Mitochondrial aconitase binds to the 3' untranslated region of the mouse hepatitis virus genome.
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.
Department of Pathology and Laboratory Medicine, Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA.
S K
SK
Nanda
J L
JL
Leibowitz
D020413:3' Untranslated Regions / Q000378:metabolism*; D000154:Aconitate Hydratase / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000032:analysis; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D007501:Iron / Q000494:pharmacology; D013058:Mass Spectrometry; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D006517:Murine hepatitis virus / Q000235:genetics* / Q000502:physiology; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D020218:Response Elements; D014779:Virus Replication / Q000187:drug effects
D020413:3' Untranslated Regions; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D007501:Iron; D000154:Aconitate Hydratase