GENETIC-ANALYSIS OF THE INTERNAL RIBOSOME ENTRY SEGMENT OF BOVINE VIRAL DIARRHEA VIRUS

Pestiviruses and hepaciviruses are atypical members of the Flavivirida e 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 cano nical initiation factors, the mode of internal ribosome entry in pesti viruses and hepaciviruses resembles prokaryotic translation initiation . To identify functionally important elements within the bovine viral diarrhea virus (BVDV) internal ribosome entry segment (IREs), we carri ed out a mutational analysis of the 5' untranslated region (5' UTR) of BVDV cloned in the intercistronic region of a bicistronic reporter pl asmid. IRES function was assessed in a bicistronic transcript by inser ting the 5' 901 nucleotides of BVDV genome, which correspond to the 38 5 nucleotides of the 5' UTR and 515 nucleotides of the open reading fr ame (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 transl ation analyses showed that stem-loops la and Ib in the 5' UTR were com pletely 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, in cluding domains II, IIIa, IIIc, and IIId, caused nearly 10-fold reduct ions of in vivo IRES activity. The tolerance of structural modificatio ns within the distal portion of domain IIIb and IIIe correlated with a low level of sequence conservation in these regions among pestiviruse s. The 5' boundary of the IRES resides at the 5' end of stem-loop II n ear nucleotide 75. The 3' of the IRES extends into the 5' end of the p olyprotein ORF because removal of the N-pro coding region reduced tran slation by 21%. (C) 1998 Academic Press.