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Journal of Virology, December 1999, p. 10303-10309, Vol. 73, No. 12
Institute for Molecular
Virology1 and Howard Hughes Medical
Institute,2 University of
Wisconsin
Received 10 June 1999/Accepted 30 August 1999
The universal membrane association of positive-strand RNA virus RNA
replication complexes is implicated in their function, but the
intracellular membranes used vary among viruses. Brome mosaic virus
(BMV) encodes two mutually interacting RNA replication proteins: 1a,
which contains RNA capping and helicase-like domains, and the
polymerase-like 2a protein. In cells from the natural plant hosts of
BMV, 1a and 2a colocalize on the endoplasmic reticulum (ER). 1a and 2a
also direct BMV RNA replication and subgenomic mRNA synthesis in the
yeast Saccharomyces cerevisiae, but whether the
distribution of 1a, 2a, and active replication complexes in yeast
duplicates that in plant cells has not been determined. For yeast
expressing 1a and 2a and replicating BMV genomic RNA3, we used
double-label confocal immunofluorescence to define the localization of
1a, 2a, and viral RNA and to explore the determinants of replication
complex targeting. As in plant cells, 1a and 2a colocalized on and were
retained on the yeast ER, with no detectable accumulation in the Golgi
apparatus. 1a and 2a were distributed over most of the ER surface, with
strongest accumulation on the perinuclear ER. In vivo labeling with
bromo-UTP showed that the sites of 1a and 2a accumulation were the
sites of nascent viral RNA synthesis. In situ hybridization showed that
completed viral RNA products accumulated predominantly in the immediate
vicinity of replication complexes but that some, possibly more mature
cells also accumulated substantial viral RNA in the surrounding
cytoplasm distal to replication complexes. Additionally, we find that
1a localizes to the ER when expressed in the absence of other viral factors. These results show that BMV RNA replication in yeast duplicates the normal localization of replication complexes, reveal the
intracellular distribution of RNA replication products, and show that
1a is at least partly responsible for the ER localization and retention
of the RNA replication complex.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Brome Mosaic Virus RNA Replication Proteins 1a and
2a Colocalize and 1a Independently Localizes on the Yeast
Endoplasmic Reticulum
and
Madison, Madison, Wisconsin 53706
*
Corresponding author. Mailing address: Institute for
Molecular Virology, University of Wisconsin
Madison, 1525 Linden Dr., Madison, WI 53706-1596. Phone: (608) 263-5916. Fax: (608) 265-9214. E-mail: ahlquist{at}facstaff.wisc.edu.
Present address: DuPont, Wilmington, DE 19880-0015.
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