Efficient translation initiation is required for replication of bovine viral diarrhea virus subgenomic replicons

An internal ribosome entry site (IRES) mediates translation initiation of b ovine viral diarrhea virus (BVDV) RNA, Studies have suggested that a portio n of the N-pro open. reading frame (ORF) is required, although its ex-act f unction has not been defined. Here ne show that a subgenomic (sg) BVDV RNA in which the NS3 ORF is preceded only bu the 5' nontranslated region did no t replicate to detectable levels following transfection. However, RNA synth esis and cytopathic effects were observed following serial passage in the p resence of a noncytopathic helper virus. Five sg clones derived from the pa ssaged virus contained an identical, silent substitution near the beginning of the NS3 coding sequence (G400U), as well as additional mutations. Four of the reconstructed mutant RNAs replicated in transfected cells, and in vi tro translation shelved increased levels of NS3 for the mutant RNAs compare d to that of wild-type (wt) MetNS3. To more precisely dissect the role of t hese mutations, we constructed two sg derivatives: ad3,10, which contains o nly the G400U mutation, and ad3,7, with silent substitutions designed to mi nimize RNA secondary structure downstream of the initiator AUG, Both RNAs r eplicated and were translated in vitro to similar levels. Moreover, ad3.7 a nd ad3,10, but not fft MetNS3, formed toeprints downstream of the initiator AUG codon in an assay for detecting the binding of 40S ribosomal subunits and 43S ribosomal complexes to the IRES, These results suggest that a lack of stable RNA secondary structure(s), rather than a specific RNA sequence, immediately downstream of the initiator AUG is important for optimal transl ation initiation of pestivirus RNAs.