IDENTIFICATION OF AN ALTERNATIVE OPEN READING FRAME (HIDDEN GENE) STRINGENTLY REQUIRED FOR INFECTIVITY OF POLIOVIRUS CDNA CLONES

Translation of the uncappped poliovirus RNA starts at the AUG triplet spanning positions 741-743, and proceeds uninterrupted for almost the entire length of the genome. Such a cap-independent mechanism of inter nal initiation of translation determines that a long, extra-cistronic region extends between the 5'-end and the main open reading frame (ORF ). We have identified 10 short ORFs initiated by the alternative trans lation initiation codons ACG, AUA, and GUG in the 5'-terminal extra-ci stronic region (5'-ECR) of poliovirus RNA. Mutations introduced in all but one of these mini-cistrons had no effect on the infectivity of fu ll-length cDNA clones, except when they modified a ''hidden frame'' sp anning between nucleotides 157-192 (starting triplet: ACG). The mini c istron 157-192 is conserved in position, length and sequence in the ge nome of all types and strains of poliovirus. Adaptation to rat (Lansi ng) or mouse (variant of Sabin 2) is accompanied by a consistent patte rn of changes in the primary sequence of this ''hidden frame''. The su bstitutions that abrogated the infectivity of cDNA clones were not exp ected to modify the predicted secondary structure of the 5' ECR, and t hey did not alter the ability of the IRES to direct internal initiatio n of translation in bi-cistronic mRNAs. The infectivity of the mutated poliovirus cDNAs could be complemented in trans by co-transfecting th e target COS-1 cells with an expression vector containing just the 5'- ECR of poliovirus type 2 (Lansing strain). The infectivity of poliovir us cDNA could be restored by co-transfecting short RNA transcripts of the wt 5'-ECR (Lansing), suggesting that the complementation in trans indeed requires the expression of the helper cDNA clone.