title: Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase.
abstract: Efficient replication of hepatitis C virus (HCV) replicons in cell culture is associated with specific sequences not generally observed in vivo. These cell culture adaptive mutations dramatically increase the frequency with which replication is established in vitro. However, replicons derived from HCV isolates that have been shown to replicate in chimpanzees do not replicate in cell culture even when these adaptive mutations are introduced. To better understand this apparent paradox, we performed a gain-of-function screen to identify sequences that could confer cell culture replication competence to replicons derived from chimpanzee infectious HCV isolates. We found that residue 470 in domain II of the NS3 helicase is a critical determinant in cell culture adaptation. Substitutions in residue 470 when combined with the NS5A-S232I adaptive mutation are both necessary and sufficient to confer cell culture replication to otherwise inactive replicons, including those derived from genotype 1b HCV-BK and genotype 1a HCV-H77 isolates. The specific substitution at residue 470 required for replication is context-dependent, with R470M and P470L being optimal for the activity of HCV-BK and HCV-H77 replicons, respectively. Together these data indicate that mutations in the NS3 helicase domain II act in concert with previously identified adaptive mutations and predict that introduction of compatible residues at these positions can confer cell culture replication activity to diverse HCV isolates.
authors.affiliation: Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486, USA.
authors.forename: Jay A
authors.initials: JA
authors.lastname: Grobler
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Markel
authors.forename: John F
authors.initials: JF
authors.lastname: Fay
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Graham
authors.forename: Amy L
authors.initials: AL
authors.lastname: Simcoe
authors.forename: Steve W
authors.initials: SW
authors.lastname: Ludmerer
authors.forename: Edward M
authors.initials: EM
authors.lastname: Murray
authors.forename: Giovanni
authors.initials: G
authors.lastname: Migliaccio
authors.forename: Osvaldo A
authors.initials: OA
authors.lastname: Flores
mesh_terms: D002478:Cells, Cultured; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D009154:Mutation; D020365:RNA Helicases / Q000502:physiology; D017361:Viral Nonstructural Proteins / Q000502:physiology*; D014779:Virus Replication / Q000235:genetics; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D020365:RNA Helicases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases