Ding suggests the hypothesis that the stem-loop structures formed in the 59 UTR of the X/P mRNA play a key role in the regulation of translation of the X protein of bornaviruses. Further studies are needed to verify the mechanism of regulation of translation of X from the X/P mRNA of the non-mammalian bornaviruses, via the predicted stem-loop structures of the various genotypes. Our study also provided an CP21 interesting finding regarding the interaction between the X and P proteins. We showed that intergenotypic interaction between X and P is found for all the genotypes, except for the X protein of RBV. Interestingly, although the sequence of the putative binding site between X and P in RBV appeared to be distinct from those in BDV and ABV, RBV P seemed to bind efficiently to the X proteins of both BDV and ABV and was translocated to the cytoplasm in the transfected cells. On the other hand, the X protein of RBV could not precipitate the P proteins of BDV and ABV5, and only a weak interaction was detected between RBV X and ABV4 P. This finding suggests that the functional constraints on the P protein during bornavirus evolution might have been stronger than those on the X protein. While the function of the P protein might be limited to acting as the polymerase of the viruses, the X protein may have been evolved to play several other roles in viral replication as previously reported [31]. In addition, considering that RBV X could not interact with the P protein of other genotypes, it is tempting to speculate that the X protein of RBV has evolved or has been evolving a specific function. The fact that the mitochondrial distribution of the X proteins is observed only in the BDV X-transfected cells (Figure S3) also support thehypothesis that the bornavirus X proteins of different genotypes may have been evolved differently. The co-evolutionary MedChemExpress KS-176 analysis of the X and P proteins of 1531364 bornaviruses may provide interest insights into the genotype-specific evolution of the viral proteins. In conclusion, we found the fundamental functions of the X and P proteins for the replication of bornaviruses may have been conserved during their evolution. Comparative study of the various genotypes of bornaviruses may be important for understanding not only the pathogenesis of the viruses but also the inter-species transmission of bornaviruses. Further studies using isolated viruses of different genotypes are needed to achieve a comprehensive analysis of the evolutionary relationships between the genotypes, as well as for the virological characterization of bornaviruses.Supporting InformationFigure S1 Nuclear export of RBV and ABV P proteins by X proteins from different genotype. Expression plasmids for the indicated bornavirus X and P proteins were transfected into OL cells. Subcellular localizations of the recombinant proteins were detected by immunofluorescence assay using anti-Flag (X: green) and -HA (P: red) antibodies. Merged images with DAPI staining are shown. Scale bars are 10 mm. (TIF) Figure S2 Sequence comparison of the C-terminal regions of bornavirus N proteins. Amino acids identical to the BDV sequence 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. (TIF) Figure SSubcellular localization of bornavirus X proteins. Expression plasmids for the bornavirus X proteins were transfected into OL (A) and QT6 (B) cells. Forty-eight hours.Ding suggests the hypothesis that the stem-loop structures formed in the 59 UTR of the X/P mRNA play a key role in the regulation of translation of the X protein of bornaviruses. Further studies are needed to verify the mechanism of regulation of translation of X from the X/P mRNA of the non-mammalian bornaviruses, via the predicted stem-loop structures of the various genotypes. Our study also provided an interesting finding regarding the interaction between the X and P proteins. We showed that intergenotypic interaction between X and P is found for all the genotypes, except for the X protein of RBV. Interestingly, although the sequence of the putative binding site between X and P in RBV appeared to be distinct from those in BDV and ABV, RBV P seemed to bind efficiently to the X proteins of both BDV and ABV and was translocated to the cytoplasm in the transfected cells. On the other hand, the X protein of RBV could not precipitate the P proteins of BDV and ABV5, and only a weak interaction was detected between RBV X and ABV4 P. This finding suggests that the functional constraints on the P protein during bornavirus evolution might have been stronger than those on the X protein. While the function of the P protein might be limited to acting as the polymerase of the viruses, the X protein may have been evolved to play several other roles in viral replication as previously reported [31]. In addition, considering that RBV X could not interact with the P protein of other genotypes, it is tempting to speculate that the X protein of RBV has evolved or has been evolving a specific function. The fact that the mitochondrial distribution of the X proteins is observed only in the BDV X-transfected cells (Figure S3) also support thehypothesis that the bornavirus X proteins of different genotypes may have been evolved differently. The co-evolutionary analysis of the X and P proteins of 1531364 bornaviruses may provide interest insights into the genotype-specific evolution of the viral proteins. In conclusion, we found the fundamental functions of the X and P proteins for the replication of bornaviruses may have been conserved during their evolution. Comparative study of the various genotypes of bornaviruses may be important for understanding not only the pathogenesis of the viruses but also the inter-species transmission of bornaviruses. Further studies using isolated viruses of different genotypes are needed to achieve a comprehensive analysis of the evolutionary relationships between the genotypes, as well as for the virological characterization of bornaviruses.Supporting InformationFigure S1 Nuclear export of RBV and ABV P proteins by X proteins from different genotype. Expression plasmids for the indicated bornavirus X and P proteins were transfected into OL cells. Subcellular localizations of the recombinant proteins were detected by immunofluorescence assay using anti-Flag (X: green) and -HA (P: red) antibodies. Merged images with DAPI staining are shown. Scale bars are 10 mm. (TIF) Figure S2 Sequence comparison of the C-terminal regions of bornavirus N proteins. Amino acids identical to the BDV sequence 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. (TIF) Figure SSubcellular localization of bornavirus X proteins. Expression plasmids for the bornavirus X proteins were transfected into OL (A) and QT6 (B) cells. Forty-eight hours.
