An Essential Secondary Structure in the 3’ Untranslated Region of the Mouse Hepatitis Virus Genome

1998 ◽  
pp. 297-302 ◽  
Author(s):  
Bilan Hsue ◽  
Paul S. Masters
Keyword(s):  
Secondary Structure ◽  
Untranslated Region ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Virus Genome

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 Secondary Structural Elements within the 3′ Untranslated Region of Mouse Hepatitis Virus Strain JHM Genomic RNA

2001 ◽  
Vol 75 (24) ◽  
pp. 12105-12113 ◽  
Author(s):  
Qi Liu ◽  
Reed F. Johnson ◽  
Julian L. Leibowitz
Keyword(s):  
Secondary Structure ◽  
Protein Binding ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Rna Replication ◽  
Secondary Structures ◽  
Host Protein ◽  
Wild Type ◽  
Base Pairing ◽  
Stem Loop

ABSTRACT Previously, we characterized two host protein binding elements located within the 3′-terminal 166 nucleotides of the mouse hepatitis virus (MHV) genome and assessed their functions in defective-interfering (DI) RNA replication. To determine the role of RNA secondary structures within these two host protein binding elements in viral replication, we explored the secondary structure of the 3′-terminal 166 nucleotides of the MHV strain JHM genome using limited RNase digestion assays. Our data indicate that multiple stem-loop and hairpin-loop structures exist within this region. Mutant and wild-type DIssEs were employed to test the function of secondary structure elements in DI RNA replication. Three stem structures were chosen as targets for the introduction of transversion mutations designed to destroy base pairing structures. Mutations predicted to destroy the base pairing of nucleotides 142 to 136 with nucleotides 68 to 74 exhibited a deleterious effect on DIssE replication. Destruction of base pairing between positions 96 to 99 and 116 to 113 also decreased DI RNA replication. Mutations interfering with the pairing of nucleotides 67 to 63 with nucleotides 52 to 56 had only minor effects on DIssE replication. The introduction of second complementary mutations which restored the predicted base pairing of positions 142 to 136 with 68 to 74 and nucleotides 96 to 99 with 116 to 113 largely ameliorated defects in replication ability, restoring DI RNA replication to levels comparable to that of wild-type DIssE RNA, suggesting that these secondary structures are important for efficient MHV replication. We also identified a conserved 23-nucleotide stem-loop structure involving nucleotides 142 to 132 and nucleotides 68 to 79. The upstream side of this conserved stem-loop is contained within a host protein binding element (nucleotides 166 to 129).

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 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.

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.

scholarly journals Effect of Mutations in the Mouse Hepatitis Virus 3′(+)42 Protein Binding Element on RNA Replication

2005 ◽  
Vol 79 (23) ◽  
pp. 14570-14585 ◽  
Author(s):  
Reed F. Johnson ◽  
Min Feng ◽  
Pinghua Liu ◽  
Jason J. Millership ◽  
Boyd Yount ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Protein Binding ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Rna Replication ◽  
Host Protein ◽  
Reverse Genetic System ◽  
Wild Type ◽  
Mobility Shift ◽  
Ribonucleoprotein Complexes

ABSTRACT The mouse hepatitis virus (MHV) genome's 3′ untranslated region contains cis-acting sequences necessary for replication. Studies of MHV and other coronaviruses have indicated a role for RNA secondary and tertiary elements in replication. Previous work in our laboratory has identified four proteins which form ribonucleoprotein complexes with the 3′-terminal 42 nucleotides [3′(+)42] of the MHV genome. Defective interfering (DI) RNA replication assays have demonstrated a role for the 3′(+)42 host protein binding element in the MHV life cycle. Using gel mobility shift RNase T1 protection assays and secondary structure modeling, we have characterized a possible role for RNA secondary structure in host protein binding to the 3′-terminal 42-nucleotide element. Additionally we have identified a role for the 3′-terminal 42-nucleotide host protein binding element in RNA replication and transcription using DI RNA replication assays and targeted recombination and by directly constructing mutants in this protein binding element using a recently described MHV reverse genetic system. DI RNA replication assays demonstrated that mutations in the 3′(+)42 host protein binding element had a deleterious effect on the accumulation of DI RNA. When the identical mutations were directly inserted into the MHV genome, most mutant genomes were viable but formed smaller plaques than the wild-type parent virus. One mutant was not viable. This mutant directed the synthesis of genome-sized negative-sense RNA approximately as efficiently as the wild type did but had a defect in subgenomic mRNA synthesis. These results point to a potential role for sequences at the extreme 3′ end of the MHV genome in subgenomic RNA synthesis.

Sign in / Sign up

Export Citation Format

Share Document