STABLE PROTEIN-RNA INTERACTION INVOLVES THE TERMINAL DOMAINS OF BLUETONGUE VIRUS MESSENGER-RNA, BUT NOT THE TERMINALLY CONSERVED SEQUENCES

The interaction of bluetongue virus (BTV) proteins with viral RNA was investigated in vitro by means of a biochemical approach. By subjectin g cytoplasmic extracts from virus-infected baby hamster kidney cells a nd in vitro synthesized radiolabeled RNA to ultraviolet cross-linking assays, we demonstrated that, of all the BTV proteins, NS2 becomes mos t intimately associated with the labeled viral RNA. Competition bindin g studies indicated that NS2 has the greatest affinity for the 3' regi on of the viral transcripts. By analyzing the binding efficiency of NS 2 to mutant RNA transcripts which lacked the fully conserved 5'- and/o r 3'-terminal hexanucleotides, we have established that these sequence s are not necessary for optimal binding, The specificity of the NS2-RN A interaction was investigated by competition experiments with unlabel ed 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 o bserved, it did not bind to the dsRNA. (C) 1997 Academic Press.