INFECTIOUS-BRONCHITIS VIRUS NUCLEOCAPSID PROTEIN BINDS RNA SEQUENCES IN THE 3'-TERMINUS OF THE GENOME

The infectious bronchitis virus (IBV) nucleocapsid protein was express ed as a bacterial fusion protein which differed from the native protei n 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 en d of the IBV genome. At greater concentrations of sodium chloride, the native and fusion nucleocapsid proteins similarly bound to G RNA, rep resenting the terminal 1805 3' nt of the genome, whereas bovine serum albumin and allantoic fluid protein did not bind to labeled G RNA. Com petitive gel shift assays with labeled G RNA indicated that the protei n 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 intera ction with IBV G RNA to approximately the same extent as homologous un labeled G RNA, whereas reactions with bovine liver RNA and yeast tRNA were considerably weaker. Whereas yeast tRNA did not inhibit the inter action with the labeled large G RNA, interactions of the fusion protei n 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 latt er 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 pr otein with RNA probably requires specific sequences and/or structures that are present on the genome, and may represent a common mechanism u sed by similar viral nucleoproteins whose functions depend on binding to RNA. (C) 1996 Academic Press, Inc.