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.

Altered pathogenicity, immunogenicity, tissue tropism and 3′-7kb region sequence of an avian infectious bronchitis coronavirus strain after serial passage in embryos

“…Until now, the corresponding protein and its IBV function have not been clear. However, it is hypothesized that the sequences in the IBV 3 -UTR are involved in regulating viral RNA replication and transcription [56]. In addition, Sapats et al [57] reported that shorter forms of the 3 -UTR in the Australian N1/88, Q3/88, and V18/91 strains are associated with a decrease in virulence.…”

Molecular Interactions in the Assembly of Coronaviruses

“…6) (Peng et al, 1995a). Although all these studies consistently attribute a major role in RNA binding to the central portion of the coronaviral N protein, the interaction of the IBV N protein with the 3 0 UTR of IBV RNA mentioned previously was mapped to the amino-and carboxy-terminal domains of the molecule (Zhou and Collisson, 2000). 3 0 UTR RNA-binding activity was also assigned to the amino-terminal domain of the SARS-CoV N protein on the basis of studies using nuclear magnetic resonance spectroscopy (Huang et al, 2004).…”

The Molecular Biology of Coronaviruses

“…The RNA-binding capability of the MHV N protein has been mapped to domain 2 of this molecule (Masters, 1992;Nelson and Stohlman, 1993). However, for IBV, two separate RNA-binding sites have been found to map, respectively, to amino-and carboxy-terminal fragments of N protein (Zhou and Collisson, 2000), and RNA-binding activity has been reported for a fragment of the SARS-CoV N protein containing parts of domains 1 and 2 (Huang et al, 2004b).…”