Analysis of hepatitis A virus translation in a T7 polymerase-expressing cell line.
Hepatitis A virus (HAV) exhibits several characteristics which distinguish it from other picornaviruses, including slow growth in cell culture even after adaptation, and lack of host-cell protein synthesis shut-down. Like other picornaviruses, HAV contains a long 5' nontranslated region (NTR) incorporating an internal ribosomal entry site (IRES), which directs cap-independent translation. We compared HAV IRES-initiated translation with translation initiated by the structurally similar encephalomyocarditis virus (EMCV) IRES, using plasmids in which each of the 5'NTRs is linked in-frame with the chloramphenicol acetyltransferase (CAT) gene. Translation was assessed in an HAV-permissive cell line which constitutively expresses T7 RNA polymerase and transcribes high levels of uncapped RNA from these plasmids following transfection. RNAs containing the EMCV IRES were efficiently translated in these cells, while those containing the HAV IRES were translated very poorly. Analysis of translation of these RNAs in the presence of poliovirus protein 2A, which shuts down cap-dependent translation, demonstrated that their translation was cap independent. Our results suggest that the HAV IRES may function poorly in these cells, and that inefficient translation may contribute to the exceptionally slow replication cycle characteristic of cell culture-adapted HAV.
Department of Medicine, University of North Carolina, Chapel Hill.
L E
LE
Whetter
S P
SP
Day
E A
EA
Brown
O
O
Elroy-Stein
S M
SM
Lemon
D000818:Animals; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase; D003546:Cysteine Endopeptidases / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis* / Q000235:genetics; D006507:Hepatovirus / Q000378:metabolism*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012315:RNA Caps; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D014162:Transfection; D014764:Viral Proteins
D012315:RNA Caps; D011994:Recombinant Proteins; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus