title: Generation of a cell culture-adapted hepatitis C virus with longer half life at physiological temperature.
abstract: BACKGROUND
  We previously reported infectious HCV clones that contain the convenient reporters, green fluorescent protein (GFP) and Renilla luciferase (Rluc), in the NS5a-coding sequence. Although these viruses were useful in monitoring viral proliferation and screening of anti-HCV drugs, the infectivity and yield of the viruses were low.
  
  
  RESULTS
  In order to obtain a highly efficient HCV cultivation system, we transfected Huh7.5.1 cells [1] with JFH 5a-GFP RNA and then cultivated cells for 20 days. We found a highly infectious HCV clone containing two cell culture-adapted mutations. Two cell culture-adapted mutations which were responsible for the increased viral infectivity were located in E2 and p7 protein coding regions. The viral titer of the variant was ∼100-fold higher than that of the parental virus. The mutation in the E2 protein increased the viability of virus at 37°C by acquiring prolonged interaction capability with a HCV receptor CD81. The wild-type and p7-mutated virus had a half-life of ∼2.5 to 3 hours at 37°C. In contrast, the half-life of viruses, which contained E2 mutation singly and combination with the p7 mutation, was 5 to 6 hours at 37°C. The mutation in the p7 protein, either singly or in combination with the E2 mutation, enhanced infectious virus production about 10-50-fold by facilitating an early step of virion production.
  
  
  CONCLUSIONS
  The mutation in the E2 protein generated by the culture system increases virion viability at 37°C. The adaptive mutation in the p7 protein facilitates an earlier stage of virus production, such as virus assembly and/or morphogenesis. These reporter-containing HCV viruses harboring adaptive mutations are useful in investigations of the viral life cycle and for developing anti-viral agents against HCV.
authors.affiliation: Molecular Virology Laboratory, POSTECH Biotech Center, Department of Life Science, Pohang University of Science and Technology, Pohang, Kyungbuk, Republic of Korea.
authors.forename: Chon Saeng
authors.initials: CS
authors.lastname: Kim
authors.forename: Sun Ju
authors.initials: SJ
authors.lastname: Keum
authors.forename: Sung Key
authors.initials: SK
authors.lastname: Jang
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D006207:Half-Life; D016174:Hepacivirus / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D013696:Temperature; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D014774:Virulence
chemical_list: D017931:DNA Primers; D014764:Viral Proteins