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Article

Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111

1
Harvard Program in Virology, Harvard Medical School, Boston, MA 02115, USA
2
Department of Organismic and Evolutionary Biology, FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138, USA
3
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
4
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA
5
Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD 21702, USA
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Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA 02120, USA
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Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA
8
La Jolla Institute for Immunology, La Jolla, CA 92037, USA
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Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, CA 92037, USA
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Scripps Translational Science Institute, La Jolla, CA 92037, USA
11
Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
These authors contributed equally to this work.
Viruses 2020, 12(1), 105; https://doi.org/10.3390/v12010105
Received: 10 December 2019 / Revised: 9 January 2020 / Accepted: 13 January 2020 / Published: 15 January 2020
(This article belongs to the Collection Advances in Ebolavirus, Marburgvirus, and Cuevavirus Research)
For highly pathogenic viruses, reporter assays that can be rapidly performed are critically needed to identify potentially functional mutations for further study under maximal containment (e.g., biosafety level 4 [BSL-4]). The Ebola virus nucleoprotein (NP) plays multiple essential roles during the viral life cycle, yet few tools exist to study the protein under BSL-2 or equivalent containment. Therefore, we adapted reporter assays to measure NP oligomerization and virion-like particle (VLP) production in live cells and further measured transcription and replication using established minigenome assays. As a proof-of-concept, we examined the NP-R111C substitution, which emerged during the 2013–2016 Western African Ebola virus disease epidemic and rose to high frequency. NP-R111C slightly increased NP oligomerization and VLP budding but slightly decreased transcription and replication. By contrast, a synthetic charge-reversal mutant, NP-R111E, greatly increased oligomerization but abrogated transcription and replication. These results are intriguing in light of recent structures of NP oligomers, which reveal that the neighboring residue, K110, forms a salt bridge with E349 on adjacent NP molecules. By developing and utilizing multiple reporter assays, we find that the NP-111 position mediates a complex interplay between NP’s roles in protein structure, virion budding, and transcription and replication. View Full-Text
Keywords: Ebola virus; nucleoprotein; budding; oligomerization; reporter assays; viral evolution Ebola virus; nucleoprotein; budding; oligomerization; reporter assays; viral evolution
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MDPI and ACS Style

Lin, A.E.; Diehl, W.E.; Cai, Y.; Finch, C.L.; Akusobi, C.; Kirchdoerfer, R.N.; Bollinger, L.; Schaffner, S.F.; Brown, E.A.; Saphire, E.O.; Andersen, K.G.; Kuhn, J.H.; Luban, J.; Sabeti, P.C. Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111. Viruses 2020, 12, 105. https://doi.org/10.3390/v12010105

AMA Style

Lin AE, Diehl WE, Cai Y, Finch CL, Akusobi C, Kirchdoerfer RN, Bollinger L, Schaffner SF, Brown EA, Saphire EO, Andersen KG, Kuhn JH, Luban J, Sabeti PC. Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111. Viruses. 2020; 12(1):105. https://doi.org/10.3390/v12010105

Chicago/Turabian Style

Lin, Aaron E., William E. Diehl, Yingyun Cai, Courtney L. Finch, Chidiebere Akusobi, Robert N. Kirchdoerfer, Laura Bollinger, Stephen F. Schaffner, Elizabeth A. Brown, Erica O. Saphire, Kristian G. Andersen, Jens H. Kuhn, Jeremy Luban, and Pardis C. Sabeti 2020. "Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111" Viruses 12, no. 1: 105. https://doi.org/10.3390/v12010105

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