Translation initiation of a bicistronic mRNA of Borna disease virus: a 16-kDa phosphoprotein is initiated at an internal start codon.

We examined translational initiation of a bicistronic 0.8-kb mRNA of Borna disease virus (BDV) using a cDNA clone of the mRNA. Upon transfection with the clone, COS-7 cells produced a 16-kDa protein (P'), in addition to the previously identified products of BDV, 24- (P) and 14.5-kDa proteins. The 16-kDa product was detected by anti-P monoclonal antibody and was shown to exist in BDV-infected cell lines as well as in infected animal brain cells. Transient expression analysis of mutated cDNA clones encoding the BDV 0.8-kb mRNA revealed that the 16-kDa protein was initiated at the second AUG codon on the same open reading frame of the P protein. The mutational analysis also demonstrated that the first AUG within the 0.8-kb mRNA is not optimal, although the signal contains a better Kozak's motif. These results demonstrated the presence of three functional AUG codons in the smallest mRNA of BDV and also suggested that a leaky scanning mechanism is involved in translational initiation at AUG codons downstream of the bicistronic mRNA of BDV. Furthermore, the 16-kDa protein was located in the BDV-specific nuclear foci and was found to associate with the other viral proteins in BDV-infected cells, demonstrating an important role of the novel identified BDV protein in viral replication.