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

Virus Res. 2000 Mar;67(1):31-9. doi: 10.1016/s0168-1702(00)00126-x.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Blotting, Western
  • Cattle
  • Coronavirus / genetics
  • Coronavirus / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Genome, Viral*
  • Nucleocapsid / isolation & purification
  • Nucleocapsid / metabolism*
  • Nucleocapsid Proteins*
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA, Viral / metabolism*

Substances

  • Nucleocapsid Proteins
  • RNA, Viral
  • nucleocapsid protein, Coronavirus