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Journal of Virology, August 2000, p. 6911-6921, Vol. 74, No. 15
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Characterization of an Essential RNA Secondary
Structure in the 3' Untranslated Region of the Murine Coronavirus
Genome
Bilan
Hsue,1,2
Toinette
Hartshorne,3 and
Paul
S.
Masters1,2,*
Wadsworth Center for Laboratories and
Research, New York State Department of Health,1
and Department of Biomedical Sciences, University at Albany,
State University of New York,2 Albany, New York
12201, and Center for Immunology and Microbial Disease,
Albany Medical College, Albany, New York 122083
Received 13 March 2000/Accepted 8 May 2000
We have previously identified a functionally essential bulged
stem-loop in the 3' untranslated region of the positive-stranded RNA
genome of mouse hepatitis virus. This 68-nucleotide structure is
composed of six stem segments interrupted by five bulges, and its
structure, but not its primary sequence, is entirely conserved in the
related bovine coronavirus. The functional importance of individual
stem segments of this stem-loop was characterized by genetic analysis
using targeted RNA recombination. We also examined the effects of stem
segment mutations on the replication of mouse hepatitis virus defective
interfering RNAs. These studies were complemented by enzymatic and
chemical probing of the stem-loop. Taken together, our results
confirmed most of the previously proposed structure, but they revealed
that the terminal loop and an internal loop are larger than originally
thought. Three of the stem segments were found to be essential for
viral replication. Further, our results suggest that the stem segment
at the base of the stem-loop is an alternative base-pairing structure
for part of a downstream, and partially overlapping, RNA pseudoknot
that has recently been shown to be necessary for bovine coronavirus replication.
*
Corresponding author. Mailing address: David Axelrod
Institute, Wadsworth Center, NYSDOH, New Scotland Ave., P.O. Box 22002, Albany, NY 12201-2002. Phone: (518) 474-1283. Fax: (518) 473-1326. E-mail: masters{at}wadsworth.org.
Journal of Virology, August 2000, p. 6911-6921, Vol. 74, No. 15
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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