Identification of an alternative open reading frame ("hidden gene"?) stringently required for infectivity of poliovirus cDNA clones.
Translation of the uncapped 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 internal 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 translation initiation codons ACG, AUA, and GUG in the 5'-terminal extra-cistronic region (5'-ECR) of poliovirus RNA. Mutations introduced in all but one of these mini-cistrons had no effect on the infectivity of full-length cDNA clones, except when they modified a "hidden frame" spanning between nucleotides 157-192 (starting triplet: ACG). The mini cistron 157-192 is conserved in position, length and sequence in the genome of all types and strains of poliovirus. Adaptation to rat (Lansing) or mouse (variant of Sabin 2) is accompanied by a consistent pattern of changes in the primary sequence of this "hidden frame". The substitutions that abrogated the infectivity of cDNA clones were not expected to modify the predicted secondary structure of the 5'-ECR, and they did not alter the ability of the IRES to direct internal initiation of translation in bi-cistronic mRNAs. The infectivity of the mutated poliovirus cDNAs could be complemented in trans by co-transfecting the target COS-1 cells with an expression vector containing just the 5'-ECR of poliovirus type 2 (Lansing strain). The infectivity of poliovirus 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.
Pierangeli
A
A
Department of Cellular and Developmental Biology, University of Rome La Sapienza, Italy.
Bucci
M
M
Forzan
M
M
Pagnotti
P
P
Equestre
M
M
Pérez Bercoff
R
R
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D002855:Chromatography, Thin Layer; D018387:Codon, Initiator / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry; D015967:Gene Expression Regulation, Viral; D005796:Genes / Q000235:genetics*; D005816:Genetic Complementation Test; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames / Q000235:genetics*; D017354:Point Mutation; D011051:Poliomyelitis / Q000235:genetics*; D017955:Poliovirus / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000737:chemistry; D011863:Radioimmunoassay; D051381:Rats; D017423:Sequence Analysis, RNA; D014158:Transcription, Genetic; D014162:Transfection / Q000235:genetics; D014709:Vero Cells
D018387:Codon, Initiator; D018076:DNA, Complementary; D012367:RNA, Viral