scholarly journals An Intrinsically Disordered Peptide from Ebola Virus VP35 Controls Viral RNA Synthesis by Modulating Nucleoprotein-RNA Interactions

Cell Reports ◽  
2015 ◽  
Vol 11 (3) ◽  
pp. 376-389 ◽  
Author(s):  
Daisy W. Leung ◽  
Dominika Borek ◽  
Priya Luthra ◽  
Jennifer M. Binning ◽  
Manu Anantpadma ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Ebola Virus ◽  
Viral Rna ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Peptide

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.

scholarly journals Differences in Viral RNA Synthesis but Not Budding or Entry Contribute to the In Vitro Attenuation of Reston Virus Compared to Ebola Virus

2020 ◽  
Vol 8 (8) ◽  
pp. 1215
Author(s):  
Bianca S. Bodmer ◽  
Josephin Greßler ◽  
Marie L. Schmidt ◽  
Julia Holzerland ◽  
Janine Brandt ◽  
...  
Keyword(s):  
Life Cycle ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Severe Disease ◽  
Case Fatality ◽  
Viral Rna ◽  
Pathogenic Potential ◽  
Rnp Complex ◽  
Reston Virus

Most filoviruses cause severe disease in humans. For example, Ebola virus (EBOV) is responsible for the two most extensive outbreaks of filovirus disease to date, with case fatality rates of 66% and 40%, respectively. In contrast, Reston virus (RESTV) is apparently apathogenic in humans, and while transmission of RESTV from domestic pigs to people results in seroconversion, no signs of disease have been reported in such cases. The determinants leading to these differences in pathogenicity are not well understood, but such information is needed in order to better evaluate the risks posed by the repeated spillover of RESTV into the human population and to perform risk assessments for newly emerging filoviruses with unknown pathogenic potential. Interestingly, RESTV and EBOV already show marked differences in their growth in vitro, with RESTV growing slower and reaching lower end titers. In order to understand the basis for this in vitro attenuation of RESTV, we used various life cycle modeling systems mimicking different aspects of the virus life cycle. Our results showed that viral RNA synthesis was markedly slower when using the ribonucleoprotein (RNP) components from RESTV, rather than those for EBOV. In contrast, the kinetics of budding and entry were indistinguishable between these two viruses. These data contribute to our understanding of the molecular basis for filovirus pathogenicity by showing that it is primarily differences in the robustness of RNA synthesis by the viral RNP complex that are responsible for the impaired growth of RESTV in tissue culture.

scholarly journals The Ebola Virus VP30-NP Interaction Is a Regulator of Viral RNA Synthesis

2016 ◽  
Vol 12 (10) ◽  
pp. e1005937 ◽  
Author(s):  
Robert N. Kirchdoerfer ◽  
Crystal L. Moyer ◽  
Dafna M. Abelson ◽  
Erica Ollmann Saphire
Keyword(s):  
Rna Synthesis ◽  
Ebola Virus ◽  
Viral Rna

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 Staufen1 Interacts with Multiple Components of the Ebola Virus Ribonucleoprotein and Enhances Viral RNA Synthesis

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Jingru Fang ◽  
Colette Pietzsch ◽  
Palaniappan Ramanathan ◽  
Rodrigo I. Santos ◽  
Philipp A. Ilinykh ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Binding Proteins ◽  
Ebola Virus ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Critical Role ◽  
Host Factors ◽  
Viral Rna ◽  
Virion Protein ◽  
Public Health Importance

ABSTRACTEbola virus (EBOV) genome and mRNAs contain long, structured regions that could hijack host RNA-binding proteins to facilitate infection. We performed RNA affinity chromatography coupled with mass spectrometry to identify host proteins that bind to EBOV RNAs and identified four high-confidence proviral host factors, including Staufen1 (STAU1), which specifically binds both 3′ and 5′ extracistronic regions of the EBOV genome. We confirmed that EBOV infection rate and production of infectious particles were significantly reduced in STAU1-depleted cells. STAU1 was recruited to sites of EBOV RNA synthesis upon infection and enhanced viral RNA synthesis. Furthermore, STAU1 interacts with EBOV nucleoprotein (NP), virion protein 30 (VP30), and VP35; the latter two bridge the viral polymerase to the NP-coated genome, forming the viral ribonucleoprotein (RNP) complex. Our data indicate that STAU1 plays a critical role in EBOV replication by coordinating interactions between the viral genome and RNA synthesis machinery.IMPORTANCEEbola virus (EBOV) is a negative-strand RNA virus with significant public health importance. Currently, no therapeutics are available for Ebola, which imposes an urgent need for a better understanding of EBOV biology. Here we dissected the virus-host interplay between EBOV and host RNA-binding proteins. We identified novel EBOV host factors, including Staufen1, which interacts with multiple viral factors and is required for efficient viral RNA synthesis.

scholarly journals Variation around the dominant viral genome sequence contributes to viral load and outcome in patients with Ebola virus disease

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Xiaofeng Dong ◽  
Jordana Munoz-Basagoiti ◽  
Natasha Y. Rickett ◽  
Georgios Pollakis ◽  
William A. Paxton ◽  
...  
Keyword(s):  
Viral Load ◽  
Genome Sequence ◽  
Protein Function ◽  
Viral Protein ◽  
Viral Genome ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Virus Disease ◽  
Viral Rna ◽  
Ebola Virus Disease

Abstract Background Viral load is a major contributor to outcome in patients with Ebola virus disease (EVD), with high values leading to a fatal outcome. Evidence from the 2013–2016 Ebola virus (EBOV) outbreak indicated that different genotypes of the virus can have different phenotypes in patients. Additionally, due to the error-prone nature of viral RNA synthesis in an individual patient, the EBOV genome exists around a dominant viral genome sequence. The minor variants within a patient may contribute to the overall phenotype in terms of viral protein function. To investigate the effects of these minor variants, blood samples from patients with acute EVD were deeply sequenced. Results We examine the minor variant frequency between patients with acute EVD who survived infection with those who died. Non-synonymous differences in viral proteins were identified that have implications for viral protein function. The greatest frequency of substitution was identified at three codon sites in the L gene—which encodes the viral RNA-dependent RNA polymerase (RdRp). Recapitulating this in an assay for virus replication, these substitutions result in aberrant viral RNA synthesis and correlate with patient outcome. Conclusions Together, these findings support the notion that in patients who survived EVD, in some cases, the genetic variability of the virus resulted in deleterious mutations that affected viral protein function, leading to reduced viral load. Such mutations may also lead to persistent strains of the virus and be associated with recrudescent infections.

scholarly journals Structural insight into Marburg virus nucleoprotein-RNA complex formation

2021 ◽  
Author(s):  
Yoko Fujita-Fujiharu ◽  
Yukihiko Sugita ◽  
Yuki Takamatsu ◽  
Kazuya Houri ◽  
Manabu Igarashi ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Ebola Virus ◽  
Mutational Analysis ◽  
Marburg Virus ◽  
Viral Rna ◽  
Structural Basis ◽  
Close Relative ◽  
Rna Complex ◽  
Key Residues ◽  
Viral Genomic Rna

The nucleoprotein (NP) of Marburg virus (MARV), a close relative of Ebola virus (EBOV), encapsidates the single-stranded, negative-sense viral genomic RNA (vRNA) to form the helical NP-RNA complex. The NP-RNA complex serves as a scaffold for the assembly of the nucleocapsid that is responsible for viral RNA synthesis. Although appropriate interactions among NPs and RNA are required for the formation of nucleocapsid, the structural basis of the helical assembly remains largely elusive. Here, we show the structure of the MARV NP-RNA complex determined using cryo-electron microscopy at a resolution of 3.1 angstrom. The structures of the asymmetric unit, a complex of an NP and six RNA nucleotides, was very similar to that of EBOV, suggesting that both viruses share common mechanisms for the nucleocapsid formation. Structure-based mutational analysis of both MARV and EBOV NPs identified key residues for the viral RNA synthesis as well as the helical assembly. Importantly, most of the residues identified were conserved in both viruses. These findings provide a structural basis for understanding the nucleocapsid formation and contribute to the development of novel antivirals against MARV and EBOV.

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 VP30 and nucleoprotein interactions modulate viral RNA synthesis

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Wei Xu ◽  
Priya Luthra ◽  
Chao Wu ◽  
Jyoti Batra ◽  
Daisy W. Leung ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Ebola Virus ◽  
Viral Rna

scholarly journals The two-stage interaction of Ebola virus VP40 with nucleoprotein results in a switch from viral RNA synthesis to virion assembly/budding

2020 ◽  
Author(s):  
Linjuan Wu ◽  
Dongning Jin ◽  
Dan Wang ◽  
Xuping Jing ◽  
Peng Gong ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Virus Assembly ◽  
Rna Virus ◽  
Critical Role ◽  
Viral Rna ◽  
Hydrophobic Core ◽  
Virion Assembly ◽  
N Terminus

AbstractEbola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40–NP interaction within IBs, sequestering VP40 within IBs, reducing VP40–VLP egress, abolishing the incorporation of NC-like structures into VP40–VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.

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