The natural sequence variations of the nucleocapsid genes of the Gray, Arkansas99 (Ark99), and Holland52 (Holl52) strains of infectious bronchitis virus (IBV) were determined. These were compared with previously published sequencing data of other IBV strains, as well as other coronaviruses, in order to correlate the serological and evolutionary relationship of coronaviruses. IBV nucleotide sequence alignment shows that overall the sequences are highly conserved, with homologies from 91.1 to 96.5%. However, there are also two regions (730 to 800 and 1138 to 1166) that appear to be even more highly conserved. Overall, the nucleocapsid protein is highly variable both in size and composition between coronavirus major antigenic groups but is conserved within these groups. A phylogenetic tree of the nucleocapsid protein of various coronaviruses indicates that the coronaviruses fall into distinct groups that correspond to the three major antigenic groups; however, a phylogenetic tree of the IBV nucleocapsid shows that this does not hold true for the type specific antigenic groups of IBV.
The infectious bronchitis virus (IBV) nucleocapsid protein was expressed as a bacterial fusion protein which differed from the native protein only in the addition of six amino terminus histidine residues. Using RNA overlay protein blot assays, the recombinant protein was shown to bind to RNA fragments specific for the positive sense 3' noncoding end of the IBV genome. At greater concentrations of sodium chloride, the native and fusion nucleocapsid proteins similarly bound to G RNA, representing the terminal 1805 3' nt of the genome, whereas bovine serum albumin and allantoic fluid protein did not bind to labeled G RNA. Competitive gel shift assays with labeled G RNA indicated that the protein interacted with several unlabeled RNA representing sequences at the 3' noncoding end of the IBV genome. Cache Valley virus (a bunyavirus) mRNA transcribed from the small segment cDNA also inhibited the interaction with IBV G RNA to approximately the same extent as homologous unlabeled G RNA, whereas reactions with bovine liver RNA and yeast tRNA were considerably weaker. Whereas yeast tRNA did not inhibit the interaction with the labeled large G RNA, interactions of the fusion protein with EF, a region from 78 to 217 nt from the 3' terminus of the IBV genome, were also apparently weaker than interactions with fragment CD which consisted of the 3' terminal 155 nt. On a molar basis, the latter interacted in an identical nature to a RNA consisting of CD and an additional 1053 nt of plasmid sequences. Compared to bovine liver RNA, unlabeled G specifically inhibited binding to the two smaller labeled IBV fragments in gel shift assays. The binding of IBV nucleocapsid protein with RNA probably requires specific sequences and/or structures that are present on the genome, and may represent a common mechanism used by similar viral nucleoproteins whose functions depend on binding to RNA.
Previous studies on infectious bronchitis virus (IBV) cDNA have identified a region of about 184 bases in the 3' non-coding terminus of both the U.S. prototype strain (Beaudette) and a Japanese strain (KB8523), that was not present in an antigenically closely related U.S. strain, Massachusetts (Mass) 41 (Boursnell et al., 1985; Sutou et al., 1988). In order to investigate the origin and function of this region and its occurrence in nature, the cDNA sequences of the 3' non-coding regions of three additional strains of IBV, Gray, Arkansas (Ark) 99 and Holland (Holl) 52, were determined and compared to the sequences of the Beaudette, KB8523 and Mass41 strains. Not only was this Urich sequence absent from the 3' non-coding region of the Mass41 strain, it was also highly variable, especially in comparison to the highly conserved 3' non coding region downstream of this sequence. Computer analyses of the sequences adjacent to this hypervariable region (HVR) showed that the 3' end of the IBV 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 were not only conserved but these sequences upstream and downstream of the HVR also formed mirrored images.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.