High affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus RNA

The nucleocapsid (N) protein of mouse hepatitis virus (MHV) is the major vi rion structural protein. It associates with both viral genomic RNA and subg enomic mRNAs and has structural and nonstructural roles in replication incl uding viral RNA-dependent RNA transcription, genome replication, encapsidat ion and translation. These processes all involve RNA-protein interactions b etween the N protein and viral RNAs. To better understand the RNA-binding p roperties of this multifunctional protein, the N protein was expressed in E scherichia coli as a chimeric protein fused to glutathione-S-transferase (G ST). 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 bin ding activities with a dissociation constant (K-d) 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 177-2 31 which bound viral RNA with a K-d of 32 nM. A consensus viral sequence re cognized by the N protein was inferred from these studies; AAUCYAAAC was id entified to be the potential minimum ligand for the N protein. Although the core UCYAA sequence is often tandemly repeated in viral genomes, ligands c ontaining one or more repeats of UCYAA showed no difference in binding to t he N protein. Together these data demonstrate a high-affinity, specific int eraction between the N protein and a conserved RNA sequence present at the 5'-ends of MHV mRNA.