A selection system for functional internal ribosome entry site (IRES) elements: Analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES

Picornavirus internal ribosome entry site (IRES) elements direct cap-indepe ndent internal initiation of protein synthesis within mammalian cells. Thes e RNA elements (about 450 nt) contain extensive secondary structure includi ng a hairpin loop with a conserved GNRA motif, Such loops are important in RNA-RNA and RNA-protein interactions. Plasmids that express dicistronic mRN As of the structure GUS/IRES/HOOK have been constructed. The HOOK sequence encodes a cell-surface-targeted protein (sFv); the translation of this open reading frame within mammalian cells from these dicistronic mRNAs requires a functional IRES element. Cells that express the sFv can be selected from nonexpressing cells. A pool of up to 256 mutant encephalomyocarditis virus IRES elements was generated by converting the wild-type hairpin loop seque nce (GCGA) to NNNN, Following transfection of this pool of mutants into COS -7 cells, plasmids were recovered from selected sFv-expressing cells. These DNAs were amplified in Escherichia coli and transfected again into COS-7 c ells for further cycles to enrich for plasmids encoding functional IRES ele ments. The sequence of individual selected IRES elements was determined. Al l functional IRES elements had a tetraloop with a 3' terminal A residue. Op timal IRES activity, assayed in vitro and within cells, was obtained from p lasmids encoding an IRES with the hairpin loop sequence fitting a RNRA cons ensus, In contrast, IRES elements containing YCYA tetraloops were severely defective.