Analysis of the nucleocapsid protein of IBV and its association with a hypervariable region in the 3' end of the genome

Abstract

The natural sequence variations of the nucleocapsid genes and 3' noncoding regions of the Gray, Ark99 and Holl52 strains of IBV were determined and compared to the Mass41, Beau and KB8523 strains. The nucleocapsid protein was highly conserved and also shared conserved amino acids with other coronaviruses. In contrast, the 3' non-coding region contained a U-rich hypervariable region (HVR) immediately downstream of the nucleocapsid gene. This U-rich sequence was absent from 3' non-coding region of the Mass41 strain, and was also highly variable, especially in comparison to the highly conserved 3' non-coding region downstream of this sequence. Computer analyses of the sequences within and adjacent to this HVR showed that the 3' end of the genome was highly conserved downstream of this region, with 94.3 to 97.8% similarity. However, the similarities for the HVR ranged from 53.2% between Holl52 and Ark99, to 92.8% between Beaudette and Gray. The flanking sequences upstream and downstream of the HVR were conserved and also formed mirrored images. The association of the IBV nucleocapsid protein with RNA from portions of the 3' end of the genome was examined by northwestern blot analysis and gel shift assays. The present studies have shown that the nucleocapsid protein was able to bind with a high affinity to RNA corresponding to the 3' end of the IBV genome. All the positive and negative sense IBV-derived RNAs used in this study were capable of binding to the nucleocapsid protein, and apparently all the positive sense IBV RNAs had similar affinity for the nucleocapsid protein.