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

Fang, Jingru; Pietzsch, Colette; Tsaprailis, George; Crynen, Gogce; Cho, Kelvin Frank; Ting, Alice Y.; Bukreyev, Alexander; Torre, Juan Carlos de la; Saphire, Erica Ollmann

doi:10.1016/j.celrep.2022.110544

Cited by 12 publications

(14 citation statements)

References 57 publications

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“…We did not observe any significant reduction in cell viability even at the highest (10 µM) concentration of CC-90009 used (CC 50 > 10 µM). Importantly, our recent work has shown a potent inhibitory effect of CC-90009 against Zaire Ebola virus growth in cell culture (100-fold reduction in viral titer with 1 μM CC-90009 treatment postinfection) ( 46 ). As a comparison, the SARS-CoV-2 polymerase inhibitor molnupiravir, which has been recently issued with an FDA Emergency Use Authorization for COVID-19 treatment, was reported to have an EC 50 = 300 nM against SARS-CoV2 in cultured cells ( 47 ).…”

Section: Discussionmentioning

confidence: 99%

Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host-directed antivirals

Fang

Pietzsch

Witwit

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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Completion of the Lassa virus (LASV) life cycle critically depends on the activities of the virally encoded, RNA-dependent RNA polymerase in replication and transcription of the viral RNA genome in the cytoplasm of infected cells. The contribution of cellular proteins to these processes remains unclear. Here, we applied proximity proteomics to define the interactome of LASV polymerase in cells under conditions that recreate LASV RNA synthesis. We engineered a LASV polymerase-biotin ligase (TurboID) fusion protein that retained polymerase activity and successfully biotinylated the proximal proteome, which allowed the identification of 42 high-confidence LASV polymerase interactors. We subsequently performed a small interfering RNA (siRNA) screen to identify those interactors that have functional roles in authentic LASV infection. As proof of principle, we characterized eukaryotic peptide chain release factor subunit 3a (eRF3a/GSPT1), which we found to be a proviral factor that physically associates with LASV polymerase. Targeted degradation of GSPT1 by a small-molecule drug candidate, CC-90009, resulted in strong inhibition of LASV infection in cultured cells. Our work demonstrates the feasibility of using proximity proteomics to illuminate and characterize yet-to-be-defined host-pathogen interactome, which can reveal new biology and uncover novel targets for the development of antivirals against highly pathogenic RNA viruses.

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Section: Discussionmentioning

confidence: 99%

Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host-directed antivirals

Fang

Pietzsch

Witwit

et al. 2022

Proc. Natl. Acad. Sci. U.S.A.

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“…Transfection and activity quantification with Pol1-based monocistronic EBOV minigenome system was adapted from 47 and described previously 68 . Transfection with the T7 pol-based EBOV bicistronic minigenome system was adapted from 65 .…”

Section: Methodsmentioning

confidence: 99%

“…Co-IP reactions were performed as previously described 68 . HEK 293T cells (1×10 6 cells/well) were seeded in 6-wells plates.…”

Section: Methodsmentioning

confidence: 99%

“…Immunofluorescence staining and confocal light microscopy: Fixation and staining was performed as described 68 .…”

Section: Figures and Figure Legendsmentioning

confidence: 99%

“…EBOV minigenome assays: Transfection and activity quantification with Pol1-based monocistronic EBOV minigenome system was adapted from 47 and described previously 68 .…”

Section: Virus Infection With Ebov-gfp-∆vp30mentioning

confidence: 99%

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Spatial and functional arrangement of Ebola virus polymerase inside phase-separated viral factories

Fang

Castillon

Phan

et al. 2022

Preprint

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Ebola virus (EBOV) infection induces formation of membrane-less, cytoplasmic compartments termed viral factories, in which multiple viral proteins gather and from which diverse viral biogenesis arises. Key to viral factory function is recruitment of the EBOV polymerase, a multifunctional machine that mediates replication and expression of the viral RNA genome. Here we show that intracellularly reconstituted EBOV viral factories are biomolecular condensates, with composition-dependent internal dynamics of exchange that likely facilitates viral replication. We report that EBOV viral factories display either droplet-like or network-like morphology, which could be influenced by multivalent intermolecular interactions between viral proteins. Within the viral factory, we found EBOV polymerase is not uniformly distributed, but instead clusters into foci. The distance between these foci increases when viral replication is enabled. This unique view of EBOV propagation suggests a form-to-function relationship that describes how physical properties and internal structures of biomolecular condensates influence and regulate viral biogenesis.

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Harnessing high-throughput OMICS in emerging zoonotic virus preparedness and response activities

Loeb,

Lemaille,

Frederick

et al. 2024

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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

Cited by 12 publications

References 57 publications

Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host-directed antivirals

Proximity interactome analysis of Lassa polymerase reveals eRF3a/GSPT1 as a druggable target for host-directed antivirals

Spatial and functional arrangement of Ebola virus polymerase inside phase-separated viral factories

Harnessing high-throughput OMICS in emerging zoonotic virus preparedness and response activities

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