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HBV RNA pre-genome encodes specific motifs that mediate interactions with the viral core protein that promote nucleocapsid assembly

Nature Microbiology volume 2, Article number: 17098 (2017) | Download Citation

Abstract

Formation of the hepatitis B virus nucleocapsid is an essential step in the viral lifecycle, but its assembly is not fully understood. We report the discovery of sequence-specific interactions between the viral pre-genome and the hepatitis B core protein that play roles in defining the nucleocapsid assembly pathway. Using RNA SELEX and bioinformatics, we identified multiple regions in the pre-genomic RNA with high affinity for core protein dimers. These RNAs form stem-loops with a conserved loop motif that trigger sequence-specific assembly of virus-like particles (VLPs) at much higher fidelity and yield than in the absence of RNA. The RNA oligos do not interact with preformed RNA-free VLPs, so their effects must occur during particle assembly. Asymmetric cryo-electron microscopy reconstruction of the T = 4 VLPs assembled in the presence of one of the RNAs reveals a unique internal feature connected to the main core protein shell via lobes of density. Biophysical assays suggest that this is a complex involving several RNA oligos interacting with the C-terminal arginine-rich domains of core protein. These core protein–RNA contacts may play one or more roles in regulating the organization of the pre-genome during nucleocapsid assembly, facilitating subsequent reverse transcription and acting as a nucleation complex for nucleocapsid assembly.

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Acknowledgements

The authors acknowledge the UK MRC (MR/N021517/1) and the Universities of Leeds and York for financial support for parts of this work, which was also supported in part by grants from the Wellcome Trust (089311/Z/09/Z, 090932/Z/09/Z and 106692). P.G.S. and R.Tw. also acknowledge the Wellcome Trust for financial support for virus work (Joint Investigator Award nos. 110145 and 110146) and R.Tw. acknowledges funding via a Royal Society Leverhulme Trust Senior Research Fellowship (LT130088) and EPSRC grant EP/K028286/1 for R.J.B. E.C.D. acknowledges funding via an Early Career Leverhulme Trust Fellowship (ECF-2013-019). A.Z. acknowledges funding from National Institutes of Health grant R01-AI118933. The authors also thank the eBIC for collection time on the Titan Krios microscopes.

Author information

Author notes

    • Nikesh Patel
    •  & Simon J. White

    These authors contributed equally to this work.

Affiliations

  1. Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

    • Nikesh Patel
    • , Simon J. White
    • , Rebecca F. Thompson
    • , Daniel P. Maskell
    • , Roman Tuma
    • , Neil A. Ranson
    •  & Peter G. Stockley
  2. Departments of Biology and Mathematics & York Centre for Complex Systems Analysis, University of York, York YO10 5DD, UK

    • Richard Bingham
    • , Eva U. Weiß
    • , Eric C. Dykeman
    •  & Reidun Twarock
  3. Department of Molecular & Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405, USA

    • Adam Zlotnick

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Contributions

P.G.S. and R.Tw. conceived the project. N.P. performed smFCS and photobleaching experiments and, with R.Tu., analysed the data. S.J.W., R.F.T. and N.A.R. collected and analysed the cryo-EM data. S.J.W. performed SELEX and R.B., E.D., E.U.W. and R.Tw. analysed the resulting sequences to identify the PSs. N.P., S.J.W. and D.P.M. purified HBV VLPs. A.Z. provided reagents. All authors contributed to the writing and editing of the manuscript.

Competing interests

A.Z. is a co-founder and consultant of Assembly BioSciences. Research in the Zlotnick laboratory is supported by the NIH and Assembly. No Assembly BioSciences employee contributed to A.Z.'s contribution to this work.

Corresponding authors

Correspondence to Reidun Twarock or Neil A. Ranson or Peter G. Stockley.

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DOI

https://doi.org/10.1038/nmicrobiol.2017.98