Replication of hepatitis A viruses with chimeric 5' nontranslated regions.
The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.
Jia
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Department of Microbiology and Molecular Genetics, University of California, Irvine 92717, USA.
Tesar
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Summers
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Ehrenfeld
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D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis; D002460:Cell Line; D002678:Chimera; D017931:DNA Primers; D004686:Encephalomyocarditis virus / Q000235:genetics*; D006507:Hepatovirus / Q000235:genetics* / Q000502:physiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014779:Virus Replication
D017931:DNA Primers; D012367:RNA, Viral