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 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.
Nelson
G W
GW
Departments of Molecular Microbiology and Immunology and Neurology(2), USC School of Medicine, 2011 Zonal Avenue, Los Angeles, CA 90033-1054, USA.
Stohlman
S A
SA
Tahara
S M
SM
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
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