Serine-Arginine Protein Kinase 1 Regulates Ebola Virus Transcription

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.

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Serine-arginine protein kinase 1 regulates Ebola virus transcription

10.1101/717371 ◽

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.

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Ebola Virus Transcription Activator VP30 Is a Zinc-Binding Protein

Journal of Virology ◽

10.1128/jvi.77.5.3334-3338.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 3334-3338 ◽

Cited By ~ 51

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.

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Role of Protein Phosphatase 1 in Dephosphorylation of Ebola Virus VP30 Protein and Its Targeting for the Inhibition of Viral Transcription

Journal of Biological Chemistry ◽

10.1074/jbc.m114.575050 ◽

2014 ◽

Vol 289 (33) ◽

pp. 22723-22738 ◽

Cited By ~ 51

Author(s):

Philipp A. Ilinykh ◽

Bersabeh Tigabu ◽

Andrey Ivanov ◽

Tatiana Ammosova ◽

Yuri Obukhov ◽

...

Keyword(s):

Protein Phosphatase ◽

Ebola Virus ◽

Protein Phosphatase 1 ◽

Viral Transcription

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Kaposi's Sarcoma-Associated Herpesvirus-Encoded Protein Kinase and Its Interaction with K-bZIP

Journal of Virology ◽

10.1128/jvi.01473-06 ◽

2006 ◽

Vol 81 (3) ◽

pp. 1072-1082 ◽

Cited By ~ 53

Author(s):

Yoshihiro Izumiya ◽

Chie Izumiya ◽

Albert Van Geelen ◽

Don-Hong Wang ◽

Kit S. Lam ◽

...

Keyword(s):

Dna Replication ◽

Protein Kinase ◽

Viral Protein ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Viral Transcription ◽

Viral Dna ◽

Viral Dna Replication ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The oncogenic herpesvirus, Kaposi's sarcoma-associated herpesvirus, also identified as human herpesvirus 8, contains genes producing proteins that control transcription and influence cell signaling. Open reading frame 36 (ORF36) of this virus encodes a serine/threonine protein kinase, which is designated the viral protein kinase (vPK). Our recent efforts to elucidate the role of vPK in the viral life cycle have focused on identifying viral protein substrates and determining the effects of vPK-mediated phosphorylation on specific steps in viral replication. The vPK gene was transcribed into 4.2-kb and 3.6-kb mRNAs during the early and late phases of viral reactivation. vPK is colocalized with viral DNA replication/transcription compartments as marked by a polymerase processivity factor, and K-bZIP, a protein known to bind the viral DNA replication origin (Ori-Lyt) and to regulate viral transcription. The vPK physically associated with and strongly phosphorylated K-bZIP at threonine 111, a site also recognized by the cyclin-dependent kinase Cdk2. Both K-bZIP and vPK were corecruited to viral promoters targeted by K-bZIP as well as to the Ori-Lyt region. Phosphorylation of K-bZIP by vPK had a negative impact on K-bZIP transcription repression activity. The extent of posttranslational modification of K-bZIP by sumoylation, a process that influences its repression function, was decreased by vPK phosphorylation at threonine 111. Our data thus identify a new role of vPK as a modulator of viral transcription.

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Role of Ebola Virus VP30 in Transcription Reinitiation

Journal of Virology ◽

10.1128/jvi.01395-08 ◽

2008 ◽

Vol 82 (24) ◽

pp. 12569-12573 ◽

Cited By ~ 61

Author(s):

Miguel J. Martínez ◽

Nadine Biedenkopf ◽

Valentina Volchkova ◽

Bettina Hartlieb ◽

Nathalie Alazard-Dany ◽

...

Keyword(s):

Gene Transcription ◽

Reverse Genetics ◽

Ebola Virus ◽

Replication Cycle ◽

Phosphorylation Sites ◽

Virus Transcription ◽

Transcription Reinitiation

ABSTRACT VP30 is a phosphoprotein essential for the initiation of Ebola virus transcription. In this work, we have studied the effect of mutations in VP30 phosphorylation sites on the ebolavirus replication cycle by using a reverse genetics system. We demonstrate that VP30 is involved in reinitiation of gene transcription and that this activity is affected by mutations at the phosphorylation sites.

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AMP-Activated Protein Kinase and Host Defense against Infection

International Journal of Molecular Sciences ◽

10.3390/ijms19113495 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3495 ◽

Cited By ~ 12

Author(s):

Prashanta Silwal ◽

Jin Kim ◽

Jae-Min Yuk ◽

Eun-Kyeong Jo

Keyword(s):

Protein Kinase ◽

Ebola Virus ◽

Viral Infections ◽

Parasitic Infections ◽

Host Defenses ◽

Adaptive Immune ◽

Pathological Conditions ◽

Ampk Activity ◽

Amp Activated Protein Kinase

5′-AMP-activated protein kinase (AMPK) plays diverse roles in various physiological and pathological conditions. AMPK is involved in energy metabolism, which is perturbed by infectious stimuli. Indeed, various pathogens modulate AMPK activity, which affects host defenses against infection. In some viral infections, including hepatitis B and C viral infections, AMPK activation is beneficial, but in others such as dengue virus, Ebola virus, and human cytomegaloviral infections, AMPK plays a detrimental role. AMPK-targeting agents or small molecules enhance the antiviral response and contribute to the control of microbial and parasitic infections. In addition, this review focuses on the double-edged role of AMPK in innate and adaptive immune responses to infection. Understanding how AMPK regulates host defenses will enable development of more effective host-directed therapeutic strategies against infectious diseases.

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The Ebola Virus Nucleoprotein Recruits the Nuclear RNA Export Factor NXF1 into Inclusion Bodies to Facilitate Viral Protein Expression

Cells ◽

10.3390/cells9010187 ◽

2020 ◽

Vol 9 (1) ◽

pp. 187 ◽

Cited By ~ 7

Author(s):

Lisa Wendt ◽

Janine Brandt ◽

Bianca S. Bodmer ◽

Sven Reiche ◽

Marie Luisa Schmidt ◽

...

Keyword(s):

Life Cycle ◽

Protein Expression ◽

Viral Protein ◽

Inclusion Bodies ◽

Ebola Virus ◽

Rna Binding ◽

Nuclear Rna ◽

Rna Export ◽

Nuclear Rna Export

Ebola virus (EBOV) causes severe outbreaks of viral hemorrhagic fever in humans. While virus-host interactions are promising targets for antivirals, there is only limited knowledge regarding the interactions of EBOV with cellular host factors. Recently, we performed a genome-wide siRNA screen that identified the nuclear RNA export factor 1 (NXF1) as an important host factor for the EBOV life cycle. NXF1 is a major component of the nuclear mRNA export pathway that is usurped by many viruses whose life cycles include nuclear stages. However, the role of NXF1 in the life cycle of EBOV, a virus replicating in cytoplasmic inclusion bodies, remains unknown. In order to better understand the role of NXF1 in the EBOV life cycle, we performed a combination of co-immunoprecipitation and double immunofluorescence assays to characterize the interactions of NXF1 with viral proteins and RNAs. Additionally, using siRNA-mediated knockdown of NXF1 together with functional assays, we analyzed the role of NXF1 in individual aspects of the virus life cycle. With this approach we identified the EBOV nucleoprotein (NP) as a viral interaction partner of NXF1. Further studies revealed that NP interacts with the RNA-binding domain of NXF1 and competes with RNA for this interaction. Co-localization studies showed that RNA binding-deficient, but not wildtype NXF1, accumulates in NP-derived inclusion bodies, and knockdown experiments demonstrated that NXF1 is necessary for viral protein expression, but not for viral RNA synthesis. Finally, our results showed that NXF1 interacts with viral mRNAs, but not with viral genomic RNAs. Based on these results we suggest a model whereby NXF1 is recruited into inclusion bodies to promote the export of viral mRNA:NXF1 complexes from these sites. This would represent a novel function for NXF1 in the life cycle of cytoplasmically replicating viruses, and may provide a basis for new therapeutic approaches against EBOV, and possibly other emerging viruses.

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