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

Mutations in the 5' nontranslated RNA (5'NTR) of an attenuated, cell c ulture-adapted hepatitis A virus (HAV), HM175/P16, enhance growth in c ultured African green monkey kidney (BS-C-I) cells but not in fetal rh esus 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 HA V internal ribosomal entry site (IRES), we compared the translational activities of the 5'NTRs of wild-type and HM175/P16 viruses in two sta bly transformed cell lines (BT7-H and FRhK-T7) which constitutively ex press cytoplasmic bacteriophage T7 RNA polymerase and which are derive d from BS-C-1 and FRhK-4 cells, respectively, Translational activity w as assessed by monitoring expression of a reporter protein, chloramphe nicol acetyltransferase (CAT), following transfection with plasmid DNA s containing bicistronic T7 transcriptional units of the form lucifera se-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 o r encephalomyocarditis virus, confirming the low activity of the HAV I RES, However, in BT7-H cells, transcripts containing the 5'NTR of HM17 5/P16 expressed CAT with four- to fivefold greater efficiency than tra nscripts containing the 5'NTR of wild-type virus, This translational e nhancement 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 cell s (a T7 polymerase-expressing cell line derived from human hepatoblast oma cells) or in vitro in rabbit reticulocyte lysates, These results d emonstrate that mutations in the 5'NTR of a cell culture-adapted HAV e nhance viral replication by facilitating cap-independent translation i n a cell-type-specific fashion and support the concept that picornavir al host range is determined in part by differences in cellular transla tion initiation factors.