A brome mosaic virus intergenic RNA3 replication signal functions with viral replication protein 1a to dramatically stabilize RNA in vivo

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-lik e superfamily, encodes two RNA replication proteins, The 1a protein has put ative helicase and RNA-capping domains, whereas 2a contains a polymerase-li ke domain Saccharomyces cerevisae expressing 1a and 2a is capable of replic ating a BMV RNA3 template produced by in vivo transcription of a DNA copy o f RNA3, Although insufficient for RNA3 replication, the expression of 1a pr otein alone results in a dramatic and specific stabilization of the RNA3 te mplate in yeast. As one step toward understanding la-induced stabilization of RNA3, the interactions involved, and its possible relation to RNA replic ation, we have identified the cis-acting sequences required for this effect . We find that 1a-induced stabilization is mediated by a 150- to 190-base s egment of the RNA3 intergenic region corresponding to a previously identifi ed enhancer of RNA3 replication. Moreover, this segment is sufficient to co nfer 1a-induced stability on a heterologous beta-globin RNA. Within this in tergenic segment, partial deletions that inhibited 1a-induced stabilization in yeast expressing 1a alone resulted in parallel decreases in the levels of negative- and positive-strand RNA3 replication products in yeast express ing 1a and 2a, In particular, a small deletion encompassing a motif corresp onding to the box B element of RNA polymerase III promoters dramatically re duced the ability of RNAs to respond to 1a or 1a and 2a. These and other fi ndings suggest that 1a-induced stabilization likely reflects an early templ ate selection step in BMV RNA replication.