1887

Abstract

The gene encoding the nucleoprotein (N) and PCR- derived subfragments from viral haemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus, were overexpressed in BL21(DE) transformed by recombinant expression vector pET-14b containing N and PCR-generated subfragment 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 re- natured. 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.

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/content/journal/jgv/10.1099/0022-1317-79-1-47
1998-01-01
2022-05-23
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