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Journal of Virology, April 2001, p. 3352-3362, Vol. 75, No. 7
Department of Pathology and Laboratory
Medicine, Texas A&M University System Health Science Center,
College Station, Texas 77843-1114
Received 14 September 2000/Accepted 4 January 2001
Mouse hepatitis virus (MHV), a member of the
Coronaviridae, contains a polyadenylated positive-sense
single-stranded genomic RNA which is 31 kb long. MHV replication and
transcription take place via the synthesis of negative-strand RNA
intermediates from a positive-strand genomic template. A
cis-acting element previously identified in the 3'
untranslated region binds to trans-acting host factors from
mouse fibroblasts and forms at least three RNA-protein complexes. The
largest RNA-protein complex formed by the cis-acting element and the lysate from uninfected mouse fibroblasts has a molecular weight of about 200 kDa. The complex observed in gel shift
assays has been resolved by second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis into four proteins of
approximately 90, 70, 58, and 40 kDa after RNase treatment. Specific
RNA affinity chromatography also has revealed the presence of a 90-kDa
protein associated with RNA containing the cis-acting element bound to magnetic beads. The 90-kDa protein has been purified from uninfected mouse fibroblast crude lysates. Protein microsequencing identified the 90-kDa protein as mitochondrial aconitase. Antibody raised against purified mitochondrial aconitase recognizes the RNA-protein complex and the 90-kDa protein, which can be released from
the complex by RNase digestion. Furthermore, UV cross-linking studies
indicate that highly purified mitochondrial aconitase binds
specifically to the MHV 3' protein-binding element. Increasing the
intracellular level of mitochondrial aconitase by iron supplementation resulted in increased RNA-binding activity in cell extracts and increased virus production as well as viral protein synthesis at early
hours of infection. These results are particularly interesting in terms
of identification of an RNA target for mitochondrial aconitase, which
has a cytoplasmic homolog, cytoplasmic aconitase, also known as iron
regulatory protein 1, a well-recognized RNA-binding protein. The
binding properties of mitochondrial aconitase and the functional
relevance of RNA binding appear to parallel those of cytoplasmic aconitase.
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.7.3352-3362.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Mitochondrial Aconitase Binds to the 3'
Untranslated Region of the Mouse Hepatitis Virus Genome
*
Corresponding author. Mailing address: Dept. of
Pathology and Laboratory Medicine, Texas A&M University System Health
Science Center, 208 Reynolds Medical Building, 1114 TAMU, College
Station, TX 77843-1114. Phone: (979) 845-7288. Fax: (979) 862-1299. E-mail: jleibowitz{at}tamu.edu.
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