Hopkins, FR, Álvarez-Rodríguez, B, Heath, GR orcid.org/0000-0001-6431-2191 et al. (5 more authors) (2022) The Native Orthobunyavirus Ribonucleoprotein Possesses a Helical Architecture. mBio. ISSN 2161-2129
Abstract
The Bunyavirales order is the largest group of negative-sense RNA viruses, containing many lethal human pathogens for which approved anti-infective measures are not available. The bunyavirus genome consists of multiple negative-sense RNA segments enwrapped by the virus-encoded nucleocapsid protein (NP), which together with the viral polymerase form ribonucleoproteins (RNPs). RNPs represent substrates for RNA synthesis and virion assembly, which require inherent flexibility, consistent with the appearance of RNPs spilled from virions. These observations have resulted in conflicting models describing the overall RNP architecture. Here, we purified RNPs from Bunyamwera virus (BUNV), the prototypical orthobunyavirus. The lengths of purified RNPs imaged by negative staining resulted in 3 populations of RNPs, suggesting that RNPs possess a consistent method of condensation. Employing microscopy approaches, we conclusively show that the NP portion of BUNV RNPs is helical. Furthermore, we present a pseudo-atomic model for this portion based on a cryo-electron microscopy average at 13 Å resolution, which allowed us to fit the BUNV NP crystal structure by molecular dynamics. This model was confirmed by NP mutagenesis using a mini-genome system. The model shows that adjacent NP monomers in the RNP chain interact laterally through flexible N- and C-terminal arms only, with no longitudinal helix-stabilizing interactions, thus providing a potential model for the molecular basis for RNP flexibility. Excessive RNase treatment disrupts native RNPs, suggesting that RNA was key in maintaining the RNP structure. Overall, this work will inform studies on bunyaviral RNP assembly, packaging, and RNA replication, and aid in future antiviral strategies.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2022 Hopkins et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Physics and Astronomy (Leeds) > Molecular & Nanoscale Physics The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Crystallography (Leeds) |
Funding Information: | Funder Grant number Wellcome Trust 108466/Z/15/Z Wellcome Trust 090932/Z/09/Z MRC (Medical Research Council) MR/T016159/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 30 Jun 2022 13:25 |
Last Modified: | 25 Jun 2023 23:01 |
Status: | Published online |
Publisher: | American Society for Microbiology |
Identification Number: | 10.1128/mbio.01405-22 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:188160 |