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Journal of Virology, April 1999, p. 2622-2632, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

A Brome Mosaic Virus Intergenic RNA3 Replication Signal Functions with Viral Replication Protein 1a To Dramatically Stabilize RNA In Vivo

Michael L. Sullivan1 and Paul Ahlquist1,2,*

Institute for Molecular Virology1 and Howard Hughes Medical Institute,2 University of Wisconsin---Madison, Madison, Wisconsin 53706

Received 9 October 1998/Accepted 23 December 1998

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication proteins. The 1a protein has putative helicase and RNA-capping domains, whereas 2a contains a polymerase-like domain. Saccharomyces cerevisiae expressing 1a and 2a is capable of replicating a BMV RNA3 template produced by in vivo transcription of a DNA copy of RNA3. Although insufficient for RNA3 replication, the expression of 1a protein alone results in a dramatic and specific stabilization of the RNA3 template in yeast. As one step toward understanding 1a-induced stabilization of RNA3, the interactions involved, and its possible relation to RNA replication, 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 segment of the RNA3 intergenic region corresponding to a previously identified enhancer of RNA3 replication. Moreover, this segment is sufficient to confer 1a-induced stability on a heterologous beta -globin RNA. Within this intergenic 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 expressing 1a and 2a. In particular, a small deletion encompassing a motif corresponding to the box B element of RNA polymerase III promoters dramatically reduced the ability of RNAs to respond to 1a or 1a and 2a. These and other findings suggest that 1a-induced stabilization likely reflects an early template selection step in BMV RNA replication.

* Corresponding author. Mailing address: Institute for Molecular Virology, University of Wisconsin---Madison, 1525 Linden Dr., Madison, WI 53706. Phone: (608) 262-5916. Fax: (608) 265-9214. E-mail address: ahlquist{at}facstaff.wisc.edu.

Journal of Virology, April 1999, p. 2622-2632, Vol. 73, No. 4
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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