The nucleocapsid (N) protein of mouse hepatitis virus (MHV) is the major virion structural protein. It associates with both viral genomic RNA and subgenomic mRNAs and has structural and non-structural roles in replication including viral RNA-dependent RNA transcription, genome replication, encapsidation and translation. These processes all involve RNA–protein interactions between the N protein and viral RNAs. To better understand the RNA-binding properties of this multifunctional protein, the N protein was expressed in as a chimeric protein fused to glutathione--transferase (GST). Biochemical analyses of RNA-binding properties were performed on full-length and partial N protein segments to define the RNA-binding domain. The full-length N protein and the GST–N protein fusion product had similar binding activities with a dissociation constant ( ) of 14 nM when the MHV 5′-leader sequence was used as ligand. The smallest N protein fragment which retained RNA-binding activity was a 55 aa segment containing residues 177–231 which bound viral RNA with a of 32 nM. A consensus viral sequence recognized by the N protein was inferred from these studies; AAAC was identified to be the potential minimum ligand for the N protein. Although the core UCYAA sequence is often tandemly repeated in viral genomes, ligands containing one or more repeats of UCYAA showed no difference in binding to the N protein. Together these data demonstrate a high-affinity, specific interaction between the N protein and a conserved RNA sequence present at the 5′-ends of MHV mRNA.


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