Coexpressed in the cells, whereas the X protein of RBV was seemed to slightly affect the nuclear distribution of ABV4 P (Figure S1). We determined finally whether the proteins of non-mammalian bornaviruses can participate in the polymerase activity of BDV using a minireplicon system [28], which can ML 264 custom synthesis reconstitute recombinant BDV nucleocapsids containing an artificial, genome-like reporter RNA (minigenome) following transfection ofConserved Interaction of Bornavirus ProteinsFigure 2. Sequence comparisons among bornavirus genotypes. (A) Nucleotide sequence alignment of the region between the N and X ORFs. The stop and start codons of the BDV N and X genes, respectively, are shown. The start codon of the BDV uORF and the proposed uORF of ABV5 are indicated in red. The S2 transcription initiation and T1 termination sites of BDV and their corresponding sequences in non-mammalian bornaviruses are underlined. (B and C) Amino acid sequence alignments of X (B) and P (C) among bornavirus genotypes. Amino acids identical to the BDV sequences are indicated by dots. Gaps are indicated by dashes. Sequences identical in four and three genotypes are indicated by asterisks and dots, respectively, above the sequences. The regions of NLS, NES and 101043-37-2 manufacturer putative binding sites for each protein are underlined. doi:10.1371/journal.pone.0051161.gexpression plasmids encoding BDV N, P, L, and the minigenome. Previous studies have demonstrated that BDV X can inhibit the viral polymerase activity in the minireplicon system [29]. We carried out the minireplicon assay using P expression plasmids of the non-mammalian bornaviruses instead of the BDV P plasmid. In addition, the assays were performed in the presence or absence of the X proteins from ABV and RBV. At 48 h post-transfection, BDV polymerase activity was determined by expression of chloramphenicol acetyltransferase (CAT), detected by ELISA. As shown in Figure 7, interestingly, the P proteins of ABV4 and 5, butnot RBV, could initiate the polymerase activity of BDV up to 40 of the level of BDV P. A recombinant RBV P, which was added the five amino acid residues, Trp-Asp-Ile-Ile-Pro, important for the interaction with BDV N of BDV P (Figure 2C) [30] in the Cterminus, also failed to induce the CAT activity in the transfected cells (Figure 7, RBV-B). This result suggested that the lack of the activation of the minireplicon by RBV P might not be due to the limited interaction between RBV P and BDV N. In addition, the X proteins of ABVs also appeared to reduce markedly the polymerase activity of BDV, as does BDV X (Figure 7). On theConserved Interaction of Bornavirus ProteinsTable 1. Amino acid sequence homologies of X proteins and phosphoproteins among bornavirus genotypes.DiscussionIn this study, we investigated the functional interaction of the X and P proteins of various bornaviruses to understand the evolutionary relationships among the genotypes. Sequence analysis revealed that the regions corresponding to the 59 UTR of the putative 1379592 X/P mRNA of ABV5 and RBV are classified into the BDV- and ABV2/4-type, respectively. The putative 59 UTR of ABV5 was shown to be almost of the same length as that of mammalian bornavirus, whereas RBV contained an ABV2/4-type deletion in this region (Figure 2). In a previous study, we revealed using phylogenetic analysis that RBV may have branched before the separation of ABV and BDV [12], suggesting that additional sequences have been inserted into the 59 UTR of the X/P mRNA of the BDV/.Coexpressed in the cells, whereas the X protein of RBV was seemed to slightly affect the nuclear distribution of ABV4 P (Figure S1). We determined finally whether the proteins of non-mammalian bornaviruses can participate in the polymerase activity of BDV using a minireplicon system [28], which can reconstitute recombinant BDV nucleocapsids containing an artificial, genome-like reporter RNA (minigenome) following transfection ofConserved Interaction of Bornavirus ProteinsFigure 2. Sequence comparisons among bornavirus genotypes. (A) Nucleotide sequence alignment of the region between the N and X ORFs. The stop and start codons of the BDV N and X genes, respectively, are shown. The start codon of the BDV uORF and the proposed uORF of ABV5 are indicated in red. The S2 transcription initiation and T1 termination sites of BDV and their corresponding sequences in non-mammalian bornaviruses are underlined. (B and C) Amino acid sequence alignments of X (B) and P (C) among bornavirus genotypes. Amino acids identical to the BDV sequences are indicated by dots. Gaps are indicated by dashes. Sequences identical in four and three genotypes are indicated by asterisks and dots, respectively, above the sequences. The regions of NLS, NES and putative binding sites for each protein are underlined. doi:10.1371/journal.pone.0051161.gexpression plasmids encoding BDV N, P, L, and the minigenome. Previous studies have demonstrated that BDV X can inhibit the viral polymerase activity in the minireplicon system [29]. We carried out the minireplicon assay using P expression plasmids of the non-mammalian bornaviruses instead of the BDV P plasmid. In addition, the assays were performed in the presence or absence of the X proteins from ABV and RBV. At 48 h post-transfection, BDV polymerase activity was determined by expression of chloramphenicol acetyltransferase (CAT), detected by ELISA. As shown in Figure 7, interestingly, the P proteins of ABV4 and 5, butnot RBV, could initiate the polymerase activity of BDV up to 40 of the level of BDV P. A recombinant RBV P, which was added the five amino acid residues, Trp-Asp-Ile-Ile-Pro, important for the interaction with BDV N of BDV P (Figure 2C) [30] in the Cterminus, also failed to induce the CAT activity in the transfected cells (Figure 7, RBV-B). This result suggested that the lack of the activation of the minireplicon by RBV P might not be due to the limited interaction between RBV P and BDV N. In addition, the X proteins of ABVs also appeared to reduce markedly the polymerase activity of BDV, as does BDV X (Figure 7). On theConserved Interaction of Bornavirus ProteinsTable 1. Amino acid sequence homologies of X proteins and phosphoproteins among bornavirus genotypes.DiscussionIn this study, we investigated the functional interaction of the X and P proteins of various bornaviruses to understand the evolutionary relationships among the genotypes. Sequence analysis revealed that the regions corresponding to the 59 UTR of the putative 1379592 X/P mRNA of ABV5 and RBV are classified into the BDV- and ABV2/4-type, respectively. The putative 59 UTR of ABV5 was shown to be almost of the same length as that of mammalian bornavirus, whereas RBV contained an ABV2/4-type deletion in this region (Figure 2). In a previous study, we revealed using phylogenetic analysis that RBV may have branched before the separation of ABV and BDV [12], suggesting that additional sequences have been inserted into the 59 UTR of the X/P mRNA of the BDV/.
