Shepherd, D.A., Ariza, A. orcid.org/0000-0003-4364-823X, Edwards, T.A. et al. (3 more authors) (2014) Probing Bunyavirus N protein oligomerisation using mass spectrometry. Rapid Communications in Mass Spectrometry, 28 (7). pp. 793-800. ISSN 0951-4198
Abstract
RATIONALE
Bunyaviruses have become a major threat to both humans and livestock in Europe and the Americas. The nucleocapsid (N) protein of these viruses is key to the replication cycle and knowledge of the N oligomerisation state is central to understanding the viral lifecycle and for development of therapeutic strategies.
METHODS
Bunyamwera virus and Schmallenberg virus N proteins (BUNV-N and SBV-N) were expressed recombinantly in E. coli as hexahistidine-SUMO-tagged fusions, and the tag removed subsequently. Noncovalent nano-electrospray ionisation mass spectrometry was conducted in the presence and absence of short RNA oligonucleotides. Instrumental conditions were optimised for the transmission of intact protein complexes into the gas phase. The resulting protein-protein and protein-RNA complexes were identified and their stoichiometries verified by their mass. Collision-induced dissociation tandem mass spectrometry was used in cases of ambiguity.
RESULTS
Both BUNV-N and SBV-N proteins reassembled into N-RNA complexes in the presence of RNA; however, SBV-N formed a wider range of complexes with varying oligomeric states. The N:RNA oligomers observed were consistent with a model of assembly via stepwise addition of N proteins. Furthermore, upon mixing the two proteins in the presence of RNA no heteromeric complexes were observed, thus revealing insights into the specificity of oligomerisation.
CONCLUSIONS
Noncovalent mass spectrometry has provided the first detailed analysis of the co-populated oligomeric species formed by these important viral proteins and revealed insights into their assembly pathways. Using this technique has also enabled comparisons to be made between the two N proteins.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2014 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, (http://creativecommons.org/licenses/by/4.0/) which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
Keywords: | Amino Acid Sequence; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Nucleocapsid Proteins; Protein Multimerization; Recombinant Proteins; Sequence Alignment |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 05 Jul 2023 13:09 |
Last Modified: | 05 Jul 2023 13:09 |
Status: | Published |
Publisher: | Wiley |
Refereed: | Yes |
Identification Number: | 10.1002/rcm.6841 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:201222 |