Characterization of the P Protein-Binding Domain on the 10-Kilodalton Protein of Borna Disease Virus

Abstract

The Borna disease virus (BDV) is the prototype member of the Bornaviridae, and it replicates in the cell nucleus. The BDV p24P and p40N proteins carry nuclear localization signals (NLS) and are found in the nuclei of infected cells. The BDV p10 protein does not have an NLS, but it binds with P and/or N and is translocated to the nucleus. Hence, p10 may play a role in the replication of BDV in the cell nucleus. Here, we show that the P-binding domain is located in the N terminus of p10 and that S 3 and L 16 are important for the interaction.The Borna disease virus (BDV) belongs to a new family of enveloped, negative, nonsegmented, single-stranded RNA viruses. The virus causes central nervous system disease in diverse species. It replicates and transcribes in the nuclei of infected cells (1, 2), and hence, viral proteins found in the nucleus may be important to the virus life cycle. Recently, members of our group (5) and others (8,11) reported the expression of a 10-kDa protein, p10, which is encoded by open reading frame x1 (ORFx1) (nucleotides [nt] 1223 to 1486). p10 is found in the nucleus and the cytoplasm of BDV-infected cells, although it does not carry a nuclear localization signal (NLS) (5,8,11). Transient transfection studies have shown that, alone, p10 is localized in the cytoplasm but that upon cotransfection with the viral P protein, p24, or the viral N protein, p40, it is imported to the nucleus (5, 8). The p40 and the p24 proteins both carry an NLS (4, 9, 10) and are found in the nucleus of BDV-infected cells (4, 6, 9). In vitro proteinprotein studies have revealed the direct interaction of p10 with p40 (5) and with p24 (8). The nuclear translocation of the p10 protein after interaction with p40 and/or p24 suggests that p10 also may play a role in BDV replication in the nucleus. Here, we report on our study to characterize the site on p10 wherein interaction with p24 occurs.A series of prokaryotic and eukaryotic expression vectors expressing full-length and mutant p10 were constructed ( Fig. 1A; Table 1) by the use of specific primers and PCR. The cDNA of ORFx1 previously cloned into the pGEX4T-3 vector to yield the pGEX-ORFx1 plasmid (5) was used as the template in the PCR. The amplified DNA fragments tagged with a sequence coding for the FLAG epitope were digested by EcoRI and XhoI and cloned into the pET-30b vector. This was in frame and downstream of the sequence encoding the His and S tags. The amplified DNA fragments were also cloned into the pcDNA3 eukaryotic expression vector under the control of the Cytomegalovirus intermediate promoter. Sequencing (7) was performed to ensure that the constructs did not have any errors.Inclusion of the FLAG epitope at the 3Ј end ensured detection of the expressed recombinant proteins. A commercially available anti-FLAG monoclonal antibody was used throughout this study to detect the full-length and the p10 mutants. As shown in Fig. 1B, the p10 fusion protein (Fig. 1B, lanes 1 and 2) expressed by the pET-ORFx1-FLAG plasmid was detected by our p10-specific ...

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

“…2C, X was efficiently precipitated in the cells transfected with S1, S2, and S3 mutants but not in the others. This result was consistent with those in previous studies (26,48) and demonstrated that the cytoplasmic localization of BDV P is correlated with the binding capacity between X and P. This observation suggested that interaction with BDV X inhibits nuclear localization of P in transfected cells.…”

“…This fact suggested that conformational change of P via binding to X may be necessary for concealing the NLSs of P. On the other hand, as described above, it may also be possible that X/P contains NES in the sequences. Although previous studies (26,40,48) and our own have not detected nuclear export activity of X/P, it is likely that the activity does not emerge until X and P generate protein complexes leading to the covering of their NLSs. A similar case may have been demonstrated in BDV N protein; the nuclear export activity of the N protein was clearly demonstrated only in the observation of p38N, which translationally lacks the NLS (18,19).…”

“…In addition, coexpression of BDV proteins also shows nuclear distribution of the protein complexes in transfected cells (18,26,40). These results suggested that nuclear direction of the viral proteins must be essential for the viral replication and transcription in infected cells.…”

“…Interaction with BDV X determines the intracellular distribution of P. Recent studies have demonstrated that X directly binds to P with its short amino acid domain in the N terminus (26,48). These observations raised a possibility that the interaction with BDV X influences the intracellular localization of P. Thus, we first generated amino acid deletion or substitution mutants in the P-binding domain of X by using the GFP-fused X/P expression plasmid and analyzed the subcellular localization of P ( Fig.…”

“…Therefore, it is possible that binding between X and P results in masking of the NLSs in both the proteins and leads to cytoplasmic retention of the protein complexes. In fact, the P-binding region in X completely overlaps with the NLS of X (26,48). However, NLSs of P are not directly covered by binding to X; the NLSs of P are found in both the N and C termini of the protein, whereas the X-binding domain is situated in the central region of P (40).…”