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Development of a Chimeric Replicon System for Phenotypic Analysis of Ns3 Protease Sequences from HCV Clinical Isolates

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.