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

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.

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HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3′ Untranslated Region and Enhances Hepatitis C Virus Replication

Journal of Virology ◽

10.1128/jvi.01714-15 ◽

2015 ◽

Vol 89 (22) ◽

pp. 11356-11371 ◽

Cited By ~ 32

Author(s):

Shivaprasad Shwetha ◽

Anuj Kumar ◽

Ranajoy Mullick ◽

Deeptha Vasudevan ◽

Nilanjan Mukherjee ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Replication ◽

Untranslated Region ◽

Rna Synthesis ◽

Binding Protein ◽

Viral Rna ◽

Host Protein ◽

Polypyrimidine Tract ◽

Polypyrimidine Tract Binding Protein

ABSTRACTHuR is a ubiquitous, RNA binding protein that influences the stability and translation of several cellular mRNAs. Here, we report a novel role for HuR, as a regulator of proteins assembling at the 3′ untranslated region (UTR) of viral RNA in the context of hepatitis C virus (HCV) infection. HuR relocalizes from the nucleus to the cytoplasm upon HCV infection, interacts with the viral polymerase (NS5B), and gets redistributed into compartments of viral RNA synthesis. Depletion in HuR levels leads to a significant reduction in viral RNA synthesis. We further demonstrate that the interaction of HuR with the 3′ UTR of the viral RNA affects the interaction of two host proteins, La and polypyrimidine tract binding protein (PTB), at this site. HuR interacts with La and facilitates La binding to the 3′ UTR, enhancing La-mediated circularization of the HCV genome and thus viral replication. In addition, it competes with PTB for association with the 3′ UTR, which might stimulate viral replication. Results suggest that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viral RNA replication. Our study unravels a novel strategy of regulation of HCV replication through an interplay of host and viral proteins, orchestrated by HuR.IMPORTANCEHepatitis C virus (HCV) is highly dependent on various host factors for efficient replication of the viral RNA. Here, we have shown how a host factor (HuR) migrates from the nucleus to the cytoplasm and gets recruited in the protein complex assembling at the 3′ untranslated region (UTR) of HCV RNA. At the 3′ UTR, it facilitates circularization of the viral genome through interaction with another host factor, La, which is critical for replication. Also, it competes with the host protein PTB, which is a negative regulator of viral replication. Results demonstrate a unique strategy of regulation of HCV replication by a host protein through alteration of its subcellular localization and interacting partners. The study has advanced our knowledge of the molecular mechanism of HCV replication and unraveled the complex interplay between the host factors and viral RNA that could be targeted for therapeutic interventions.

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

Journal of Virology ◽

10.1128/jvi.74.22.10571-10580.2000 ◽

2000 ◽

Vol 74 (22) ◽

pp. 10571-10580 ◽

Cited By ~ 13

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.

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Polypyrimidine-Tract-Binding Protein Affects Transcription but Not Translation of Mouse Hepatitis Virus RNA

Virology ◽

10.1006/viro.2002.1675 ◽

2002 ◽

Vol 303 (1) ◽

pp. 58-68 ◽

Cited By ~ 39

Author(s):

Keum S. Choi ◽

Pei-yong Huang ◽

Michael M.C. Lai

Keyword(s):

Hepatitis Virus ◽

Binding Protein ◽

Mouse Hepatitis Virus ◽

Polypyrimidine Tract ◽

Polypyrimidine Tract Binding ◽

Polypyrimidine Tract Binding Protein ◽

Virus Rna

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Polypyrimidine Tract-Binding Protein Binds to the Leader RNA of Mouse Hepatitis Virus and Serves as a Regulator of Viral Transcription

Journal of Virology ◽

10.1128/jvi.73.1.772-777.1999 ◽

1999 ◽

Vol 73 (1) ◽

pp. 772-777 ◽

Cited By ~ 58

Author(s):

Hsin-Pai Li ◽

Peiyong Huang ◽

Sungmin Park ◽

Michael M. C. Lai

Keyword(s):

Binding Site ◽

Hepatitis Virus ◽

Binding Protein ◽

Mouse Hepatitis Virus ◽

Peptide Sequencing ◽

Regulatory Sequences ◽

Polypyrimidine Tract ◽

Reporter System ◽

Polypyrimidine Tract Binding ◽

Polypyrimidine Tract Binding Protein

ABSTRACT A cellular protein, previously described as p55, binds specifically to the plus strand of the mouse hepatitis virus (MHV) leader RNA. We have purified this protein and determined by partial peptide sequencing that it is polypyrimidine tract-binding protein (PTB) (also known as heterogeneous nuclear ribonucleoprotein [hnRNP] I), a nuclear protein which shuttles between the nucleus and cytoplasm. PTB plays a role in the regulation of alternative splicing of pre-mRNAs in normal cells and translation of several viruses. By UV cross-linking and immunoprecipitation studies using cellular extracts and a recombinant PTB, we have established that PTB binds to the MHV plus-strand leader RNA specifically. Deletion analyses of the leader RNA mapped the PTB-binding site to the UCUAA pentanucleotide repeats. Using a defective-interfering RNA reporter system, we have further shown that the PTB-binding site in the leader RNA is critical for MHV RNA synthesis. This and our previous study (H.-P. Li, X. Zhang, R. Duncan, L. Comai, and M. M. C. Lai, Proc. Natl. Acad. Sci. USA 94:9544–9549, 1997) combined thus show that two cellular hnRNPs, PTB and hnRNP A1, bind to the transcription-regulatory sequences of MHV RNA and may participate in its transcription.

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