INTERNAL ENTRY OF RIBOSOMES IS DIRECTED BY THE 5'-NONCODING REGION OFCLASSICAL SWINE FEVER VIRUS AND IS DEPENDENT ON THE PRESENCE OF AN RNA PSEUDOKNOT UPSTREAM OF THE INITIATION CODON

Bicistronic RNAs containing the 373-nucleotide-long 5' nontranslated r egion (NTR) of the classical swine fever virus (CSFV) genome as interc istronic spacer were used to show the presence of an internal ribosome entry site (IRES) in the 5' end of the CSFV genome. By coexpression o f the poliovirus 2A protease it was demonstrated that the CSFV 5' NTR- driven translation is independent of the presence of functional eukary otic initiation factor eIF-4F. Deletion analysis indicated that the 5' border of the IRES is located between nucleotides 28 and 66. The role of a proposed pseudoknot structure at the 3' end of the CSFV 5' NTR i n IRES-mediated translation was investigated by site-directed mutagene sis. Mutant RNAs that had lost the ability to base pair in stem II of the pseudoknot were translationally inactive. Translation to wild-type levels could be restored through the introduction bf compensatory com plementary base changes that repaired base pairing in stem II. In addi tion, we showed that the AUG codon, which is located 7 nucleotides ups tream of the polyprotein initiation site and is conserved in pestiviru ses, could not be used to initiate translation. Also, an AUG codon int roduced downstream of the polyprotein initiation site was not recogniz ed as an initiation site by ribosomes. These data suggest that after i nternal entry on the CSFV 5' NTR, ribosomal scanning for the initiatio n codon is limited to a small region.