title: Hepatitis C virus RNA recombination in cell culture.
abstract: OBJECTIVE
  The Hepatitis C virus (HCV) exhibits large genetic diversity, both on a global scale and at the level of the infected individual. A major underlying mechanism of the observed sequence differences is error-prone virus replication by the viral RNA polymerase NS5B. In addition, based on phylogenetic comparisons of patient-derived HCV sequences, there is evidence of HCV recombination. However, to date little is known about the frequency by which recombination events occur in HCV and under what conditions recombination may become important in HCV evolution. We, therefore, aimed to set up an experimental model system that would allow us to analyze and to characterize recombination events during HCV replication.
  
  
  METHODS
  A neomycin-selectable, HCV replicon-based recombination detection system was established. HCV replicons were mutated within either the neomycin-phosphotransferase gene or the NS5B polymerase. Upon co-transfection of hepatic cells lines, recombination between the mutated sites is necessary to restore the selectable phenotype.
  
  
  RESULTS
  Recombinants were readily detected with frequencies correlating to the distance between the mutations. The recombinant frequency normalized to a crossover range of one nucleotide was around 4 × 10(-8).
  
  
  CONCLUSIONS
  An experimental system to select for HCV recombinants in cell culture was successfully established. It allowed deriving first estimates of recombinant frequencies. Based on these, recombination in HCV seems rare. However, due to the rapid virus turnover and the large number of HCV-infected liver cells in vivo, it is expected that recombination will be of biological importance when strong selection pressures are operative.
authors.affiliation: Department of Virology, Saarland University, D-66421 Hamburg, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Reiter
authors.forename: Gemma
authors.initials: G
authors.lastname: Pérez-Vilaró
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Scheller
authors.forename: Leonardo Bruno
authors.initials: LB
authors.lastname: Mina
authors.forename: Juana
authors.initials: J
authors.lastname: Díez
authors.forename: Andreas
authors.initials: A
authors.lastname: Meyerhans
mesh_terms: D006528:Carcinoma, Hepatocellular; D045744:Cell Line, Tumor; D019143:Evolution, Molecular; D016174:Hepacivirus / Q000235:genetics* / Q000254:growth & development; D006526:Hepatitis C / Q000821:virology*; D006801:Humans; D019868:Kanamycin Kinase / Q000235:genetics; D008113:Liver Neoplasms; D009154:Mutation / Q000235:genetics; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics*; D011995:Recombination, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase
chemical_list: D019868:Kanamycin Kinase; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase