@article{mbs:/content/journal/jgv/10.1099/0022-1317-81-1-181, author = "Nelson, Gary W. and Stohlman, Stephen A. and Tahara, Stanley M.", title = "High affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus RNA", journal= "Journal of General Virology", year = "2000", volume = "81", number = "1", pages = "181-188", doi = "https://doi.org/10.1099/0022-1317-81-1-181", url = "https://www.microbiologyresearch.org/content/journal/jgv/10.1099/0022-1317-81-1-181", publisher = "Microbiology Society", issn = "1465-2099", type = "Journal Article", 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.", }