title: High affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus RNA.
abstract: 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 Escherichia coli as a chimeric protein fused to glutathione-S-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 (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 177-231 which bound viral RNA with a K(d) of 32 nM. A consensus viral sequence recognized by the N protein was inferred from these studies; AAUCYAAAC 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.
authors.affiliation: Departments of Molecular Microbiology and Immunology and Neurology(2), USC School of Medicine, 2011 Zonal Avenue, Los Angeles, CA 90033-1054, USA.
authors.forename: G W
authors.initials: GW
authors.lastname: Nelson
authors.forename: S A
authors.initials: SA
authors.lastname: Stohlman
authors.forename: S M
authors.initials: SM
authors.lastname: Tahara
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics / Q000378:metabolism*; D019251:Nucleocapsid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019590:Nucleocapsid Proteins; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D008024:Ligands; D019590:Nucleocapsid Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; C099600:nucleocapsid protein, Hepatitis virus; D005982:Glutathione Transferase