Journal of General Virology, Vol 79, 47-50, Copyright © 1998 by Society for General Microbiology
An RNA-binding domain in the viral haemorrhagic septicaemia virus nucleoprotein
T Said, H Bruley, A Lamoureux and M Bremont
Unite de Virologie et Immunologie Moleculaires, Institut National de la Recherche Agronomique, jouy-en-Josas, France.
The gene encoding the nucleoprotein (N) and PCR-derived subfragments from
viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus, were
overexpressed in Escherichia coli BL21(DE3) transformed by recombinant
expression vector pET-14b containing N and PCR-generated sub-fragment cDNAs
under the control of the T7 RNA polymerase promoter. Following induction
with IPTG, recombinant His- tagged proteins were expressed, purified by
affinity metal chelation chromatography under denaturing conditions and
renatured. Protein blots were hybridized with various 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 more accurate approaches were
undertaken: (i) a gel retardation assay of RNA and purified protein
complexes was done; and (ii) RNA-protein complexes were cross-linked by UV
light and analysed on a denaturing polyacrylamide gel. All these
experiments led us to conclude that the middle part of N is the domain
which interacts with RNA, despite the absence of homology with known
consensus amino acid sequences of other RNA-binding proteins.
