title: Mutations within the 5' nontranslated RNA of cell culture-adapted hepatitis A virus which enhance cap-independent translation in cultured African green monkey kidney cells.
abstract: Mutations in the 5' nontranslated RNA (5'NTR) of an attenuated, cell culture-adapted hepatitis A virus (HAV), HM175/P16, enhance growth in cultured African green monkey kidney (BS-C-1) cells but not in fetal rhesus monkey kidney (FRhK-4) cells (S. P. Day, P. Murphy, E. A. Brown, and S. M. Lemon, J. Virol. 66: 6533-6540, 1992). To determine whether these mutations enhance cap-independent translation directed by the HAV internal ribosomal entry site (IRES), we compared the translational activities of the 5'NTRs of wild-type and HM175/P16 viruses in two stably transformed cell lines (BT7-H and FRhK-T7) which constitutively express cytoplasmic bacteriophage T7 RNA polymerase and which are derived from BS-C-1 and FRhK-4 cells, respectively. Translational activity was assessed by monitoring expression of a reporter protein, chloramphenicol acetyltransferase (CAT), following transfection with plasmid DNAs containing bicistronic T7 transcriptional units of the form luciferase-5'NTR-CAT. In both cell types, transcripts containing the 5'NTR of HM175/P16 expressed CAT at levels that were 50- to 100-fold lower than transcripts containing the IRES elements of Sabin type 1 poliovirus or encephalomyocarditis virus, confirming the low activity of the HAV IRES. However, in BT7-H cells, transcripts containing the 5'NTR of wild-type virus. This translational enhancement was due to additive effects of a UU deletion at nucleotides 203 and 204 and a U-to-G substitution at nucleotide 687 of HM175/P16. These mutations did not enhance translation in FRhK-T7 or Huh-T7 cells (a T7 polymerase-expressing cell line derived from human hepatoblastoma cells) or in vitro in rabbit reticulocyte lysates. These results demonstrate that mutations in the 5'NTR of a cell culture-adapted HAV enhance viral replication by facilitating cap-independent translation in a cell-type-specific fashion and support the concept that picornaviral host range is determined in part by differences in cellular translation initiation factors.
authors.affiliation: Department of Medicine, University of North Carolina at Chapel Hill 27599-7030, USA.
authors.forename: D E
authors.initials: DE
authors.lastname: Schultz
authors.forename: M
authors.initials: M
authors.lastname: Honda
authors.forename: L E
authors.initials: LE
authors.lastname: Whetter
authors.forename: K L
authors.initials: KL
authors.lastname: McKnight
authors.forename: S M
authors.initials: SM
authors.lastname: Lemon
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D004279:DNA, Viral; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D006507:Hepatovirus / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000166:cytology; D008253:Macaca mulatta; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D014764:Viral Proteins
chemical_list: D004279:DNA, Viral; D012315:RNA Caps; D012367:RNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases