Global phosphoproteomic analysis of Ebola virions reveals a novel role for VP35 phosphorylation-dependent regulation of genome transcription

Abstract

Ebola virus (EBOV) causes severe human disease with a high case fatality rate. The balance of evidence implies that the virus circulates in bats. The molecular basis for host–viral interactions, including the role for phosphorylation during infections, is largely undescribed. To address this, and to better understand the biology of EBOV, the phosphorylation of EBOV proteins was analyzed in virions purified from infected monkey Vero-E6 cells and bat EpoNi/22.1 cells using high-resolution mass spectrometry. All EBOV structural proteins were detected with high coverage, along with phosphopeptides. Phosphorylation sites were identified in all viral structural proteins. Comparison of EBOV protein phosphorylation in monkey and bat cells showed only partial overlap of phosphorylation sites, with shared sites found in NP, VP35, and VP24 proteins, and no common sites in the other proteins. Three-dimensional structural models were built for NP, VP35, VP40, GP, VP30 and VP24 proteins using available crystal structures or by de novo structure prediction to elucidate the potential role of the phosphorylation sites. Phosphorylation of one of the identified sites in VP35, Thr-210, was demonstrated to govern the transcriptional activity of the EBOV polymerase complex. Thr-210 phosphorylation was also shown to be important for VP35 interaction with NP. This is the first study to compare phosphorylation of all EBOV virion proteins produced in primate versus bat cells, and to demonstrate the role of VP35 phosphorylation in the viral life cycle. The results uncover a novel mechanism of EBOV transcription and identify novel targets for antiviral drug development.

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Acknowledgements

This work was supported by NIH Research Grants U19AI109664 (AB and SN), 1P50HL118006 (SN), 1R01HL125005 (SN), 5G12MD007597 (SN) and P30AI117970 (SN). We thank Dr. Ryan Leib (Stanford University) for generating and providing FASTA database containing EBOV proteins that was concatenated with proteins of African green monkey and common contaminants. We thank Dr. Christian Drosten (University of Bönn, Germany) for providing EpoNi/22.1 bat cells. We thank Dr. Yoshihiro Kawaoka (University of Wisconsin) for providing the plasmids expressing EBOV NP, VP35, L, VP30, and the T7 polymerase and Dr. Christopher Basler (Georgia State University) for providing Sendai virus, ISG54 promoter-firefly luciferase reporter plasmid, and IFN-ß promoter firefly luciferase reporter plasmid. We also thank Mr. Adam Rink for editing the manuscript.

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AI, PR, PAI, XL and TA performed experiments and analyzed the data. CP modeled protein structures and MP conducted MD simulations. AI, PR, CP, AB and SN wrote the manuscript. GKA contributed interpretation of the data. AB and SN conceived the project. All authors read and approved the final manuscript.

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Correspondence to Alexander Bukreyev or Sergei Nekhai.

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Ivanov, A., Ramanathan, P., Parry, C. et al. Global phosphoproteomic analysis of Ebola virions reveals a novel role for VP35 phosphorylation-dependent regulation of genome transcription. Cell. Mol. Life Sci. 77, 2579–2603 (2020). https://doi.org/10.1007/s00018-019-03303-1

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Keywords

  • Ebola virus
  • Phosphorylation
  • Transcription
  • Replication