Out the bornavirus X expression plasmid, using Lipofectamine 2000 (Invitrogen). 48 h later, the cells were lysed and cell lysates were prepared for CAT assays. CAT activity was quantified with a CAT ELISA (Roche) according to the manufacturer’s directions.Results Sequence K162 web Comparison of the X and P Genes of Mammalian, Avian and Reptile BornavirusesPrevious studies indicated that ABV may feature sequence diversity in the region between the N and X genes [15,18]. In a recent study, we detected persistent infection by ABV5 of an Eclectus parrot (Eclectus roratus), suffering from the feather picking disease [18]. Sequence analysis revealed that the upstream region of the X ORF of ABV5 is almost the same length as that of BDV, although the putative uORF of ABV5 is 9 nucleotides (nt) shorter than that of BDV (Figure 2A). In addition, we reported the identification of RBV N and X/P mRNA sequences in a cDNA library derived from a Bitis gabonica venom gland [12,20]. Interestingly, in comparison with BDV, the sequence between the N and X ORFs of RBV appeared to have a 21 nt deletion (Figure 2A). These findings suggested that these genotypes may be useful tools for analysis of the functional interaction between X and P among bornavirus genotypes. We aligned the amino acid sequences of X and P of BDV, ABV4, ABV5 and RBV. Consistent with phylogenetic analyses [9,12], the X and P proteins of ABV5 showed a relatively high similarity with those of BDV (Figure 2B and C). In contrast, RBV seems to be distant from the other genotypes. The amino acid sequence identities and similarities among the genotypes are shown in Table 1. Previous studies revealed that the BDV X and P proteins contain several signal sequences, including nuclear localization (NLS) and nuclear export (NES) signals, putative binding sites for each other and phosphorylation sites [21?6]. The BDV P protein has two proline-rich NLSs, located in the Nand C-terminal regions [23,24]. A proline residue is substituted in the corresponding regions of ABV4 and 5 (Figure 2C). On the other hand, RBV has only one and three proline residues in the Nand C-terminal regions, respectively. In addition, the methioninerich NES of the BDV P protein seems to be relatively highly conserved among the ABVs, but not in RBV (Figure 1C). The putative binding sites of the X and P proteins also seem to have evolved differently in RBV, compared to other genotypesInter-genotypic Interaction between the X and P Proteins of Vertebrate BornavirusesTo understand the evolutionary relationship between bornavirus genotypes, we next examined the inter-genotypic interaction between the X and P proteins. 293T cells were transfected with the X and P expression plasmids with various combinations of the different genotypes and protein interactions were detected by immunoprecipitation analysis using anti-HA antibody. Interestingly, the P protein of all genotypes, including RBV, was shown to efficiently precipitate the X proteins of BDV and the ABVs (Figure 6A, B, C). In contrast, the inter-genotypic interaction of the RBV X protein was detected only in cells co-transfected with ABV4 P after a long exposure image of the 3PO chemical information membrane (Figure 6D). Although the weak interaction between RBV X and ABV4 P may be due to the over-expression experiment of the recombinant proteins, similar results were obtained in the experiment using immunofluorescence assay; the RBV P protein was translocated to the cytoplasm when the BDV and ABV X proteins were.Out the bornavirus X expression plasmid, using Lipofectamine 2000 (Invitrogen). 48 h later, the cells were lysed and cell lysates were prepared for CAT assays. CAT activity was quantified with a CAT ELISA (Roche) according to the manufacturer’s directions.Results Sequence Comparison of the X and P Genes of Mammalian, Avian and Reptile BornavirusesPrevious studies indicated that ABV may feature sequence diversity in the region between the N and X genes [15,18]. In a recent study, we detected persistent infection by ABV5 of an Eclectus parrot (Eclectus roratus), suffering from the feather picking disease [18]. Sequence analysis revealed that the upstream region of the X ORF of ABV5 is almost the same length as that of BDV, although the putative uORF of ABV5 is 9 nucleotides (nt) shorter than that of BDV (Figure 2A). In addition, we reported the identification of RBV N and X/P mRNA sequences in a cDNA library derived from a Bitis gabonica venom gland [12,20]. Interestingly, in comparison with BDV, the sequence between the N and X ORFs of RBV appeared to have a 21 nt deletion (Figure 2A). These findings suggested that these genotypes may be useful tools for analysis of the functional interaction between X and P among bornavirus genotypes. We aligned the amino acid sequences of X and P of BDV, ABV4, ABV5 and RBV. Consistent with phylogenetic analyses [9,12], the X and P proteins of ABV5 showed a relatively high similarity with those of BDV (Figure 2B and C). In contrast, RBV seems to be distant from the other genotypes. The amino acid sequence identities and similarities among the genotypes are shown in Table 1. Previous studies revealed that the BDV X and P proteins contain several signal sequences, including nuclear localization (NLS) and nuclear export (NES) signals, putative binding sites for each other and phosphorylation sites [21?6]. The BDV P protein has two proline-rich NLSs, located in the Nand C-terminal regions [23,24]. A proline residue is substituted in the corresponding regions of ABV4 and 5 (Figure 2C). On the other hand, RBV has only one and three proline residues in the Nand C-terminal regions, respectively. In addition, the methioninerich NES of the BDV P protein seems to be relatively highly conserved among the ABVs, but not in RBV (Figure 1C). The putative binding sites of the X and P proteins also seem to have evolved differently in RBV, compared to other genotypesInter-genotypic Interaction between the X and P Proteins of Vertebrate BornavirusesTo understand the evolutionary relationship between bornavirus genotypes, we next examined the inter-genotypic interaction between the X and P proteins. 293T cells were transfected with the X and P expression plasmids with various combinations of the different genotypes and protein interactions were detected by immunoprecipitation analysis using anti-HA antibody. Interestingly, the P protein of all genotypes, including RBV, was shown to efficiently precipitate the X proteins of BDV and the ABVs (Figure 6A, B, C). In contrast, the inter-genotypic interaction of the RBV X protein was detected only in cells co-transfected with ABV4 P after a long exposure image of the membrane (Figure 6D). Although the weak interaction between RBV X and ABV4 P may be due to the over-expression experiment of the recombinant proteins, similar results were obtained in the experiment using immunofluorescence assay; the RBV P protein was translocated to the cytoplasm when the BDV and ABV X proteins were.
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