Genetic analysis of the internal ribosome entry segment of bovine viral diarrhea virus

Pestiviruses and hepaciviruses are atypical members of the Flaviviridae due to their unique biological properties, including the utilization of internal ribosome entry for translation initiation. In contrast to internal initiation in picornaviruses, which depends on numerous canonical initiation factors, the mode of internal ribosome entry in pestiviruses and hepaciviruses resembles prokaryotic translation initiation. To identify functionally important elements within the bovine viral diarrhea virus (BVDV) internal ribosome entry segment (IREs), we carried out a mutational analysis of the 5' untranslated region (5' UTR) of BVDV cloned in the intercistronic region of a bicistronic reporter plasmid. IRES function was assessed in a bicistronic transcript by inserting the 5' 901 nucleotides of BVDV genome, which correspond to the 385 nucleotides of the 5' UTR and 515 nucleotides of the open reading frame (ORF) encoding for N-pro and 4 amino acids from the capsid protein. The resulting N-pro-luciferase fusion encoded by the 3' cistron was cleaved efficiently to release the luciferase reporter, In vivo translation analyses showed that stem-loops la and Ib in the 5' UTR were completely dispensable for efficient translation, whereas stem-loop IIIe and the hairpin end of IIIb were only partially required. In contrast, deletions or insertions in any of other four stem-loop structures, including domains II, IIIa, IIIc, and IIId, caused nearly 10-fold reductions of in vivo IRES activity. The tolerance of structural modifications within the distal portion of domain IIIb and IIIe correlated with a low level of sequence conservation in these regions among pestiviruses. The 5' boundary of the IRES resides at the 5' end of stem-loop II near nucleotide 75. The 3' of the IRES extends into the 5' end of the polyprotein ORF because removal of the N-pro coding region reduced translation by 21%. (C) 1998 Academic Press.