Assessment of the function and intergenus-compatibility of Ebola and Lloviu virus proteins

Kämper, Lennart; Zierke, Lukas; Schmidt, Marie Luisa; Müller, Andreas; Wendt, Lisa; Brandt, Janine; Hartmann, Eric; Braun, Stefanie; Holzerland, Julia; Groseth, Allison; Hoenen, Thomas

doi:10.1099/jgv.0.001261

Cited by 10 publications

(3 citation statements)

References 68 publications

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“…However, our results also clearly show that it is the origin of the RNP proteins, rather than regulatory RNA elements in the genome termini, or their compatibility with the RNP proteins, that is responsible for this difference. Interestingly, EBOV RNP proteins also show a higher efficiency of viral RNA synthesis in comparison to other newly discovered filoviruses with potentially restricted pathogenicity, for example, when comparing them with RNP proteins from the related LLOV [ 36 ]. Again, this is seen irrespective of whether the RNPs are acting on an EBOV minigenome or a LLOV minigenome.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2020

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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.

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Section: Discussionmentioning

confidence: 99%

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

et al. 2020

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“…LLOV minigenome assays were performed as described previously ( 40 ). Briefly, HEK 293T cells were transfected with the PolII-driven LLOV minigenome (pCAGGS-LLOV-vRNA-hrluc; 125 ng), pCAGGS-LLOV-L (500 ng), pCAGGS-LLOV-VP30 (37.5 ng), and pCAGGS-LLOV-VP35 (62.5 ng), as well as the different myc-tagged NP mutants or NP-WT (62.5 ng), using Transit LT-1, according to manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Cryoelectron microscopic structure of the nucleoprotein–RNA complex of the European filovirus, Lloviu virus

Fujita-Fujiharu

Sugita

et al. 2023

PNAS Nexus

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Lloviu virus (LLOV) is a novel filovirus detected in Schreiber’s bats in Europe. The isolation of the infectious LLOV from bats has raised public health concerns. However, the virological and molecular characteristics of LLOV remain largely unknown. The nucleoprotein (NP) of LLOV encapsidates the viral genomic RNA to form a helical NP-RNA complex, which acts as a scaffold for nucleocapsid formation and de novo viral RNA synthesis. In this study, using single-particle cryo-electron microscopy, we determined two structures of the LLOV NP-RNA helical complex, comprising a full-length and a C-terminally truncated NP. The two helical structures were identical, demonstrating that the N-terminal region determines the helical arrangement of the NP. The LLOV NP-RNA protomers displayed a structure similar to that in the Ebola and Marburg virus, but the spatial arrangements in the helix differed. Structure-based mutational analysis identified amino acids involved in the helical assembly and viral RNA synthesis. These structures advance our understanding of the filovirus nucleocapsid formation, and provide a structural basis for the development of anti-filoviral therapeutics.

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“…For validation of CAD knockdown efficiency and analyses of coIP input and lysates, samples were subjected to SDS-PAGE and Western blotting as previously described [34]. FLAG-tagged CAD was detected using a monoclonal anti-FLAG antibody (1:2000), while NP, wild type CAD, and GAPDH were detected using anti-NP (1:1000), anti-CAD (1:250), and anti-GAPDH (1:1000) antibodies.…”

Section: Western Blottingmentioning

confidence: 99%

The Cellular Protein CAD is Recruited into Ebola Virus Inclusion Bodies by the Nucleoprotein NP to Facilitate Genome Replication and Transcription

Brandt

Wendt

Bodmer

et al. 2020

Cells

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Ebola virus (EBOV) is a zoonotic pathogen causing severe hemorrhagic fevers in humans and non-human primates with high case fatality rates. In recent years, the number and extent of outbreaks has increased, highlighting the importance of better understanding the molecular aspects of EBOV infection and host cell interactions to control this virus more efficiently. Many viruses, including EBOV, have been shown to recruit host proteins for different viral processes. Based on a genome-wide siRNA screen, we recently identified the cellular host factor carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) as being involved in EBOV RNA synthesis. However, mechanistic details of how this host factor plays a role in the EBOV life cycle remain elusive. In this study, we analyzed the functional and molecular interactions between EBOV and CAD. To this end, we used siRNA knockdowns in combination with various reverse genetics-based life cycle modelling systems and additionally performed co-immunoprecipitation and co-immunofluorescence assays to investigate the influence of CAD on individual aspects of the EBOV life cycle and to characterize the interactions of CAD with viral proteins. Following this approach, we could demonstrate that CAD directly interacts with the EBOV nucleoprotein NP, and that NP is sufficient to recruit CAD into inclusion bodies dependent on the glutaminase (GLN) domain of CAD. Further, siRNA knockdown experiments indicated that CAD is important for both viral genome replication and transcription, while substrate rescue experiments showed that the function of CAD in pyrimidine synthesis is indeed required for those processes. Together, this suggests that NP recruits CAD into inclusion bodies via its GLN domain in order to provide pyrimidines for EBOV genome replication and transcription. These results define a novel mechanism by which EBOV hijacks host cell pathways in order to facilitate genome replication and transcription and provide a further basis for the development of host-directed broad-spectrum antivirals.

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Assessment of the function and intergenus-compatibility of Ebola and Lloviu virus proteins

Cited by 10 publications

References 68 publications

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

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

Cryoelectron microscopic structure of the nucleoprotein–RNA complex of the European filovirus, Lloviu virus

The Cellular Protein CAD is Recruited into Ebola Virus Inclusion Bodies by the Nucleoprotein NP to Facilitate Genome Replication and Transcription

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