scholarly journals Cell Proteins TIA-1 and TIAR Interact with the 3′ Stem-Loop of the West Nile Virus Complementary Minus-Strand RNA and Facilitate Virus Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 11989-12000 ◽  
Author(s):  
W. Li ◽  
Y. Li ◽  
N. Kedersha ◽  
P. Anderson ◽  
M. Emara ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Rna Binding ◽  
Peptide Sequencing ◽  
Viral Rna ◽  
Cell Protein ◽  
Affinity Column ◽  
Rna Recognition Motif ◽  
The West ◽  
West Nile ◽  
Stem Loop

ABSTRACT It was reported previously that four baby hamster kidney (BHK) proteins with molecular masses of 108, 60, 50, and 42 kDa bind specifically to the 3′-terminal stem-loop of the West Nile virus minus-stand RNA [WNV 3′(−) SL RNA] (P. Y. Shi, W. Li, and M. A. Brinton, J. Virol. 70:6278-6287, 1996). In this study, p42 was purified using an RNA affinity column and identified as TIAR by peptide sequencing. A 42-kDa UV-cross-linked viral RNA-cell protein complex formed in BHK cytoplasmic extracts incubated with the WNV 3′(−) SL RNA was immunoprecipitated by anti-TIAR antibody. Both TIAR and the closely related protein TIA-1 are members of the RNA recognition motif (RRM) family of RNA binding proteins. TIA-1 also binds to the WNV 3′(−) SL RNA. The specificity of these viral RNA-cell protein interactions was demonstrated using recombinant proteins in competition gel mobility shift assays. The binding site for the WNV 3′(−) SL RNA was mapped to RRM2 on both TIAR and TIA-1. However, the dissociation constant (Kd ) for the interaction between TIAR RRM2 and the WNV 3′(−) SL RNA was 1.5 × 10−8, while that for TIA-1 RRM2 was 1.12 × 10−7. WNV growth was less efficient in murine TIAR knockout cell lines than in control cells. This effect was not observed for two other types of RNA viruses or two types of DNA viruses. Reconstitution of the TIAR knockout cells with TIAR increased the efficiency of WNV growth, but neither the level of TIAR nor WNV replication was as high as in control cells. These data suggest a functional role for TIAR and possibly also for TIA-1 during WNV replication.

scholarly journals An RNA Thermometer Activity of the West Nile Virus Genomic 3′-Terminal Stem-Loop Element Modulates Viral Replication Efficiency during Host Switching

Viruses ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 104 ◽  
Author(s):  
Alexandra Meyer ◽  
Marie Freier ◽  
Tobias Schmidt ◽  
Katja Rostowski ◽  
Juliane Zwoch ◽  
...  
Keyword(s):  
Viral Replication ◽  
Rna Binding ◽  
Rna Replication ◽  
Viral Rna ◽  
Cell Protein ◽  
Host Switching ◽  
West Nile ◽  
Stem Loop ◽  
Mosquito Cells ◽  
Rna Thermometer

The 3′-terminal stem-loop (3′SL) of the RNA genome of the flavivirus West Nile (WNV) harbors, in its stem, one of the sequence elements that are required for genome cyclization. As cyclization is a prerequisite for the initiation of viral replication, the 3′SL was proposed to act as a replication silencer. The lower part of the 3′SL is metastable and confers a structural flexibility that may regulate the switch from the linear to the circular conformation of the viral RNA. In the human system, we previously demonstrated that a cellular RNA-binding protein, AUF1 p45, destabilizes the 3′SL, exposes the cyclization sequence, and thus promotes flaviviral genome cyclization and RNA replication. By investigating mutant RNAs with increased 3′SL stabilities, we showed the specific conformation of the metastable element to be a critical determinant of the helix-destabilizing RNA chaperone activity of AUF1 p45 and of the precision and efficiency of the AUF1 p45-supported initiation of RNA replication. Studies of stability-increasing mutant WNV replicons in human and mosquito cells revealed that the cultivation temperature considerably affected the replication efficiencies of the viral RNA variants and demonstrated the silencing effect of the 3′SL to be temperature dependent. Furthermore, we identified and characterized mosquito proteins displaying similar activities as AUF1 p45. However, as the RNA remodeling activities of the mosquito proteins were found to be considerably lower than those of the human protein, a potential cell protein-mediated destabilization of the 3′SL was suggested to be less efficient in mosquito cells. In summary, our data support a model in which the 3′SL acts as an RNA thermometer that modulates flavivirus replication during host switching.

scholarly journals Energetics of RNA binding by the West Nile virus RNA triphosphatase

FEBS Letters ◽  
2006 ◽  
Vol 580 (3) ◽  
pp. 867-877 ◽  
Author(s):  
Ines Benzaghou ◽  
Isabelle Bougie ◽  
Frédéric Picard-Jean ◽  
Martin Bisaillon
Keyword(s):  
West Nile Virus ◽  
Rna Binding ◽  
The West ◽  
West Nile ◽  
Virus Rna ◽  
Rna Triphosphatase

scholarly journals West Nile Virus Methyltransferase Catalyzes Two Methylations of the Viral RNA Cap through a Substrate-Repositioning Mechanism

2008 ◽  
Vol 82 (9) ◽  
pp. 4295-4307 ◽  
Author(s):  
Hongping Dong ◽  
Suping Ren ◽  
Bo Zhang ◽  
Yangsheng Zhou ◽  
Francesc Puig-Basagoiti ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Binding Site ◽  
Binding Sites ◽  
Biochemical Analysis ◽  
Rna Binding ◽  
Binding Pocket ◽  
Viral Rna ◽  
West Nile ◽  
Rna Molecules ◽  
Gtp Binding

ABSTRACT Flaviviruses encode a single methyltransferase domain that sequentially catalyzes two methylations of the viral RNA cap, GpppA-RNA→m7GpppA-RNA→m7GpppAm-RNA, by using S-adenosyl-l-methionine (SAM) as a methyl donor. Crystal structures of flavivirus methyltransferases exhibit distinct binding sites for SAM, GTP, and RNA molecules. Biochemical analysis of West Nile virus methyltransferase shows that the single SAM-binding site donates methyl groups to both N7 and 2′-O positions of the viral RNA cap, the GTP-binding pocket functions only during the 2′-O methylation, and two distinct sets of amino acids in the RNA-binding site are required for the N7 and 2′-O methylations. These results demonstrate that flavivirus methyltransferase catalyzes two cap methylations through a substrate-repositioning mechanism. In this mechanism, guanine N7 of substrate GpppA-RNA is first positioned to SAM to generate m7GpppA-RNA, after which the m7G moiety is repositioned to the GTP-binding pocket to register the 2′-OH of the adenosine with SAM, generating m7GpppAm-RNA. Because N7 cap methylation is essential for viral replication, inhibitors designed to block the pocket identified for the N7 cap methylation could be developed for flavivirus therapy.

scholarly journals Interaction between the Cellular Protein eEF1A and the 3′-Terminal Stem-Loop of West Nile Virus Genomic RNA Facilitates Viral Minus-Strand RNA Synthesis

2007 ◽  
Vol 81 (18) ◽  
pp. 10172-10187 ◽  
Author(s):  
William G. Davis ◽  
Jerry L. Blackwell ◽  
Pei-Yong Shi ◽  
Margo A. Brinton
Keyword(s):  
West Nile Virus ◽  
Rna Synthesis ◽  
Cell Protein ◽  
Minus Strand ◽  
Genomic Rna ◽  
West Nile ◽  
Stem Loop ◽  
Transfected Cells ◽  
Infected Cells

ABSTRACT RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3′(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3′(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3′ SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3′ SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3′ SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3′ end of the genome and the RC. eEF1A bound with similar efficiencies to the 3′-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

scholarly journals The Host Factor AUF1 p45 Supports Flavivirus Propagation by Triggering the RNA Switch Required for Viral Genome Cyclization

2017 ◽  
Vol 92 (6) ◽  
Author(s):  
Susann Friedrich ◽  
Susanne Engelmann ◽  
Tobias Schmidt ◽  
Grit Szczepankiewicz ◽  
Sandra Bergs ◽  
...  
Keyword(s):  
Viral Replication ◽  
Viral Genome ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Viral Rna ◽  
West Nile ◽  
Rna Chaperone ◽  
Stem Loop ◽  
Content Type ◽  
Rna Switch

ABSTRACTIn previous studies, we showed that the cellular RNA-binding protein AUF1 supports the replication process of the flavivirus West Nile virus. Here we demonstrate that the protein also enables effective proliferation of dengue virus and Zika virus, indicating that AUF1 is a general flavivirus host factor. Further studies demonstrated that the AUF1 isoform p45 significantly stimulates the initiation of viral RNA replication and that the protein's RNA chaperone activity enhances the interactions of the viral 5′UAR and 3′UAR genome cyclization sequences. Most interestingly, we observed that AUF1 p45 destabilizes not only the 3′-terminal stem-loop (3′SL) but also 5′-terminal stem-loop B (SLB) of the viral genome. RNA structure analyses revealed that AUF1 p45 increases the accessibility of defined nucleotides within the 3′SL and SLB and, in this way, exposes both UAR cyclization elements. Conversely, AUF1 p45 does not modulate the fold of stem-loop A (SLA) at the immediate genomic 5′ end, which is proposed to function as a promoter of the viral RNA-dependent RNA polymerase (RdRp). These findings suggest that AUF1 p45, by destabilizing specific stem-loop structures within the 5′ and 3′ ends of the flaviviral genome, assists genome cyclization and concurrently enables the RdRp to initiate RNA synthesis. Our study thus highlights the role of a cellular RNA-binding protein inducing a flaviviral RNA switch that is crucial for viral replication.IMPORTANCEThe genusFlaviviruswithin theFlaviviridaefamily includes important human pathogens, such as dengue, West Nile, and Zika viruses. The initiation of replication of the flaviviral RNA genome requires a transformation from a linear to a cyclized form. This involves considerable structural reorganization of several RNA motifs at the genomic 5′ and 3′ ends. Specifically, it needs a melting of stem structures to expose complementary 5′ and 3′ cyclization elements to enable their annealing during cyclization. Here we show that a cellular RNA chaperone, AUF1 p45, which supports the replication of all three aforementioned flaviviruses, specifically rearranges stem structures at both ends of the viral genome and in this way permits 5′-3′ interactions of cyclization elements. Thus, AUF1 p45 triggers the RNA switch in the flaviviral genome that is crucial for viral replication. These findings represent an important example of how cellular (host) factors promote the propagation of RNA viruses.

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