Translation Initiation of a Bicistronic mRNA of Borna Disease Virus: A 16-kDa Phosphoprotein Is Initiated at an Internal Start Codon

Abstract

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.

Two Alternative Translation Mechanisms Are Responsible for the Expression of the HCV ARFP/F/Core+1 Coding Open Reading Frame

“…Different mechanisms that allow escape from an upstream initiator codon and direct initiation from internal codons have been described; these include context-dependent leaky-scanning (13,17,18,19,20,21), ribosome shunting (17,22,23,24,25,26,27), and IRES-mediated initiation (28). The internal initiation of ARFP/F/coreϩ1 translation in vivo might include features of one of these mechanisms.…”

Modulation of Borna Disease Virus Phosphoprotein Nuclear Localization by the Viral Protein X Encoded in the Overlapping Open Reading Frame

“…To generate the eukaryotic expression plasmids encoding green fluorescent protein (GFP)-fused BDV X/P, pgX, pgP, and pgX/P, BDV cDNAs were amplified by PCR with pcX/P construct (20) and were inserted into the EcoRI-BamHI site of the pEGFP-N1 vector (Clontech Laboratories, Inc., Palo Alto, Calif.). The BDV X expression plasmids, pcX and pcXf, were constructed by insertion of PCR fragments from the pcX/P plasmid into the EcoRIXhoI site of the pcDNA3 vector (Invitrogen, San Diego, Calif.).…”

“…To create the T7 promoter-containing vector, ptfX, a cDNA fragment corresponding to BDV X ORF was amplified by PCR and was inserted into the pTF1 plasmid (42). The construction of BDV P expression vectors, pcPf and pcP'f, previously referred to as pcP-FLAG and pcP'-FLAG, respectively, is described elsewhere (20). The mutant forms of these expression plasmids were generated from wild-type plasmids by using PCR amplification and recloning or a PCR-based site-directed mutagenesis technique.…”

“…1A). This construct efficiently expresses both X and P in transfected cells (20). We introduced the expression plasmids into the OL cells and investigated the intracellular distribution of the viral proteins 48 h posttransfection.…”

“…3A). The mutant plasmid, as well as the wild-type P (pcPf) and p16P'-expression plasmids (pcP'f) (20), was cotransfected with X expression plasmid, and the subcellular localization of BDV P was analyzed by anti-P antibody 48 h posttransfection. As shown in Fig.…”