RNA binding properties of Bunyamwera virus nucleocapsid protein and selective binding to an element in the 5 ' terminus of the negative-sense S segment

The genome of Bunyamwera virus (BUN) (family Bunyaviridae, genus Bunyavirus ) comprises three negative-sense RNA segments which act as transcriptional templates for the viral polymerase only when encapsidated by the nucleocaps id protein (N), Previous studies have suggested that the encapsidation sign al may reside within the 5' terminus of each segment. The BUN N protein was expressed as a 6-histidine-tagged fusion protein in Escherichia coli and p urified by metal chelate chromatography. An RNA probe containing the 5'-ter minal 32 and 3'-terminal 33 bases of the BUN S (small) genome segment was u sed to investigate binding by the N protein in vitro using gel mobility shi ft and filter binding assays. On acrylamide gels a number of discrete RNA-N complexes were resolved, and analysis of filter binding data indicated a d egree of cooperativity in N protein binding, RNA-N complexes were resistant to digestion with up to 1 mu g of RNase A per mi, Competition assays with a variety of viral and nonviral RNAs identified a region within the 5' term inus of the BUN S segment for which N had a high preference for binding. Th is site may constitute the signal for initiation of encapsidation by N.