Reporter Assays for Ebola Virus Nucleoprotein Oligomerization, Virion-Like Particle Budding, and Minigenome Activity Reveal the Importance of Nucleoprotein Amino Acid Position 111
Aaron E. Lin,
William E. Diehl,
Yingyun Cai,
Courtney L. Finch,
Chidiebere Akusobi,
Robert N. Kirchdoerfer,
Laura Bollinger,
Stephen F. Schaffner,
Elizabeth A. Brown,
Erica Ollmann Saphire,
Kristian G. Andersen,
Jens H. Kuhn,
Jeremy Luban,
Pardis C. Sabeti
Affiliations
Aaron E. Lin
Harvard Program in Virology, Harvard Medical School, Boston, MA 02115, USA
William E. Diehl
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA
Yingyun Cai
Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD 21702, USA
Courtney L. Finch
Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD 21702, USA
Chidiebere Akusobi
Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA 02120, USA
Robert N. Kirchdoerfer
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA
Laura Bollinger
Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD 21702, USA
Stephen F. Schaffner
Department of Organismic and Evolutionary Biology, FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138, USA
Elizabeth A. Brown
Department of Organismic and Evolutionary Biology, FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138, USA
Erica Ollmann Saphire
La Jolla Institute for Immunology, La Jolla, CA 92037, USA
Kristian G. Andersen
Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, CA 92037, USA
Jens H. Kuhn
Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD 21702, USA
Jeremy Luban
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA
Pardis C. Sabeti
Harvard Program in Virology, Harvard Medical School, Boston, MA 02115, USA
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.
