The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30

Kruse, Thomas; Biedenkopf, Nadine; Hertz, Emil Peter Thrane; Dietzel, Erik; Stalmann, Gertrud; López-Méndez, Blanca; Davey, Norman E.; Nilsson, Jakob; Becker, Stephan

doi:10.1016/j.molcel.2017.11.034

Cited by 83 publications

(104 citation statements)

References 23 publications

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“…VP30 phosphorylation regulates the balance between viral transcription (mRNA synthesis) and genome RNA replication, with dephosphorylated VP30 favoring transcription (Biedenkopf et al, 2013; Modrof et al, 2002). Recruitment of protein phosphatase 1 or 2A by NP leads to the dephosphorylation of VP30 and subsequently promotes transcriptional activity; other host interactors relevant to VP30 function have not yet been identified (llinykh et al, 2014; Kruse et al, 2018; Modrof et al, 2002). RBBP6 is a large multidomain protein that interacts with a variety of host molecules including RNA, the retinoblastoma protein (pRb), p53, MDM2, Zinc finger and BTB domain-containing protein 38 (ZBTB38) as well as Y-box binding protein 1 (YB1) (Baltz et al, 2012; Chibi et al, 2008; Li et al, 2007; Miotto et al, 2014; Sakai et al, 1995; Simons et al, 1997).…”

Section: Resultsmentioning

confidence: 99%

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Protein Interaction Mapping Identifies RBBP6 as a Negative Regulator of Ebola Virus Replication

Batra

Hultquist

Liu

et al. 2018

Cell

118

123

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SUMMARY Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologies that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.

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Section: Resultsmentioning

confidence: 99%

“…Upon interaction with VP30, NP recruits the host PP2A-B56 protein phosphatase to dephosphorylate VP30 and thereby promote viral transcription (Kruse et al, 2018). Disruption of the VP30-NP interaction through competitive binding to the same PPxPxY motif presumably prevents VP30 dephosphorylation and so impairs viral transcription.…”

Section: Resultsmentioning

confidence: 99%

Protein Interaction Mapping Identifies RBBP6 as a Negative Regulator of Ebola Virus Replication

Batra

Hultquist

Liu

et al. 2018

Cell

118

123

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“…fig.5). Nevertheless, to test directly whether a lack of phosphodependence was the reason for enhanced SAC silencing, we mutated the PP2A binding sequence in BubR1 to an LxxIxE sequence that binds to B56 in the same manner and with similar affinities, but, crucially, does not depend on phosphorylation (BubR1 deP-PP2A which uses an LPTIHE sequence (Kruse et al, 2018)). Fig.4e shows that BubR1 deP-PP2A cells were now able to silence the SAC in the absence of PP1-Knl1, demonstrating that PP2A-B56 is restricted from silencing the SAC due to a phosphodependent interaction with BubR1.…”

Section: Pp1-knl1 and Pp2a-b56 Use Opposite Phosphodependencies To Comentioning

confidence: 99%

PP1 and PP2A use opposite phospho-dependencies to control distinct processes at the kinetochore

Smith

Cordeiro

Davey

et al. 2019

Preprint

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PP1 and PP2A-B56 are major serine/threonine phosphatase families that achieve specificity by colocalising with substrates. At the kinetochore, however, both phosphatases localise to an almost identical molecular space and yet they still manage to regulate unique pathways and processes. By switching or modulating the positions of PP1/PP2A-B56 at kinetochores, we show that their unique downstream effects are not due to either the identity of the phosphatase or its precise location. Instead, these phosphatases signal differently because their kinetochore recruitment can be either inhibited (PP1) or enhanced (PP2A) by phosphorylation inputs. Mathematical modelling explains how these inverse phospho-dependencies elicit unique forms of crossregulation and feedback, which allows otherwise indistinguishable phosphatases to produce distinct network behaviours and control different mitotic processes. Therefore, the kinetochore uses PP1 and PP2A-B56 because their binding motifs respond to kinase inputs in opposite ways. Genome-wide motif analysis suggests that many other pathways also select for these same key features, implying that these similar catalytic enzymes may have diverged during evolution to allow opposite modes of phospho-regulation.

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“…Marburg virus, a close relative of EBOV, was shown to recruit components of the endosomal sorting complex required for transport (ESCRT) to inclusion bodies to facilitate the trafficking of nucleocapsids to the plasma membrane for viral assembly and budding [12,13]. Kinases and phosphatases such as PP2A-B56 are also known to be recruited to inclusion bodies, and are important in regulating the activity of VP30 in viral RNA synthesis, which is dependent on its phosphorylation status [14,15]. Similarly, RBBP6 appears to regulate the balance of replication and transcription by binding to VP30, and also Staufen1 was described to influence viral RNA synthesis [16,17].…”

Section: Introductionmentioning

confidence: 99%

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

Wendt

Brandt

Bodmer

et al. 2020

Cells

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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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The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30

Cited by 83 publications

References 23 publications

Protein Interaction Mapping Identifies RBBP6 as a Negative Regulator of Ebola Virus Replication

Protein Interaction Mapping Identifies RBBP6 as a Negative Regulator of Ebola Virus Replication

PP1 and PP2A use opposite phospho-dependencies to control distinct processes at the kinetochore

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

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