title: The amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with 3' genomic RNA.
abstract: Previous studies indicated that the nucleocapsid (N) protein of infectious bronchitis virus (IBV) interacted with specific sequences in the 3' non-coding region of IBV RNA. In order to identify domains in the N protein that bind to RNA, the whole protein (409 amino acids) and six overlapping fragments were expressed as fusion polypeptides with six histidine-tags. Using gel shift assays, the intact N protein and amino polypeptides, from residues 1 to 171 and residues 1 to 274, and carboxyl polypeptides, extending from residues 203 to 409 and residues 268 to 407, were found to interact with positive-stranded IBV RNA representing the 3' end of the genome. The two 32P-labeled probes that interacted with N and the amino and carboxyl fragments of N were RNA consisting of the IBV N gene and adjacent 3' non-coding terminus, and RNA consisting of the 155-nucleotide sequences at the 3' end of the 504-nt 3' untranslated region. In contrast, the polypeptide fragment from the middle region, residues 101-283, did not interact with these 3' IBV RNAs. The binding site in the amino region of N was either not present or only partially present in the first 91 residues because no interaction with RNA was observed with the polypeptide incorporating these residues. Cache Valley virus N expressed with a histidine tag, bovine serum albumin, and the basic lysozyme protein did not shift the IBV RNA. The lower molarities of the carboxyl fragment compared with residue 1-274 fragment needed for equivalent shifts suggested that the binding avidity for RNA at the carboxyl domain was greater than the amino domain.
authors.affiliation: Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, TX 77843-4467, USA.
authors.forename: M
authors.initials: M
authors.lastname: Zhou
authors.forename: E W
authors.initials: EW
authors.lastname: Collisson
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D017934:Coronavirus / Q000235:genetics / Q000378:metabolism*; D000086462:Coronavirus Nucleocapsid Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D016679:Genome, Viral; D019251:Nucleocapsid / Q000302:isolation & purification / Q000378:metabolism*; D019590:Nucleocapsid Proteins; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000378:metabolism*
chemical_list: D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D012367:RNA, Viral