Identification of the Hepatitis A Virus Internal Ribosome Entry Site: In Vivo and in Vitro Analysis of Bicistronic RNAs Containing the HAV 5′ Noncoding Region

Abstract

Hepatitis A virus (HAV), a RNA virus of positive polarity, contains a long 5′ noncoding region (5′NCR) that lacks the characteristic m7GpppN cap group of most eukaryotic messages. By creating bicistronic constructs that contain the bacterial chloramphenicol acetyltransferase gone followed by the HAV 5′NCR and the luciferase gene we have demonstrated by assaying in vitro and in vivo that ribosome entry for translation initiation occurs via binding to sequences within the HAV 5′NCR. Using mutations created within this region we have identified that the HAV internal ribosome entry site (IRES) is located downstream of nucleotide 45 and including sequences up to nucleotide 734 of the HAV 5′NCR. Translation of a number of mutant constructs both in vitro in a rabbit reticulocyte lysate and in vivo by transfection of the cDNAs into BS-C-1 cells in the presence of the recombinant vaccinia virus, vTF7-3, gave similar results. However, a 4-nucleotide insertion at base 628 showed an increased activity over wild-type when transfected into BS-C-1 cells that was not seen in vitro. This increase in activity correlated with an increase in luciferase gene product as assayed by immunoprecipitations of [ 35S]methionine radiolabeled cells. Comparison of mono- and bicistronic RNAs that were synthesized with or without a m7GpppG cap group showed a competition for ribosome binding when translated in a rabbit reticulocyte lysate system. The presence of the cap group on the RNA 5′ terminus of the RNA led to a greater ability of this RNA to translate than the RNA containing the HAV IRES.