DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: 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.

Authors:
ORCiD logo [1]; ORCiD logo [2];  [3];  [4]; ORCiD logo [5]; ORCiD logo [6];  [1]; ORCiD logo [1];  [2]; ORCiD logo [1]
  1. Univ. of Leeds (United Kingdom)
  2. Univ. of York (United Kingdom)
  3. Univ. of Würzburg (Germany); Univ. of York (United Kingdom)
  4. National Centre for Microbiology (ISCIII). Majadahonda, Madrid (Spain); Univ. of Leeds (United Kingdom)
  5. Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Brookhaven National Lab. (BNL), Upton, NY (United States)
  6. Brookhaven National Lab. (BNL), Upton, NY (United States)
Publication Date:
Research Org.:
Brookhaven National Lab. (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities Division; National Institutes of Health (NIH); National Science Foundation (NSF)
OSTI Identifier:
1875647
Report Number(s):
BNL-223145-2022-JACI
Journal ID: ISSN 2399-3642
Grant/Contract Number:  
SC0012704; P30-EB-009998; 1228549
Resource Type:
Accepted Manuscript
Journal Name:
Communications Biology
Additional Journal Information:
Journal Volume: 4; Journal Issue: 1; Journal ID: ISSN 2399-3642
Publisher:
Springer Nature
Country of Publication:
United States
Language:
English
Subject:
99 GENERAL AND MISCELLANEOUS; Cryoelectron microscopy; Hepatitis B virus; RNA

Citation Formats

Patel, Nikesh, Clark, Sam, Weiß, Eva U., Mata, Carlos P., Bohon, Jen, Farquhar, Erik R., Maskell, Daniel P., Ranson, Neil A., Twarock, Reidun, and Stockley, Peter G. In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus. United States: N. p., 2021. Web. doi:10.1038/s42003-021-02897-2.
Patel, Nikesh, Clark, Sam, Weiß, Eva U., Mata, Carlos P., Bohon, Jen, Farquhar, Erik R., Maskell, Daniel P., Ranson, Neil A., Twarock, Reidun, & Stockley, Peter G. In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus. United States. https://doi.org/10.1038/s42003-021-02897-2
Patel, Nikesh, Clark, Sam, Weiß, Eva U., Mata, Carlos P., Bohon, Jen, Farquhar, Erik R., Maskell, Daniel P., Ranson, Neil A., Twarock, Reidun, and Stockley, Peter G. Thu . "In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus". United States. https://doi.org/10.1038/s42003-021-02897-2. https://www.osti.gov/servlets/purl/1875647.
@article{osti_1875647,
title = {In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus},
author = {Patel, Nikesh and Clark, Sam and Weiß, Eva U. and Mata, Carlos P. and Bohon, Jen and Farquhar, Erik R. and Maskell, Daniel P. and Ranson, Neil A. and Twarock, Reidun and Stockley, Peter G.},
abstractNote = {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.},
doi = {10.1038/s42003-021-02897-2},
journal = {Communications Biology},
number = 1,
volume = 4,
place = {United States},
year = {Thu Dec 16 00:00:00 EST 2021},
month = {Thu Dec 16 00:00:00 EST 2021}
}

Works referenced in this record:

Antiviral therapy and resistance with hepatitis B virus infection
journal, January 2007


Protective immunisation against hepatitis B with an internal antigen of the virus
journal, October 1987

  • Murray, Kenneth; Bruce, Sandra A.; Wingfield, Paul
  • Journal of Medical Virology, Vol. 23, Issue 2
  • DOI: 10.1002/jmv.1890230202

Hepatitis B viruses: Reverse transcription a different way
journal, June 2008


Long-term safety of lamivudine treatment in patients with chronic hepatitis B
journal, December 2003


Structural organization of the hepatitis B virus minichromosome
journal, March 2001

  • Bock, C. Thomas; Schwinn, Susanne; Locarnini, Stephen
  • Journal of Molecular Biology, Vol. 307, Issue 1
  • DOI: 10.1006/jmbi.2000.4481

HBc binds to the CpG islands of HBV cccDNA and promotes an epigenetic permissive state
journal, June 2011


Assembly and Release of Hepatitis B Virus
journal, November 2015


Hepatitis B Virus Biology
journal, March 2000


HBV RNA pre-genome encodes specific motifs that mediate interactions with the viral core protein that promote nucleocapsid assembly
journal, June 2017


Aptamers come of age – at last
journal, August 2006

  • Bunka, David H. J.; Stockley, Peter G.
  • Nature Reviews Microbiology, Vol. 4, Issue 8
  • DOI: 10.1038/nrmicro1458

Packaging signals in single-stranded RNA viruses: nature’s alternative to a purely electrostatic assembly mechanism
journal, March 2013

  • Stockley, Peter G.; Twarock, Reidun; Bakker, Saskia E.
  • Journal of Biological Physics, Vol. 39, Issue 2
  • DOI: 10.1007/s10867-013-9313-0

Packaging Signals in Two Single-Stranded RNA Viruses Imply a Conserved Assembly Mechanism and Geometry of the Packaged Genome
journal, September 2013

  • Dykeman, Eric C.; Stockley, Peter G.; Twarock, Reidun
  • Journal of Molecular Biology, Vol. 425, Issue 17
  • DOI: 10.1016/j.jmb.2013.06.005

A two-stage mechanism of viral RNA compaction revealed by single molecule fluorescence
journal, April 2013

  • Borodavka, Alexander; Tuma, Roman; Stockley, Peter G.
  • RNA Biology, Vol. 10, Issue 4
  • DOI: 10.4161/rna.23838

Degenerate RNA Packaging Signals in the Genome of Satellite Tobacco Necrosis Virus: Implications for the Assembly of a T=1 Capsid
journal, October 2011

  • Bunka, David H. J.; Lane, Stephen W.; Lane, Claire L.
  • Journal of Molecular Biology, Vol. 413, Issue 1
  • DOI: 10.1016/j.jmb.2011.07.063

Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2
journal, January 2016

  • Rolfsson, Óttar; Middleton, Stefani; Manfield, Iain W.
  • Journal of Molecular Biology, Vol. 428, Issue 2
  • DOI: 10.1016/j.jmb.2015.11.014

Genomic RNA folding mediates assembly of human parechovirus
journal, February 2017


RNA-Mediated Virus Assembly: Mechanisms and Consequences for Viral Evolution and Therapy
journal, May 2019


Calicivirus VP2 forms a portal-like assembly following receptor engagement
journal, January 2019


Structure of the herpes simplex virus portal-vertex
journal, June 2018


Atomic structure of the apoptosome: mechanism of cytochrome c - and dATP-mediated activation of Apaf-1
journal, November 2015


The XFP (17-BM) beamline for X-ray footprinting at NSLS-II
journal, June 2019

  • Asuru, Awuri; Farquhar, Erik R.; Sullivan, Michael
  • Journal of Synchrotron Radiation, Vol. 26, Issue 4
  • DOI: 10.1107/S1600577519003576

Evolution of a virus-like architecture and packaging mechanism in a repurposed bacterial protein
journal, June 2021


Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome.
journal, September 1992


Core Protein Phosphorylation Modulates Pregenomic RNA Encapsidation to Different Extents in Human and Duck Hepatitis B Viruses
journal, May 2000


Roles of the Three Major Phosphorylation Sites of Hepatitis B Virus Core Protein in Viral Replication
journal, July 1999


A short cis-acting sequence is required for hepatitis B virus pregenome encapsidation and sufficient for packaging of foreign RNA.
journal, October 1990


An RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation
journal, June 1993


Mfold web server for nucleic acid folding and hybridization prediction
journal, July 2003


Convergence and coevolution of Hepatitis B virus drug resistance
journal, April 2012

  • Thai, Hong; Campo, David S.; Lara, James
  • Nature Communications, Vol. 3, Issue 1
  • DOI: 10.1038/ncomms1794

Revealing the density of encoded functions in a viral RNA
journal, February 2015

  • Patel, Nikesh; Dykeman, Eric C.; Coutts, Robert H. A.
  • Proceedings of the National Academy of Sciences, Vol. 112, Issue 7
  • DOI: 10.1073/pnas.1420812112

Host RNAs, including transposons, are encapsidated by a eukaryotic single-stranded RNA virus
journal, January 2012

  • Routh, A.; Domitrovic, T.; Johnson, J. E.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 6
  • DOI: 10.1073/pnas.1116168109

Sequence-Specific, RNA–Protein Interactions Overcome Electrostatic Barriers Preventing Assembly of Satellite Tobacco Necrosis Virus Coat Protein
journal, March 2013

  • Ford, Robert J.; Barker, Amy M.; Bakker, Saskia E.
  • Journal of Molecular Biology, Vol. 425, Issue 6
  • DOI: 10.1016/j.jmb.2013.01.004

Full-Length Hepatitis B Virus Core Protein Packages Viral and Heterologous RNA with Similarly High Levels of Cooperativity
journal, April 2010

  • Porterfield, J. Z.; Dhason, M. S.; Loeb, D. D.
  • Journal of Virology, Vol. 84, Issue 14
  • DOI: 10.1128/JVI.00586-10

Evidence that viral RNAs have evolved for efficient, two-stage packaging
journal, September 2012

  • Borodavka, A.; Tuma, R.; Stockley, P. G.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 39
  • DOI: 10.1073/pnas.1204357109

Role of Electrostatics in the Assembly Pathway of a Single-Stranded RNA Virus
journal, September 2014

  • Garmann, Rees F.; Comas-Garcia, Mauricio; Koay, Melissa S. T.
  • Journal of Virology, Vol. 88, Issue 18
  • DOI: 10.1128/JVI.01044-14

Icosahedral Packing of RNA Viral Genomes
journal, January 2005


Electrostatics and the assembly of an RNA virus
journal, June 2005


Electrostatic origin of the genome packing in viruses
journal, November 2006

  • Belyi, V. A.; Muthukumar, M.
  • Proceedings of the National Academy of Sciences, Vol. 103, Issue 46
  • DOI: 10.1073/pnas.0608311103

Physics of RNA and viral assembly
journal, March 2006


Solving a Levinthal's paradox for virus assembly identifies a unique antiviral strategy
journal, March 2014

  • Dykeman, E. C.; Stockley, P. G.; Twarock, R.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 14
  • DOI: 10.1073/pnas.1319479111

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus
journal, December 2016


Double-slit photoelectron interference in strong-field ionization of the neon dimer
journal, January 2019


Encapsidated hepatitis B virus reverse transcriptase is poised on an ordered RNA lattice
journal, July 2014

  • Wang, Joseph Che-Yen; Nickens, David G.; Lentz, Thomas B.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 31
  • DOI: 10.1073/pnas.1321424111

The Crystal Structure of the Human Hepatitis B Virus Capsid
journal, June 1999


Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy
journal, March 1997

  • Böttcher, B.; Wynne, S. A.; Crowther, R. A.
  • Nature, Vol. 386, Issue 6620
  • DOI: 10.1038/386088a0

Visualization of a 4-helix bundle in the hepatitis B virus capsid by cryo-electron microscopy
journal, March 1997

  • Conway, J. F.; Cheng, N.; Zlotnick, A.
  • Nature, Vol. 386, Issue 6620
  • DOI: 10.1038/386091a0

3.5Å cryoEM Structure of Hepatitis B Virus Core Assembled from Full-Length Core Protein
journal, September 2013


Asymmetric analysis reveals novel virus capsid features
journal, July 2019


Assembly Properties of Hepatitis B Virus Core Protein Mutants Correlate with Their Resistance to Assembly-Directed Antivirals
journal, August 2018

  • Ruan, Lu; Hadden, Jodi A.; Zlotnick, Adam
  • Journal of Virology, Vol. 92, Issue 20
  • DOI: 10.1128/jvi.01082-18

Core protein: A pleiotropic keystone in the HBV lifecycle
journal, September 2015


New high-throughput endstation to accelerate the experimental optimization pipeline for synchrotron X-ray footprinting
journal, July 2021

  • Jain, Rohit; Abel, Donald; Rakitin, Maksim
  • Journal of Synchrotron Radiation, Vol. 28, Issue 5
  • DOI: 10.1107/S1600577521005026

Safety and efficacy of lamivudine in patients with severe acute or fulminant hepatitis B, a multicenter experience
journal, April 2006


Effect of Human Leukocyte Interferon on Hepatitis B Virus Infection in Patients with Chronic Active Hepatitis
journal, September 1976

  • Greenberg, Harry B.; Pollard, Richard B.; Lutwick, Larry I.
  • New England Journal of Medicine, Vol. 295, Issue 10
  • DOI: 10.1056/NEJM197609022951001

Discovery and Mechanistic Study of Benzamide Derivatives That Modulate Hepatitis B Virus Capsid Assembly
journal, August 2017

  • Wu, Shuo; Zhao, Qiong; Zhang, Pinghu
  • Journal of Virology, Vol. 91, Issue 16
  • DOI: 10.1128/jvi.00519-17

Hepatitis B Virus Capsids Have Diverse Structural Responses to Small-Molecule Ligands Bound to the Heteroaryldihydropyrimidine Pocket
journal, April 2016

  • Venkatakrishnan, Balasubramanian; Katen, Sarah P.; Francis, Samson
  • Journal of Virology, Vol. 90, Issue 8
  • DOI: 10.1128/jvi.03058-15

Discovery of Novel Hepatitis B Virus Nucleocapsid Assembly Inhibitors
journal, December 2018


Effect of a hepatitis B virus inhibitor, NZ-4, on capsid formation
journal, January 2016


BAY 41-4109 has multiple effects on Hepatitis B virus capsid assembly
journal, January 2006

  • Stray, Stephen J.; Zlotnick, Adam
  • Journal of Molecular Recognition, Vol. 19, Issue 6
  • DOI: 10.1002/jmr.801

A heteroaryldihydropyrimidine activates and can misdirect hepatitis B virus capsid assembly
journal, May 2005

  • Stray, S. J.; Bourne, C. R.; Punna, S.
  • Proceedings of the National Academy of Sciences, Vol. 102, Issue 23
  • DOI: 10.1073/pnas.0409732102

An Intracellular Model of Hepatitis B Viral Infection: An In Silico Platform for Comparing Therapeutic Strategies
journal, December 2020

  • Fatehi, Farzad; Bingham, Richard J.; Dykeman, Eric C.
  • Viruses, Vol. 13, Issue 1
  • DOI: 10.3390/v13010011

A Novel Mouse Model for Stable Engraftment of a Human Immune System and Human Hepatocytes
journal, March 2015


A simple and general method for determining the protein and nucleic acid content of viruses by UV absorbance
journal, November 2010


RELION: Implementation of a Bayesian approach to cryo-EM structure determination
journal, December 2012


New tools for automated high-resolution cryo-EM structure determination in RELION-3
journal, November 2018

  • Zivanov, Jasenko; Nakane, Takanori; Forsberg, Björn O.
  • eLife, Vol. 7
  • DOI: 10.7554/eLife.42166

MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy
journal, February 2017

  • Zheng, Shawn Q.; Palovcak, Eugene; Armache, Jean-Paul
  • Nature Methods, Vol. 14, Issue 4
  • DOI: 10.1038/nmeth.4193

Gctf: Real-time CTF determination and correction
journal, January 2016


UCSF Chimera?A visualization system for exploratory research and analysis
journal, January 2004

  • Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.
  • Journal of Computational Chemistry, Vol. 25, Issue 13
  • DOI: 10.1002/jcc.20084

PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925

Coot model-building tools for molecular graphics
journal, November 2004

  • Emsley, Paul; Cowtan, Kevin
  • Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132
  • DOI: 10.1107/S0907444904019158

MolProbity : all-atom structure validation for macromolecular crystallography
journal, December 2009

  • Chen, Vincent B.; Arendall, W. Bryan; Headd, Jeffrey J.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 1
  • DOI: 10.1107/S0907444909042073