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Journal of Virology, April 2001, p. 3207-3219, Vol. 75, No. 7
Institute for Molecular
Virology1 and Howard Hughes Medical
Institute,2 University of
Wisconsin
Received 10 October 2000/Accepted 27 December 2000
Brome mosaic virus (BMV), a positive-strand RNA virus in the
alphavirus-like superfamily, encodes two RNA replication factors. Membrane-associated 1a protein contains a helicase-like domain and RNA
capping functions. 2a, which is targeted to membranes by 1a, contains a
central polymerase-like domain. In the absence of 2a and RNA
replication, 1a 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 1a had similar effects on
another BMV RNA replication template, we constructed a plasmid
expressing BMV genomic RNA2 in vivo. In vivo-expressed RNA2 templates
were replicated upon expression of 1a and 2a. In the absence of 2a, 1a
stabilized RNA2 and induced RNA2 to associate with membrane. Deletion
analysis demonstrated that 1a-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 1a-induced membrane
association on a nonviral RNA. However, sequences in the N-terminal
region of the 2a open reading frame enhanced 1a responsiveness of RNA2
and a chimeric RNA. A 5'-terminal RNA2 stem-loop important for RNA2 replication was essential for 1a-induced membrane association of RNA2
and, like the 1a-responsive RNA3 intergenic region, contained a
required box B motif corresponding to the T
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.7.3207-3219.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Brome Mosaic Virus Protein 1a Recruits Viral RNA2
to RNA Replication through a 5' Proximal RNA2 Signal
Madison, Madison, Wisconsin 53706
C stem-loop of host tRNAs. The level of 1a-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.
*
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.
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