The amino and carboxyl domains of the infectious bronchitis virus nucleocapsid protein interact with 3 ' genomic RNA

Previous studies indicated that the nucleocapsid (N) protein of infectious bronchitis virus (IBV) interacted with specific sequences in the 3' non-cod ing region of IBV RNA. In order to identify domains in the N protein that b ind to RNA, the whole protein (409 amino acids) and six overlapping fragmen ts 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 re sidues 203 to 409 and residues 268 to 407, were found to interact with posi tive-stranded IBV RNA representing the 3' end of the genome. The two P-32-l abeled probes that interacted with N and the amino and carboxyl fragments o f N were RNA consisting of the IBV N gene and adjacent 3' non-coding termin us, 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 RNA s. 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 Vall ey virus N expressed with a histidine tag, bovine serum albumin, and the ba sic lysozyme protein did not shift the IBV RNA. The lower molarities of the carboxyl fragment compared with residue 1-274 fragment needed for equivale nt shifts suggested that the binding avidity for RNA at the carboxyl domain was greater than the amino domain; (C) 2000 Elsevier Science B.V. All righ ts reserved.