A cis-acting element within the hepatitis A virus 5'-non-coding region required for in vitro translation.
Every picornavirus studied thus far has a sequence within the 5'-non-coding region that is required for internal ribosome binding and translation of the polyprotein. In an attempt to identify this region in hepatitis A virus we constructed a truncated hepatitis A virus (HAV) cDNA clone that contains the entire 736 bp 5' non-coding region (5'-NCR) and 754 base pairs of the viral capsid coding region (P1) under control of the SP6 promoter. In vitro transcription and translation of this transcript in a rabbit reticulocyte lysate yielded a protein product of about 29 kDa as analyzed by autoradiography following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A series of mutations of this construct have defined a minimal sequence between bases 347 and 734 in the 5'-NCR that is required for efficient in vitro translation. The deleted constructs (D 523-734 and D 632-734) showed a reduced ability to translate in the rabbit reticulocyte lysate system in comparison with the full-length 5'-NCR construct, pH1489. The translation of these deleted constructs was artificially restored by the addition of a 5'-terminal methylated cap structure, m7GpppG, to the RNA. This increase in translational efficiency could be competed away with cap analog (m7GDP) thus indicating that this region is required for cap-independent internal ribosome binding for HAV translation.
Glass
M J
MJ
Department of Cellular, Viral and Molecular Biology, University of Utah School of Medicine, Salt Lake City 84132.
Summers
D F
DF
D001483:Base Sequence; D002213:Capsid / Q000235:genetics; D002474:Cell-Free System; D003062:Codon; D004247:DNA / Q000235:genetics; D016679:Genome, Viral; D006507:Hepatovirus / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D012315:RNA Caps / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D017423:Sequence Analysis, RNA; D014158:Transcription, Genetic
D003062:Codon; D012315:RNA Caps; D012367:RNA, Viral; D004247:DNA