title: Development of a chimeric replicon system for phenotypic analysis of NS3 protease sequences from HCV clinical isolates.
abstract: BACKGROUND
  To support clinical development of HCV non-structural protein (NS) 3 protease inhibitors (PIs), phenotypic monitoring of patient isolates is a prerequisite for understanding the emergence of resistance. HCV isolates typically fail to replicate in cell culture, necessitating the use of alternative phenotyping methods.
  
  
  METHODS
  An NS3 protease chimeric replicon system was developed to monitor the phenotype of clinical isolates. The transfer of NS3 protease domain sequences from HCV-infected patients to the background of genotype (Gt) 1a-H77c, 1b-Con1 and 2a-JFH-1 lab strain replicons adapted to high-level cell culture replication was investigated.
  
  
  RESULTS
  NS3 protease sequences derived from HCV Gt 1a or Gt 1b infected patients were transferred into Gt 1a and 1b replicons, respectively. Replication was detected for 20% of Gt 1a and 75% of Gt 1b sequences. Incorporation of known cell culture adaptive change NS3-E176G improved replication of Gt 1b but not of Gt 1a sequences. Transfer of Gt 1a clinical sequences into the Gt 1b background enhanced replication and allowed phenotypic analysis of all sequences. A correlation was observed between clinical isolate sequence polymorphisms and reduced susceptibility to NS3 PI. In mixed populations containing known NS3 PI resistance changes NS3-R155K or D168E/V, sensitivity of resistance detection was ≥ 10%.
  
  
  CONCLUSIONS
  An HCV replicon capable of supporting phenotypic characterization of patient-derived HCV NS3 protease sequences was developed. Pre-existence of amino acid changes associated with NS3 PI resistance highlights the need for combination therapies in the treatment of HCV.
authors.affiliation: Bristol-Myers Squibb Research and Development, Wallingford, CT, USA. amy.sheaffer@bms.com
authors.forename: Amy K
authors.initials: AK
authors.lastname: Sheaffer
authors.forename: Min S
authors.initials: MS
authors.lastname: Lee
authors.forename: Dennis
authors.initials: D
authors.lastname: Hernandez
authors.forename: Susan
authors.initials: S
authors.lastname: Chaniewski
authors.forename: Fei
authors.initials: F
authors.lastname: Yu
authors.forename: Paul
authors.initials: P
authors.lastname: Falk
authors.forename: Jacques
authors.initials: J
authors.lastname: Friborg
authors.forename: Guangzhi
authors.initials: G
authors.lastname: Zhai
authors.forename: Fiona
authors.initials: F
authors.lastname: McPhee
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D024882:Drug Resistance, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000378:metabolism; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D050506:Mutant Chimeric Proteins / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology*; D012093:Replicon / Q000187:drug effects / Q000235:genetics*; D012680:Sensitivity and Specificity; D017361:Viral Nonstructural Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D050506:Mutant Chimeric Proteins; D011480:Protease Inhibitors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases