Stable protein-RNA interaction involves the terminal domains of bluetongue virus mRNA, but not the terminally conserved sequences

Abstract

The interaction of bluetongue virus (BTV) proteins with viral RNA was investigated in vitro by means of a biochemical approach. By subjecting cytoplasmic extracts from virus-infected baby hamster kidney cells and in vitro synthesized radiolabeled RNA to ultraviolet cross-linking assays, we demonstrated that, of all the BTV proteins, NS2 becomes most intimately associated with the labeled viral RNA. Competition binding studies indicated that NS2 has the greatest affinity for the 3' region of the viral transcripts. By analyzing the binding efficiency of NS2 to mutant RNA transcripts which lacked the fully conserved 5'- and/or 3'-terminal hexanucleotides, we have established that these sequences are not necessary for optimal binding. The specificity of the NS2-RNA interaction was investigated by competition experiments with unlabeled BTV-specific homologous and heterologous competitor RNAs as well as with viral double-stranded RNA (dsRNA). Although apparent differences in the ability of NS2 to bind to the different RNA transcripts were observed, it did not bind to the dsRNA.