Patel, N orcid.org/0000-0001-6098-3633, Clark, S, Weiß, EU et al. (7 more authors) (2021) In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus. Communications Biology, 4. 1407. ISSN 2399-3642
Abstract
The roles of RNA sequence/structure motifs, Packaging Signals (PSs), for regulating assembly of an HBV genome transcript have been investigated in an efficient in vitro assay containing only core protein (Cp) and RNA. Variants of three conserved PSs, within the genome of a strain not used previously, preventing correct presentation of a Cp-recognition loop motif are differentially deleterious for assembly of nucleocapsid-like particles (NCPs). Cryo-electron microscopy reconstruction of the T = 4 NCPs formed with the wild-type gRNA transcript, reveal that the interior of the Cp shell is in contact with lower resolution density, potentially encompassing the arginine-rich protein domains and gRNA. Symmetry relaxation followed by asymmetric reconstruction reveal that such contacts are made at every symmetry axis. We infer from their regulation of assembly that some of these contacts would involve gRNA PSs, and confirmed this by X-ray RNA footprinting. Mutation of the ε stem-loop in the gRNA, where polymerase binds in vivo, produces a poor RNA assembly substrate with Cp alone, largely due to alterations in its conformation. The results show that RNA PSs regulate assembly of HBV genomic transcripts in vitro, and therefore may play similar roles in vivo, in concert with other molecular factors.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Biological Chemistry (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Cryo EM, Image Processing (Leeds) |
Funding Information: | Funder Grant number Wellcome Trust 110145/Z/15/Z Medical Research Foundation MRF-044-0002-RG-PATEL |
Depositing User: | Symplectic Publications |
Date Deposited: | 26 Oct 2021 13:11 |
Last Modified: | 29 Sep 2022 17:40 |
Status: | Published |
Publisher: | Nature Research |
Identification Number: | 10.1038/s42003-021-02897-2 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:179608 |