scholarly journals Serine-arginine protein kinase 1 regulates Ebola virus transcription

2019 ◽  
Author(s):  
Yuki Takamatsu ◽  
Verena Krähling ◽  
Larissa Kolesnikova ◽  
Sandro Halwe ◽  
Clemens Lier ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Protein Kinase ◽  
Inclusion Bodies ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Viral Transcription ◽  
Fatal Disease ◽  
Recognition Motif ◽  
Virus Transcription ◽  
Regulatory Circuit

AbstractEbola virus (EBOV) causes a severe and often fatal disease for which no approved vaccines or antivirals are currently available. EBOV transcription requires the sequential phosphorylation and dephosphorylation of the viral transcription factor VP30. While dephosphorylation is carried out by phosphatases PP2A and PP1, the VP30-specific kinase is unknown. Here, we report that serine-arginine protein kinase 1 and 2 (SRPK1 and SRPK2) phosphorylate serine-29 of VP30, which is located in an N-terminal R26xxS29 motif. Interaction with VP30 via the R26xxS29 motif recruits SRPK1 into EBOV-induced inclusion bodies, the sites of viral RNA synthesis and an inhibitor of SRPK1/SRPK2 downregulates primary viral transcription. When the SRPK1 recognition motif of VP30 was mutated in a recombinant EBOV, virus replication was severely impaired. It is presumed that the interplay between SRPK1 and PP2A in the EBOV inclusions provides a comprehensive regulatory circuit to ensure the activity of VP30 in EBOV transcription.

scholarly journals Serine-Arginine Protein Kinase 1 Regulates Ebola Virus Transcription

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuki Takamatsu ◽  
Verena Krähling ◽  
Larissa Kolesnikova ◽  
Sandro Halwe ◽  
Clemens Lier ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Inclusion Bodies ◽  
Ebola Virus ◽  
Viral Transcription ◽  
Recognition Motif ◽  
Virus Transcription ◽  
Regulatory Circuit ◽  
Republic Of The Congo ◽  
Significant Growth Inhibition

ABSTRACT Ebola virus (EBOV) causes a severe and often fatal disease for which no approved vaccines or antivirals are currently available. EBOV VP30 has been described as a viral phosphoprotein, and nonphosphorylated VP30 is essential and sufficient to support secondary transcription in an EBOV-specific minigenome system; however, phosphorylatable serine residues near the N terminus of VP30 are required to support primary viral transcription as well as the reinitiation of VP30-mediated transcription at internal EBOV genes. While the dephosphorylation of VP30 by the cellular phosphatase PP2A was found to be mediated by nucleoprotein, the VP30-specific kinases and the role of phosphorylation remain unknown. Here, we report that serine-arginine protein kinase 1 (SRPK1) and SRPK2 phosphorylate serine 29 of VP30, which is located in an N-terminal R26xxS29 motif. Interaction with VP30 via the R26xxS29 motif recruits SRPK1 into EBOV-induced inclusion bodies, the sites of viral RNA synthesis, and an inhibitor of SRPK1/SRPK2 downregulates primary viral transcription. When the SRPK1 recognition motif of VP30 was mutated in a recombinant EBOV, virus replication was severely impaired. It is presumed that the interplay between SRPK1 and PP2A in the EBOV inclusions provides a comprehensive regulatory circuit to ensure the activity of VP30 in EBOV transcription. Thus, the identification of SRPK1 is an important mosaic stone that completes our picture of the players involved in Ebola virus transcription regulation. IMPORTANCE The largest Ebola virus (EBOV) epidemic in West Africa ever caused more than 28,000 cases and 11,000 deaths, and the current EBOV epidemic in the Democratic Republic of the Congo continues, with more than 3,000 cases to date. Therefore, it is essential to develop antivirals against EBOV. Recently, an inhibitor of the cellular phosphatase PP2A-mediated dephosphorylation of the EBOV transcription factor VP30 has been shown to suppress the spread of Ebola virus. Here, we identified the protein kinase SRPK1 as a VP30-specific kinase that phosphorylates serine 29, the same residue that is dephosphorylated by PP2A. SRPK1-mediated phosphorylation of serine 29 enabled primary viral transcription. Mutation of the SRPK1 recognition motif in VP30 resulted in significant growth inhibition of EBOV. Similarly, elevation of the phosphorylation status of serine 29 by overexpression of SRPK1 inhibited EBOV growth, highlighting the importance of reversible phosphorylation of VP30 as a potential therapeutic target.

scholarly journals RNA binding specificity of Ebola virus transcription factor VP30

RNA Biology ◽  
2016 ◽  
Vol 13 (9) ◽  
pp. 783-798 ◽  
Author(s):  
Julia Schlereth ◽  
Arnold Grünweller ◽  
Nadine Biedenkopf ◽  
Stephan Becker ◽  
Roland K. Hartmann
Keyword(s):  
Transcription Factor ◽  
Ebola Virus ◽  
Rna Binding ◽  
Binding Specificity ◽  
Virus Transcription

scholarly journals Ebola Virus Transcription Activator VP30 Is a Zinc-Binding Protein

2003 ◽  
Vol 77 (5) ◽  
pp. 3334-3338 ◽  
Author(s):  
Jens Modrof ◽  
Stephan Becker ◽  
Elke Mühlberger
Keyword(s):  
Ebola Virus ◽  
Binding Assay ◽  
Complete Loss ◽  
Zinc Binding ◽  
Viral Transcription ◽  
Zinc Ions ◽  
Transcription Activator ◽  
Virus Transcription ◽  
Viral Particles ◽  
Functional Analyses

ABSTRACT Ebola virus VP30 is an essential activator of viral transcription. In viral particles, VP30 is closely associated with the nucleocapsid complex. A conspicuous structural feature of VP30 is an unconventional zinc-binding Cys3-His motif comprising amino acids 68 to 95. By using a colorimetric zinc-binding assay we found that the VP30-specific Cys3-His motif stoichiometrically binds zinc ions in a one-to-one relationship. Substitution of the conserved cysteines and the histidine within the motif led to a complete loss of the capacity for zinc binding. Functional analyses revealed that none of the tested mutations of the proposed zinc-coordinating residues influenced binding of VP30 to nucleocapsid-like particles but, concerning its role in activating viral transcription, all resulted in a protein that was inactive.

scholarly journals Ebola virus inclusion body formation and RNA synthesis are controlled by a novel domain of NP interacting with VP35

2020 ◽  
Author(s):  
Tsuyoshi Miyake ◽  
Charlotte M. Farley ◽  
Benjamin E. Neubauer ◽  
Thomas P. Beddow ◽  
Thomas Hoenen ◽  
...  
Keyword(s):  
Life Cycle ◽  
Inclusion Body ◽  
Inclusion Bodies ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Central Domain ◽  
Body Formation ◽  
Inclusion Body Formation

AbstractEbola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection IBs display dynamic properties regarding their size and location. Also, the contents of IBs must transition prior to further viral maturation, assembly and release, implying additional steps in IB function. Interestingly, expression of the viral nucleoprotein (NP) alone is sufficient for generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30 and the RNA polymerase L. Previously we defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here we show that NP-Ct is absolutely required for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for production of infectious virus-like particles and retention of viral RNA within these particles. Furthermore, co-expression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins only VP35 is able to overcome the defect in IB formation caused by deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself, and which is mediated by a newly identified domain of NP, the “central domain” (CD).ImportanceInclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative sense RNA viruses including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.

scholarly journals Functional interactomes of the Ebola virus polymerase identified by proximity proteomics in the context of viral replication

2021 ◽  
Author(s):  
Jingru Fang ◽  
Colette Pietzsch ◽  
George Tsaprailis ◽  
Gogce Crynen ◽  
Kelvin Frank Cho ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Ebola Virus ◽  
Polymerase Activity ◽  
Host Factors ◽  
Viral Rna ◽  
Viral Transcription ◽  
Biotin Ligase ◽  
Functional Screen ◽  
Ebov Infection ◽  
Transcription And Replication

Ebola virus (EBOV) critically depends on the viral polymerase to replicate and transcribe the viral RNA genome. To examine whether interactions between EBOV polymerase and cellular and viral factors affect distinct viral RNA synthesis events, we applied proximity proteomics to define the cellular interactome of EBOV polymerase, under conditions that recapitulate viral transcription and replication. We engineered EBOV polymerase tagged with the split-biotin ligase split-TurboID, which successfully biotinylated the proximal proteome while retaining polymerase activity. We further analyzed the interactomes in an siRNA-based, functional screen and uncovered 35 host factors, which, when depleted, affect EBOV infection. We validated one host factor, eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we show physically and functionally associates with EBOV polymerase to facilitate viral transcription termination. Our work demonstrates the utility of proximity proteomics to capture the functional host-interactome of the EBOV polymerase and to illuminate host-dependent regulations of viral RNA synthesis.

scholarly journals Ebola Virus Inclusion Body Formation and RNA Synthesis Are Controlled by a Novel Domain of Nucleoprotein Interacting with VP35

2020 ◽  
Vol 94 (16) ◽  
Author(s):  
Tsuyoshi Miyake ◽  
Charlotte M. Farley ◽  
Benjamin E. Neubauer ◽  
Thomas P. Beddow ◽  
Thomas Hoenen ◽  
...  
Keyword(s):  
Life Cycle ◽  
Inclusion Body ◽  
Inclusion Bodies ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Rna Replication ◽  
Viral Rna ◽  
Central Domain ◽  
Body Formation ◽  
Inclusion Body Formation

ABSTRACT Ebola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection, IBs display dynamic properties regarding their size and location. The contents of IBs also must transition prior to further viral maturation, assembly, and release, implying additional steps in IB function. Interestingly, the expression of the viral nucleoprotein (NP) alone is sufficient for the generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30, and the RNA polymerase L. We previously defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here, we show that NP-Ct is necessary for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for the production of infectious virus-like particles (VLPs), and that defective VLPs with NP-Ct deletions are significantly reduced in viral RNA content. Furthermore, coexpression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins, only VP35 is able to overcome the defect in IB formation caused by the deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself and that is mediated by a newly identified functional domain of NP, the central domain. IMPORTANCE Inclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative-sense RNA viruses, including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.

scholarly journals RNA Binding of Ebola Virus VP30 Is Essential for Activating Viral Transcription

2016 ◽  
Vol 90 (16) ◽  
pp. 7481-7496 ◽  
Author(s):  
Nadine Biedenkopf ◽  
Julia Schlereth ◽  
Arnold Grünweller ◽  
Stephan Becker ◽  
Roland K. Hartmann
Keyword(s):  
Transcription Factor ◽  
Zinc Finger ◽  
Ebola Virus ◽  
Rna Binding ◽  
Case Fatality ◽  
Viral Rna ◽  
Viral Transcription ◽  
Viral Nucleocapsid ◽  
Support Activity ◽  
Transcription And Replication

ABSTRACTThe template for Ebola virus (EBOV) transcription and replication is the helical viral nucleocapsid composed of the viral negative-sense (−) RNA genome, which is complexed by the nucleoprotein (NP), VP35, polymerase L, VP24, and VP30. While viral replication is exerted by polymerase L and its cofactor VP35, EBOV mRNA synthesis is regulated by the viral nucleocapsid protein VP30, an essential EBOV-specific transcription factor. VP30 is a homohexameric phosphoprotein containing a nonconventional zinc finger. The transcriptional support activity of VP30 is strongly influenced by its phosphorylation state. We studied here how RNA binding contributed to VP30's function in transcriptional activation. Using a novel mobility shift assay and the 3′-terminal 154 nucleotides of the EBOV genome as a standard RNA substrate, we detected that RNA binding of VP30 was severely impaired by VP30 mutations that (i) destroy the protein's capability to form homohexamers, (ii) disrupt the integrity of its zinc finger domain, (iii) mimic its fully phosphorylated state, or (iv) alter the putative RNA binding region. RNA binding of the mutant VP30 proteins correlated strongly with their transcriptional support activity. Furthermore, we showed that the interaction between VP30 and the polymerase cofactor VP35 is RNA dependent, while formation of VP30 homohexamers and VP35 homotetramers is not. Our data indicate that RNA binding of VP30 is essential for its transcriptional support activity and stabilizes complexes of VP35/L polymerase with the (−) RNA template to favor productive transcriptional initiation in the presence of termination-active RNA secondary structures.IMPORTANCEEbola virus causes severe fevers with unusually high case fatality rates. The recent outbreak of Ebola virus in West Africa claimed more than 11,000 lives and threatened to destabilize a whole region because of its dramatic effects on the public health systems. It is currently not completely understood how Ebola virus manages to balance viral transcription and replication in the infected cells. This study shows that transcriptional support activity of the Ebola virus transcription factor VP30 is highly correlated with its ability to bind viral RNA. The interaction between VP30 and VP35, the Ebola virus polymerase cofactor, is dependent on the presence of RNA as well. Our data contribute to the understanding of the dynamic interplay between nucleocapsid proteins and the viral RNA template in order to promote viral RNA synthesis.

scholarly journals Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

2015 ◽  
Vol 89 (9) ◽  
pp. 5148-5153 ◽  
Author(s):  
Priya Luthra ◽  
David S. Jordan ◽  
Daisy W. Leung ◽  
Gaya K. Amarasinghe ◽  
Christopher F. Basler
Keyword(s):  
Rna Synthesis ◽  
Ebola Virus ◽  
Mutational Analysis ◽  
Cytoplasmic Dynein ◽  
Viral Rna ◽  
Interferon Production ◽  
Dynein Light Chain ◽  
High Affinity ◽  
Functional Consequences ◽  
Oligomerization Domain

Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

Sign in / Sign up

Export Citation Format

Share Document