E2-p7 region of the bovine viral diarrhea virus polyprotein: Processing and functional studies

The genes encoding pestivirus E2 and NS2-3 are separated by a sequence that encodes a small hydrophobic polypeptide with an apparent molecular mass of 6 to 7 kDa (p7), It has been shown that cleavage between E2 and p7 is inco mplete, resulting in proteins E2-p7, E2, and p7, We found no precursor-prod uct relationship between E2-p7 and E2, which indicates a stable nature of E 2-p7, To study the function of the E2-p7 region of the polyprotein, mutatio ns were introduced into an infectious cDNA of bovine viral diarrhea virus ( BVDV). When cleavage between E2 and p7 was abolished, viral RNA replication occurred; however, no infectious virus could be recovered. A corresponding result was obtained with a construct encompassing a large in-frame deletio n of p7. To prevent synthesis of E2-p7, a translational stop codon was intr oduced after the last codon of the E2 gene and an internal ribosome entry s ite element followed by a signal peptide coding sequence was inserted upstr eam of the p7 gene. Transfection of RNA transcribed from the bicistronic co nstruct led to the release of infectious virus particles. Thus, synthesis o f E2-p7 is not essential for the generation of infectious virions, Cell lin es constitutively expressing BVDV p7 and/or E2 were generated for complemen tation studies. Transfection of BVDV RNAs with point mutations or a deletio n in the E2-p7 region into the complementing cell lines led to the generati on of infectious virions. According to our studies, p7 as well as E2 can be complemented in trans.