AN RNA-BINDING DOMAIN IN THE VIRAL HEMORRHAGIC SEPTICEMIA VIRUS NUCLEOPROTEIN

The gene encoding the nucleoprotein (N) and PCR-derived subfragments f rom viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdoviru s, were overexpressed in Escherichia coli BL21(DE3) transformed by rec ombinant expression vector pET-14b containing N and PCR-generated subf ragment cDNAs under the control of the T7 RNA polymerase promoter. Fol lowing induction with IPTG, recombinant His-tagged proteins were expre ssed, purified by affinity metal chelation chromatography under denatu ring conditions and renatured. Protein blots were hybridized with vari ous radiolabelled nucleic acid probes. Results obtained using genomic or messenger virus RNA as a probe indicated that the middle part of N was possibly an RNA-binding domain. To confirm this observation, two m ore accurate approaches were undertaken: (i) a gel retardation assay o f RNA and purified protein complexes was done; and (ii) RNA-protein co mplexes were cross-linked by UV light and analysed on a denaturing pol yacrylamide gel. All these experiments led us to conclude that the mid dle part of N is the domain which interacts with RNA, despite the abse nce of homology with known consensus amino acid sequences of other RNA -binding proteins.