Mitochondrial Aconitase Binds to the 3′ Untranslated Region of the Mouse Hepatitis Virus Genome

Abstract

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.The coronaviruses belong to the newly established order Nidovirales (5) and have enveloped virions containing the largest known RNA virus genome. Mouse hepatitis virus (MHV) is a prototypic member of the Coronaviridae family and contains a single-stranded, positive-sense RNA genome approximately 31 kb in length (28,29,33,44). Viral proteins are translated from six to seven subgenomic mRNAs as well as from the genome. These RNAs are produced in different quantities, and their molar ratios remain constant during MHV replication. The virus-specific subgenomic and genomic RNAs make up a 3Ј-coterminal nested set (33, 62) and contain a leader sequence of approximately 70 nucleotides (nt) at the 5Ј end (27, 60). Coronaviruses perform their entire replication program in the cytoplasm of infected cells. Following uncoating, coronaviruses express the largest known replicase polyproteins, which in turn are proteolytically proc...

LRP130, a Pentatricopeptide Motif Protein with a Noncanonical RNA-Binding Domain, Is Bound In Vivo to Mitochondrial and Nuclear RNAs

“…3), supporting the conclusion that LRP130 binds mitochondrially encoded RNAs. Other mitochondrial proteins with well-characterized enzymatic activities such as glutamate dehydrogenase, aconitase, and enoyl-coenzyme A hydratase have been shown to have RNA-binding activity in vitro (1,29,37). It will be of interest to determine whether they also bind mitochondrial RNA in vivo.…”

“…By analogy to nuclear RNP complexes, and based on the paradigm set mainly through studies of the yeast Saccharomyces cerevisiae (9,36), RNA-binding proteins would also be expected to participate and/or regulate every aspect of mitochondrial mRNA metabolism. A small number of mammalian mitochondrial proteins with well-characterized enzymatic activities, such as cis-aconitase, glutamate dehydrogenase, and enoyl-coenzyme A hydratase (1,29,37), have been shown to exhibit RNA-binding activity in vitro. However, it is not known whether they bind mitochondrial RNA directly in vivo.…”

Hosting the severe acute respiratory syndrome coronavirus: specific cell factors required for infection

“…In addition to the N protein, which is also required for efficient RNA replication (Almazan et al ., 2004; Schelle et al ., 2005), several heterologous nuclear ribonucleoprotein (hnRNP) family members (hnRNPA1, PTB and SYNCRYP), known to be involved in premRNA processing, were found to bind to different regions of the (+) and (–) strand genomic RNA and to affect coronavirus replication and transcription (reviewed by Shi and Lai, 2005). Other RNA‐binding proteins have also been suggested to play a role in coronavirus replication, such as m‐aconitase (Nanda and Leibowitz, 2001) and poly‐A‐binding protein (Spagnolo and Hogue, 2000). The N proteins from MHV and SARS‐CoV were shown to interact with hnRNAP A1 (Wang and Zhang, 1999; Luo et al ., 2005).…”

Of the three tegument proteins that package mRNA in herpes simplex virions, one (VP22) transports the mRNA to uninfected cells for expression prior to viral infection

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