Protein biogenesis of the bovine viral diarrhea virus p20 protein

The role of 3' flanking RNA sequence required for autocatalytic processing of the p20 gene product of bovine viral diarrhea virus (BVDV) was investigated using recombinant transcription vectors. Three cDNA sequences, each with the same origin within the 5' noncoding region of the (BVDV) genome, were synthesized using the polymerase chain reaction, and were ligated into the Eco RI and Hind III sites of thepGEM3Zf⁽⁺⁾ transcription vector. The protein coding capacity of each cDNA in the recombinant vectors (p750, p930, and p1450) was approximately 22.5, 29, and 48kDa, respectively. Transcription from the T7 promoter and subsequent translation, of the RNA product, in rabbit reticulocyte lysates resulted in the production of a 20 kDa protein from each of the recombinant vectors. These results suggested that the sequences downstream from the p20 were not critical for autocatalytic processing. In an attempt to characterize the class of proteinase activity in this catalysis, three proteinases inhibitors, leupeptin, pepstatin A and PMSF. These inhibitors did not abrogate cleavage of the p20 protein. The cleavage may occur through an intramolecular reaction since the proteinase inhibitors did not function as substrate analogs. The tertiary structure of the p20 protein may be responsible for this phenomenon.

Oligonucleotides (20 nucleotides in length) complementary to RNA sequences of bovine viral diarrhea virus, NADL isolate, were used to assess the role of the 5' noncoding region in ribosomal accession of the recombinant transcription vector pT75'p20.In vitro translation of RNA transcripts derived from pT75'p20 resulted in synthesis of a protein with a molecular weight of 20 kDa. Six AUG triplets occur upstream of the translation initiation codon for the large ORF of the BVDV genome. Eleven complementary oligonucleotides were designed and shown to anneal to regions of the pT75'p20 RNA transcripts encompassing each of the AUG triplets. Six oligonucleotides complementary to a region spanning nucleotides 154-391 inhibited protein synthesis. These results suggest that the mechanism for translation initiation by this pestivirus may involve internal ribosome binding in a fashion reminiscent of that employed by picornaviruses and that nucleotides 154-391 of the BVDV (NADL) genome contain the ribosome entry site.