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

ABSTRACT: 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.

SUBMITTER: Lin AE

PROVIDER: S-EPMC7019320 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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

Lin Aaron E AE Diehl William E WE Cai Yingyun Y Finch Courtney L CL Akusobi Chidiebere C Kirchdoerfer Robert N RN Bollinger Laura L Schaffner Stephen F SF Brown Elizabeth A EA Saphire Erica Ollmann EO Andersen Kristian G KG Kuhn Jens H JH Luban Jeremy J Sabeti Pardis C PC

Viruses 20200115 1

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) pro ...[more]

PMID: 31952352

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Project description:Ebola virus (EBOV) causes severe hemorrhagic fever in humans and other primates with a high case fatality rate. No approved drug or vaccine of EBOV is available, which necessitates better understanding of the virus life cycle. Studies on EBOV have been hampered because experimentations involving live virus are restricted to biosafety level 4 (BSL4) laboratories. The EBOV minigenome system has provided researchers with the opportunity to study EBOV under BSL2 conditions. Here, we developed a novel EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe the EBOV minigenome. The minigenomic RNA harboring a Gaussia luciferase and hygromycin-resistant marker can replicate for months in a helper cell stably expressing viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and L proteins. Quantification of viral RNA (vRNA), cRNA, and mRNA levels of the EBOV minigenome demonstrated that the stable EBOV replicon had much-more-active minigenome replication than previously developed transient-transfection-based EBOV minigenome systems, which recapitulate viral primary transcription more than genome replication. Interestingly, minigenome replication in the stable EBOV replicon cells was insensitive to interferon treatment or RNA interference. Moreover, RNase digestion of the replicon cell lysates revealed the remarkably stable nature of the EBOV minigenomic vRNA ribonucleoprotein complex, which may help improve understanding of EBOV persistence in convalescent patients.IMPORTANCE The scope and severity of the recent Ebola outbreak in Western Africa justified a more comprehensive investigation of the causative risk group 4 agent Ebola virus (EBOV). Study of EBOV replication and antiviral development can be facilitated by developing a cell culture system that allows experimentation under biosafety level 2 conditions. Here, we developed a novel stable EBOV minigenome replicon which, to our knowledge, is the first EBOV cell culture system that can stably replicate and transcribe the EBOV minigenome. The replicon system had more-active genome replication than previously developed transient-transfection-based EBOV minigenome systems, providing a convenient surrogate system to study EBOV replication. Furthermore, self-replicating minigenomic vRNA in the replicon cells displayed strong stability in response to interferon treatment, RNA silencing, and RNase digestion, which may provide an explanation for the persistence of EBOV in survivors.

Dataset Information

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

Publications

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

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