Polioviruses containing picornavirus type 1 and/ortype 2 internal ribosomal entry site elements: genetic hybrids and theexpression of a foreign gene.

A picornavirus hybrid genome was constructed inwhich the internal ribosomal entry site (IRES) of encephalomyocarditis virus wasinserted between the 5' non-translated region and the open reading frame ofpoliovirus (PV), type 1 (Mahoney). Upon transfection into HeLa cells, the hybridRNA replicated and yielded a derivative of PV (W1-PNENPO). The PV IRES could beremoved from pPNENPO, which resulted in a hybrid picornavirus (W1-P108ENPO) inwhich the translation of the PV open reading frame normally promoted by the type1 IRES of PV was promoted by the type 2 IRES of encephalomyocarditis virus. Thisresult indicates that these elements are not likely to contain cis-actingelements necessary for PV replication or encapsidation. A foreign gene(bacterial chloramphenicol acetyltransferase, CAT) was inserted into pPNENPOcDNA between the PV and encephalomyocarditis virus IRES elements. Thedicistronic 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 andimmunoblot analyses showed that the viral RNA efficiently expressed the foreigngene in cell culture. The CAT activity diminished somewhat with each passage ofthe dicistronic virus, an observation which suggested that the inserted gene hada deleterious effect on viral replication. However, even after five viruspassages, a significant quantity of the foreign gene was still expressed.Insertion of the open reading frame of luciferase (67 kDa) resulted in an RNAspecies that replicated and expressed luciferase for up to 20 hr aftertransfection. However, this elongated RNA was not encapsidated.