In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus

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.

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Item Type:	Article
Authors/Creators:	Patel, Nikesh Clark, Sam https://orcid.org/0000-0002-6865-4452 Weiß, Eva U. Mata, Carlos P. Bohon, Jen Farquhar, Erik R. Maskell, Daniel P. Ranson, Neil A. Twarock, Reidun https://orcid.org/0000-0002-1824-2003 Stockley, Peter G.
Copyright, Publisher and Additional Information:	© The Author(s) 2021 Funding Information: We thank Prof Adam Zlotnick, Indiana University, for the gift of his Cp expression construct and advice on its purification, and Ms Leah Wells who assisted with reassembly experiments during her BSc (Honours) undergraduate research project. We thank the Medical Research Foundation for the award of a career development grant to N.P., and the UK MRC for previous grant funding to study HBV assembly (MRF-044-0002-RG-PATEL & MR/N021517/1). R.T. and P.G.S. thank The Wellcome Trust (Joint Investigator Award Nos. 110145 & 110146 to P.G.S. and R.T., respectively) for funding, and we acknowledge the financial support of The Trust of infrastructure and equipment in the Astbury Centre, University of Leeds (089311/Z/09/Z; 090932/Z/09/Z & 106692), and for their additional support, together with The University of Leeds, of the Astbury Bios-tructure Facility. R.T. acknowledges additional funding via an EPSRC Established Career Fellowship (EP/R023204/1) and a Royal Society Wolfson Fellowship (RSWF\R1\180009). Portions of this work used the XFP (17-BM) beamline at NSLS-II. Development of XFP was made possible by the National Science Foundation, Division of Biological Infrastructure (grant No. 1228549), while operations support of XFP was provided by the National Institutes of Health (grant No. P30-EB-009998). NSLS-II, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory, was supported under Contract No. DE-SC0012704. We thank DNA Sequencing & Services (MRC I PPU, School of Life Sciences, University of Dundee, Scotland, www.dnaseq.co.uk) for DNA sequencing.
Dates:	Accepted: 16 November 2021 Published: 16 December 2021
Institution:	The University of York
Academic Units:	The University of York > Faculty of Sciences (York) > Mathematics (York) The University of York > Faculty of Sciences (York) > Biology (York)
Depositing User:	Pure (York)
Date Deposited:	13 Jan 2023 17:20
Last Modified:	10 Apr 2025 23:34
Published Version:	https://doi.org/10.1038/s42003-021-02897-2
Status:	Published
Refereed:	Yes
Identification Number:	10.1038/s42003-021-02897-2
Related URLs:	http://www.scopus.com/inward/record.url?...
Open Archives Initiative ID (OAI ID):	oai:eprints.whiterose.ac.uk:195308

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Description: In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus

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In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus

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