Brome mosaic virus protein 1a recruits viral RNA2 to RNA replication through a 5′ proximal RNA2 signal

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication factors. Membrane-associated la protein contains a helicase-like domain and RNA capping functions, 2a, which is targeted to membranes by la, contains a central polymerase-like domain, In the absence of Za and RNA replication, la acts through an intergenic replication signal in BMV genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular membrane, Multiple results imply that 1a-induced RNA3 stabilization reflects interactions involved in recruiting RNA3 templates into replication, To determine if la had similar effects on another Bhn RNA replication template, we constructed a plasmid expressing BR IV genomic RNA2 in vivo, In vivo-expressed RNA2 templates were replicated upon expression of la and 2a, In the absence of 2a, la stabilized RNA2 and induced RNA2 to associate with membrane. Deletion analysis demonstrated that la-induced membrane association of RNA2 was mediated by sequences in the 5'-proximal third of RNA2, The RNA2 5' untranslated region was sufficient to confer la-induced membrane association on a nonviral RNA. However, sequences in the N-terminal region of the Za open reading frame enhanced la responsiveness of RNA2 and a chimeric RNA, A 5'-terminal RNA2 stem-loop important for RNA2 replication was essential for la-induced membrane association of RNA2 and, like the la-responsive RNA3 intergenic region, contained a required box B motif corresponding to the T PsiC stem-loop of host tRNAs, The Level of la-induced membrane association of various RNA2 mutants correlated well with their abilities to serve as replication templates, These results support and expand the conclusion that 1a-induced BMV RNA stabilization and membrane association reflect early, 1a-mediated steps in viral RNA replication.