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 Insertion of a New Transcriptional Unit into the Genome of Mouse Hepatitis Virus

1999 ◽  
Vol 73 (7) ◽  
pp. 6128-6135 ◽  
Author(s):  
Bilan Hsue ◽  
Paul S. Masters
Keyword(s):  
Relative Efficiency ◽  
Untranslated Region ◽  
Hepatitis Virus ◽  
Consensus Sequence ◽  
Mouse Hepatitis Virus ◽  
Transcriptional Unit ◽  
N Gene ◽  
Subgenomic Rna ◽  
N Protein ◽  
Intergenic Sequences

ABSTRACT The subgenomic mRNAs of the coronavirus mouse hepatitis virus (MHV) are composed of a leader sequence, identical to the 5′ 70 nucleotides of the genome, joined at distant downstream sites to a stretch of sequence that is identical to the 3′ end of the genome. The points of fusion occur at intergenic sequences (IGSs), loci on the genome that contain a tract of sequence homologous to the 3′ end of the leader RNA. We have constructed a mutant of MHV-A59 containing an extra IGS inserted into the genome immediately downstream of the 3′-most gene, that encoding the nucleocapsid (N) protein. We show that in cells infected with the mutant, there is synthesis of an additional leader-containing subgenomic RNA of the predicted size. Our study demonstrates that (i) an IGS can be a sufficient cis-acting element to dictate MHV transcription, (ii) the relative efficiency of an IGS must be influenced by factors other than the nucleotides immediately adjacent to the 5′AAUCUAAAC3′ core consensus sequence or its position relative to the 3′ end of the genome, (iii) a downstream IGS can exert a polar attenuating effect on upstream IGSs, and (iv) unknown factors prevent the insertion of large exogenous elements between the N gene and the 3′ untranslated region of MHV. These results confirm and extend conclusions previously derived from the analysis of defective interfering RNAs.

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 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 RT-PCR Assay for Detection of Enterotropic Strains of Mouse Hepatitis Virus in Faecal Samples

2021 ◽  
Author(s):  
M.R. Srinivasan ◽  
K. Vijay ◽  
A.K. Karuppannan ◽  
S. Ramesh ◽  
Y. Krishnamohan Reddy
Keyword(s):  
Viral Infections ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
N Gene ◽  
Laboratory Mice ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Faecal Samples ◽  
Retrospective Nature ◽  
Gene Template

Background: Mouse Hepatitis Virus (MHV) is one of the most important and common viral infections of laboratory mice, due to its highly contagious and subclinical nature, posing threat to the research outcomes. Periodic screening of laboratory mice for MHV is mandatory. Hence, this study was intended to develop sensitive faecal based RT-PCR assays to detect active infection of enterotropic MHV in laboratory mice. Methods: Primers targeting N-gene of MHV were selected and their sensitivity was analysed in tenfold serially diluted gene template in the presence of negative mouse faecal cDNA. Thirty-six weaned mice at the age of 6 to 18 weeks were randomly selected at different periods and the blood and faecal sample were collected for serology and RT-PCR assay respectively. RT-PCR assay in colon samples was carried out for comparison. Result: PCR assay of MHV detected as low as 4 fg of plasmid DNA. Seroprevalence of MHV is very high than the prevalence by RT-PCR assay showing its retrospective nature and also seroprevalence includes both enterotropic and polytropic strain. RT-PCR results in faecal samples are analogous with that of the colon samples, showing the reliability of antemortem testing of mice for enterotropic strain of MHV.

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 Mouse Hepatitis Virus Stem-Loop 4 Functions as a Spacer Element Required To Drive Subgenomic RNA Synthesis

2011 ◽  
Vol 85 (17) ◽  
pp. 9199-9209 ◽  
Author(s):  
D. Yang ◽  
P. Liu ◽  
D. P. Giedroc ◽  
J. Leibowitz
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Stem Loop ◽  
Subgenomic Rna

scholarly journals Translation of the psbA mRNA of Chlamydomonas reinhardtii requires a structured RNA element contained within the 5' untranslated region.

1994 ◽  
Vol 127 (6) ◽  
pp. 1537-1545 ◽  
Author(s):  
S P Mayfield ◽  
A Cohen ◽  
A Danon ◽  
C B Yohn
Keyword(s):  
Secondary Structure ◽  
Untranslated Region ◽  
Translational Regulation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Higher Plants ◽  
Consensus Sequence ◽  
Specific Protein ◽  
Rna Elements ◽  
Ribosome Association

Translational regulation is a key modulator of gene expression in chloroplasts of higher plants and algae. Genetic analysis has shown that translation of chloroplast mRNAs requires nuclear-encoded factors that interact with chloroplastic mRNAs in a message-specific manner. Using site-specific mutations of the chloroplastic psbA mRNA, we show that RNA elements contained within the 5' untranslated region of the mRNA are required for translation. One of these elements is a Shine-Dalgarno consensus sequence, which is necessary for ribosome association and psbA translation. A second element required for high levels of psbA translation is located adjacent to and upstream of the Shine-Dalgarno sequence, and maps to the location on the RNA previously identified as the site of message-specific protein binding. This second element appears to act as a translational attenuator that must be overcome to activate translation. Mutations that affect the secondary structure of these RNA elements greatly reduce the level of psbA translation, suggesting that secondary structure of these RNA elements plays a role in psbA translation. These data suggest a mechanism for translational activation of the chloroplast psbA mRNA in which an RNA element containing the ribosome-binding site is bound by message-specific RNA binding proteins allowing for increased ribosome association and translation initiation. These elements may be involved in the light-regulated translation of the psbA mRNA.

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