title: A genotype 2b NS5B polymerase with novel substitutions supports replication of a chimeric HCV 1b:2b replicon containing a genotype 1b NS3-5A background.
abstract: HCV diversity suggests that evaluation of HCV inhibitors for broad genotypic efficacy is warranted. The replicon system enables cell-culture compound efficacy evaluation against an active replication complex, and a functional replicon dependent upon a genotype 2b polymerase would augment existing cell-culture efficacy studies that are presently limited to genotype 1a, 1b, and 2a replicons. We made a chimeric Neo(r) 1b:2b replicon where genotype 2b NS5B was inserted into a genotype 1b NS3-5A background and transfected replicon RNA to generate Neo(r) cell lines. All cell lines contained novel substitutions within NS5B which were subsequently engineered into the parental 1b:2b replicon and shown to enhance replication to various degrees. A single NS5B M31I substitution enhanced replication to levels sufficiently robust to quantify sensitivity to HCV inhibitors in a transient replication assay. The M31I 1b:2b replicon was similarly sensitive to an active-site nucleoside inhibitor of NS5B as genotype 1b replicons, but was insensitive to two non-nucleoside inhibitors which were otherwise efficacious against the genotype 1b replicons. This work describes a novel HCV replicon sustained by a genotype 2b polymerase that is sufficiently robust for quantifiable analysis in a transient replication assay, and demonstrates its utility in characterizing anti-HCV compounds for cross-genotypic efficacy.
authors.affiliation: Department of Antiviral Research, Merck Research Laboratories, P.O. Box 4, West Point, PA 19486, USA.
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Graham
authors.forename: Mark
authors.initials: M
authors.lastname: Stahlhut
authors.forename: Osvaldo
authors.initials: O
authors.lastname: Flores
authors.forename: David B
authors.initials: DB
authors.lastname: Olsen
authors.forename: Daria J
authors.initials: DJ
authors.lastname: Hazuda
authors.forename: Robert L
authors.initials: RL
authors.lastname: Lafemina
authors.forename: Steven W
authors.initials: SW
authors.lastname: Ludmerer
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008958:Models, Molecular; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D012093:Replicon; D012262:Ribonucleoside Diphosphate Reductase / Q000494:pharmacology; D013045:Species Specificity; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D014779:Virus Replication
chemical_list: D017361:Viral Nonstructural Proteins; D012262:Ribonucleoside Diphosphate Reductase; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; D000241:Adenosine