Polioviruses containing picornavirus type 1 and/or type 2 internal ribosomal entry site elements: genetic hybrids and the expression of a foreign gene.
A picornavirus hybrid genome was constructed in which the internal ribosomal entry site (IRES) of encephalomyocarditis virus was inserted between the 5' non-translated region and the open reading frame of poliovirus (PV), type 1 (Mahoney). Upon transfection into HeLa cells, the hybrid RNA replicated and yielded a derivative of PV (W1-PNENPO). The PV IRES could be removed from pPNENPO, which resulted in a hybrid picornavirus (W1-P108ENPO) in which the translation of the PV open reading frame normally promoted by the type 1 IRES of PV was promoted by the type 2 IRES of encephalomyocarditis virus. This result indicates that these elements are not likely to contain cis-acting elements necessary for PV replication or encapsidation. A foreign gene (bacterial chloramphenicol acetyltransferase, CAT) was inserted into pPNENPO cDNA between the PV and encephalomyocarditis virus IRES elements. The dicistronic RNA replicated in HeLa cells and yielded a derivative of PV (W1-DICAT) with a genome 17% longer than that of wild-type PV. CAT assays and immunoblot analyses showed that the viral RNA efficiently expressed the foreign gene in cell culture. The CAT activity diminished somewhat with each passage of the dicistronic virus, an observation which suggested that the inserted gene had a deleterious effect on viral replication. However, even after five virus passages, a significant quantity of the foreign gene was still expressed. Insertion of the open reading frame of luciferase (67 kDa) resulted in an RNA species that replicated and expressed luciferase for up to 20 hr after transfection. However, this elongated RNA was not encapsidated.
Alexander
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Department of Microbiology, School of Medicine, State University of New York at Stony Brook 11794.
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Wimmer
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D001483:Base Sequence; D002678:Chimera; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis* / Q000235:genetics; D004686:Encephalomyocarditis virus / Q000235:genetics; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D010849:Picornaviridae / Q000235:genetics*; D017955:Poliovirus / Q000235:genetics; D012313:RNA / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism
C039919:RNA, recombinant; D011994:Recombinant Proteins; D012313:RNA; D015500:Chloramphenicol O-Acetyltransferase