-resistant gt6NS3p_gt1b replicon cell lines could be selected and they demonstrated drug-associated amino acid changes that have previously been reported in other HCV gts. Interestingly, no mutations were observed at A156, a position defined for boceprevir resistance in gt1 NS3p, while mutation at N122, which is rarely reported in boceprevir-resistant gt1 proteases, was frequently observed. Re-introduction of these mutations into the chimeric replicon altered their replication capacity, ranging from complete abolishment of replication (A156T) to increasing replication capacity (V36A, N122S). This report provides the first characterization of gt6 HCV resistance to boceprevir.
  
  
  CONCLUSIONS
  A chimeric HCV replicon encoding gt6 NS3 protease is sensitive to boceprevir and develops drug-resistant mutations at amino acid sites previously reported for other gts. Mutation at N122 also appears to be associated with boceprevir resistance in the gt6 NS3 protease.
authors.affiliation: School of Molecular and Biomedical Science, Adelaide, The University of Adelaide and Centre for Cancer Biology, SA Pathology, Adelaide, South Australia, Australia.
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Aloia
authors.forename: Nicholas S
authors.initials: NS
authors.lastname: Eyre
authors.forename: Stuart
authors.initials: S
authors.lastname: Black
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Bent
authors.forename: Adriana
authors.initials: A
authors.lastname: Gaeguta
authors.forename: Zhuyan
authors.initials: Z
authors.lastname: Guo
authors.forename: Sumudu K
authors.initials: SK
authors.lastname: Narayana
authors.forename: Robert
authors.initials: R
authors.lastname: Chase
authors.forename: Stephen
authors.initials: S
authors.lastname: Locarnini
authors.forename: Jill M
authors.initials: JM
authors.lastname: Carr
authors.forename: John A
authors.initials: JA
authors.lastname: Howe
authors.forename: Michael R
authors.initials: MR
authors.lastname: Beard
mesh_terms: D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use; D045744:Cell Line, Tumor; D024882:Drug Resistance, Viral / Q000235:genetics*; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000188:drug therapy / Q000821:virology*; D006801:Humans; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D011392:Proline / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use; D011995:Recombination, Genetic; D012093:Replicon; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics*; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D011392:Proline; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; 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