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

Viruses

RobertN. Kirchdoerfer¹  Chidiebere Akusobi²  KristianG. Andersen³  ElizabethA. Brown⁴  StephenF. Schaffner⁴  PardisC. Sabeti⁵  AaronE. Lin⁵  CourtneyL. Finch⁶  Yingyun Cai⁶  JensH. Kuhn⁶  Laura Bollinger⁶  EricaOllmann Saphire⁷  Jeremy Luban⁸  WilliamE. Diehl⁸ 

[1] Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA;Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA 02120, USA;Department of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, CA 92037, USA;Department of Organismic and Evolutionary Biology, FAS Center for Systems Biology, Harvard University, Cambridge, MA 02138, USA;Harvard Program in Virology, Harvard Medical School, Boston, MA 02115, USA;Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, MD 21702, USA;La Jolla Institute for Immunology, La Jolla, CA 92037, USA;Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA;

关键词: ebola virus;    nucleoprotein;    budding;    oligomerization;    reporter assays;    viral evolution;   

DOI  :  10.3390/v12010105

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown