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

2001 ◽  
Vol 75 (7) ◽  
pp. 3352-3362 ◽  
Author(s):  
Santosh K. Nanda ◽  
Julian L. Leibowitz
Keyword(s):  
Protein Complex ◽  
Untranslated Region ◽  
Rna Binding ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Sodium Dodecyl ◽  
Uninfected Mouse ◽  
Mouse Fibroblasts ◽  
Cell Extracts ◽  
Mitochondrial Aconitase

ABSTRACT Mouse hepatitis virus (MHV), a member of theCoronaviridae, 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. Acis-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.

scholarly journals SYNCRIP, a Member of the Heterogeneous Nuclear Ribonucleoprotein Family, Is Involved in Mouse Hepatitis Virus RNA Synthesis

2004 ◽  
Vol 78 (23) ◽  
pp. 13153-13162 ◽  
Author(s):  
Keum S. Choi ◽  
Akihiro Mizutani ◽  
Michael M. C. Lai
Keyword(s):  
Mammalian Cells ◽  
Rna Synthesis ◽  
Rna Binding ◽  
Hepatitis Virus ◽  
Affinity Purification ◽  
Mouse Hepatitis Virus ◽  
Viral Rna ◽  
Negative Strand

ABSTRACT Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5′ and 3′ untranslated regions (UTRs) of the viral RNA. Here, we identified another cellular protein, p70, which has been shown by UV cross-linking to bind both the positive- and negative-strand UTRs of MHV RNA specifically. We purified p70 with a a one-step RNA affinity purification procedure with the biotin-labeled 5′-UTR. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of the hnRNP family and localizes largely in the cytoplasm. The p70 was cross-linked to the MHV positive- or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to the 5′-UTR of both strands. The SYNCRIP-binding site was mapped to the leader sequence of the 5′-UTR, requiring the UCUAA repeat sequence. To investigate the functional significance of SYNCRIP in MHV replication, we expressed a full-length or a C-terminally truncated form of SYNCRIP in mammalian cells expressing the MHV receptor. The overexpression of either form of SYNCRIP inhibited syncytium formation induced by MHV infection. Furthermore, downregulation of the endogenous SYNCRIP with a specific short interfering RNA delayed MHV RNA synthesis; in contrast, overexpression or downregulation of SYNCRIP did not affect MHV translation. These results suggest that SYNCRIP may be directly involved in MHV RNA replication as a positive regulator. This study identified an additional cellular hnRNP as an MHV RNA-binding protein potentially involved in viral RNA synthesis.

scholarly journals The Leader RNA of Coronavirus Mouse Hepatitis Virus Contains an Enhancer-Like Element for Subgenomic mRNA Transcription

2000 ◽  
Vol 74 (22) ◽  
pp. 10571-10580 ◽  
Author(s):  
Yicheng Wang ◽  
Xuming Zhang
Keyword(s):  
Secondary Structure ◽  
Untranslated Region ◽  
Hepatitis Virus ◽  
Consensus Sequence ◽  
Mouse Hepatitis Virus ◽  
Mrna Transcription ◽  
Rt Pcr ◽  
Reporter System ◽  
Rna Transcription ◽  
Subgenomic Rna

ABSTRACT While the 5′ cis-acting sequence of mouse hepatitis virus (MHV) for genomic RNA replication has been determined in several defective interfering (DI) RNA systems, it remains elusive for subgenomic RNA transcription. Previous studies have shown that the leader RNA in the DI genome significantly enhances the efficiency of DI subgenomic mRNA transcription, indicating that the leader RNA is a cis-acting sequence for mRNA transcription. To further characterize thecis-acting sequence, we made a series of deletion mutants, all but one of which have an additional deletion of thecis-acting signal for replication in the 5′ untranslated region. This deletion effectively eliminated the replication of the DI-chloramphenicol acetyltransferase (CAT)-reporter, as demonstrated by the sensitive reverse transcription (RT)-PCR. The ability of these replication-minus mutants to transcribe subgenomic mRNAs was then assessed using the DI RNA-CAT reporter system. Results from both CAT activity and mRNA transcripts detected by RT-PCR showed that a 5′-proximal sequence of 35 nucleotides (nt) at nt 25 to 59 is a cis-acting sequence required for subgenomic RNA transcription, while the consensus repeat sequence of the leader RNA does not have such effect. Analyses of the secondary structure indicate that this 35-nt sequence forms two stem-loops conserved among MHVs. Deletion of this sequence abrogated transcriptional activity and disrupted the predicted stem-loops and overall RNA secondary structure at the 5′ untranslated region, suggesting that the secondary structure formed by this 35-nt sequence may facilitate the downstream consensus sequence accessible for the discontinuous RNA transcription. This may provide a mechanism by which the 5′ cis-acting sequence regulates subgenomic RNA transcription. The 5′-most 24 nt are not essential for transcription, while the 9 nt immediately downstream of the leader enhances RNA transcription. The sequence between nt 86 and 135 had little effect on transcription. This study thus defines thecis-acting transcription signal at the 5′ end of the DI genome.

scholarly journals Polypyrimidine Tract-Binding Protein Binds to the Complementary Strand of the Mouse Hepatitis Virus 3′ Untranslated Region, Thereby Altering RNA Conformation

1999 ◽  
Vol 73 (11) ◽  
pp. 9110-9116 ◽  
Author(s):  
Peiyong Huang ◽  
Michael M. C. Lai
Keyword(s):  
Conformational Change ◽  
Viral Genome ◽  
Untranslated Region ◽  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Complementary Strand ◽  
Polypyrimidine Tract ◽  
Mrna Transcription ◽  
Polypyrimidine Tract Binding Protein

ABSTRACT Mouse hepatitis virus (MHV) RNA transcription is regulated mainly by the leader and intergenic (IG) sequences. However, a previous study has shown that the 3′ untranslated region (3′-UTR) of the viral genome is also required for subgenomic mRNA transcription; deletion of nucleotides (nt) 270 to 305 from the 3′-UTR completely abolished subgenomic mRNA transcription without affecting minus-strand RNA synthesis (Y.-J. Lin, X. Zhang, R.-C. Wu, and M. M. C. Lai, J. Virol. 70:7236–7240, 1996), suggesting that the 3′-UTR affects positive-strand RNA synthesis. In this study, by UV-cross-linking experiments, we found that several cellular proteins bind specifically to the minus-strand 350 nucleotides complementary to the 3′-UTR of the viral genome. The major protein species, p55, was identified as the polypyrimidine tract-binding protein (PTB, also known as heterogeneous nuclear RNP I) by immunoprecipitation of the UV-cross-linked protein and binding of the recombinant PTB. A strong PTB-binding site was mapped to nt 53 to 149, and another weak binding site was mapped to nt 270 to 307 on the complementary strand of the 3′-UTR (c3′-UTR). Partial substitutions of the PTB-binding nucleotides reduced PTB binding in vitro. Furthermore, defective interfering (DI) RNAs harboring these mutations showed a substantially reduced ability to synthesize subgenomic mRNA. By enzymatic and chemical probing, we found that PTB binding to nt 53 to 149 caused a conformational change in the neighboring RNA region. Partial deletions within the PTB-binding sequence completely abolished the PTB-induced conformational change in the mutant RNA even when the RNA retained partial PTB-binding activity. Correspondingly, the MHV DI RNAs containing these deletions completely lost their ability to transcribe mRNAs. Thus, the conformational change in the c3′-UTR caused by PTB binding may play a role in mRNA transcription.

High affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus RNA

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 181-188 ◽  
Author(s):  
Gary W. Nelson ◽  
Stephen A. Stohlman ◽  
Stanley M. Tahara
Keyword(s):  
Rna Binding ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Full Length ◽  
Viral Rna ◽  
Intergenic Sequence ◽  
Protein Fusion ◽  
Binding Properties ◽  
N Protein ◽  
High Affinity

The nucleocapsid (N) protein of mouse hepatitis virus (MHV) is the major virion structural protein. It associates with both viral genomic RNA and subgenomic mRNAs and has structural and non-structural roles in replication including viral RNA-dependent RNA transcription, genome replication, encapsidation and translation. These processes all involve RNA–protein interactions between the N protein and viral RNAs. To better understand the RNA-binding properties of this multifunctional protein, the N protein was expressed in Escherichia coli as a chimeric protein fused to glutathione-S-transferase (GST). Biochemical analyses of RNA-binding properties were performed on full-length and partial N protein segments to define the RNA-binding domain. The full-length N protein and the GST–N protein fusion product had similar binding activities with a dissociation constant (K d) of 14 nM when the MHV 5′-leader sequence was used as ligand. The smallest N protein fragment which retained RNA-binding activity was a 55 aa segment containing residues 177–231 which bound viral RNA with a K d of 32 nM. A consensus viral sequence recognized by the N protein was inferred from these studies; AAUCYAAAC was identified to be the potential minimum ligand for the N protein. Although the core UCYAA sequence is often tandemly repeated in viral genomes, ligands containing one or more repeats of UCYAA showed no difference in binding to the N protein. Together these data demonstrate a high-affinity, specific interaction between the N protein and a conserved RNA sequence present at the 5′-ends of MHV mRNA.

scholarly journals The 3′ cis-Acting Genomic Replication Element of the Severe Acute Respiratory Syndrome Coronavirus Can Function in the Murine Coronavirus Genome

2004 ◽  
Vol 78 (14) ◽  
pp. 7846-7851 ◽  
Author(s):  
Scott J. Goebel ◽  
Jill Taylor ◽  
Paul S. Masters
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Untranslated Region ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Cis Acting ◽  
Group 3 ◽  
Group 2 ◽  
Group 1 ◽  
Murine Coronavirus

ABSTRACT The 3′ untranslated region (3′ UTR) of the genome of the severe acute respiratory syndrome coronavirus can functionally replace its counterpart in the prototype group 2 coronavirus mouse hepatitis virus (MHV). By contrast, the 3′ UTRs of representative group 1 or group 3 coronaviruses cannot operate as substitutes for the MHV 3′ UTR.

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