Cell-free hepatitis B virus capsid assembly dependent on the core protein C-terminal domain and regulated by phosphorylation

Laurie Ludgate, Kuancheng Liu, Laurie Luckenbaugh, Nicholas Streck, Stacey Eng, Christian Voitenleitner, William E. Delaney, Jianming Hu

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro. Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors.

Original languageEnglish (US)
Pages (from-to)5830-5844
Number of pages15
JournalJournal of virology
Volume90
Issue number12
DOIs
StatePublished - Jun 1 2016

Fingerprint

Virus Assembly
capsid
Hepatitis B virus
Capsid
Protein C
phosphorylation
Phosphorylation
reticulocytes
Reticulocytes
proteins
Proteins
cells
rabbits
Rabbits
RNA
Viral Core Proteins
cell free system
RNA-Binding Proteins
Cell-Free System
dephosphorylation

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

Cite this

Ludgate, Laurie ; Liu, Kuancheng ; Luckenbaugh, Laurie ; Streck, Nicholas ; Eng, Stacey ; Voitenleitner, Christian ; Delaney, William E. ; Hu, Jianming. / Cell-free hepatitis B virus capsid assembly dependent on the core protein C-terminal domain and regulated by phosphorylation. In: Journal of virology. 2016 ; Vol. 90, No. 12. pp. 5830-5844.
@article{60c2a6c5bd2f4b01b67b58ccd0ac9e8f,
title = "Cell-free hepatitis B virus capsid assembly dependent on the core protein C-terminal domain and regulated by phosphorylation",
abstract = "Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro. Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors.",
author = "Laurie Ludgate and Kuancheng Liu and Laurie Luckenbaugh and Nicholas Streck and Stacey Eng and Christian Voitenleitner and Delaney, {William E.} and Jianming Hu",
year = "2016",
month = "6",
day = "1",
doi = "10.1128/JVI.00394-16",
language = "English (US)",
volume = "90",
pages = "5830--5844",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "12",

}

Ludgate, L, Liu, K, Luckenbaugh, L, Streck, N, Eng, S, Voitenleitner, C, Delaney, WE & Hu, J 2016, 'Cell-free hepatitis B virus capsid assembly dependent on the core protein C-terminal domain and regulated by phosphorylation', Journal of virology, vol. 90, no. 12, pp. 5830-5844. https://doi.org/10.1128/JVI.00394-16

Cell-free hepatitis B virus capsid assembly dependent on the core protein C-terminal domain and regulated by phosphorylation. / Ludgate, Laurie; Liu, Kuancheng; Luckenbaugh, Laurie; Streck, Nicholas; Eng, Stacey; Voitenleitner, Christian; Delaney, William E.; Hu, Jianming.

In: Journal of virology, Vol. 90, No. 12, 01.06.2016, p. 5830-5844.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Cell-free hepatitis B virus capsid assembly dependent on the core protein C-terminal domain and regulated by phosphorylation

AU - Ludgate, Laurie

AU - Liu, Kuancheng

AU - Luckenbaugh, Laurie

AU - Streck, Nicholas

AU - Eng, Stacey

AU - Voitenleitner, Christian

AU - Delaney, William E.

AU - Hu, Jianming

PY - 2016/6/1

Y1 - 2016/6/1

N2 - Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro. Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors.

AB - Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro. Furthermore, at physiologically low HBc concentrations in RRL, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and the NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions, including its modulation by host factors.

UR - http://www.scopus.com/inward/record.url?scp=84971394435&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84971394435&partnerID=8YFLogxK

U2 - 10.1128/JVI.00394-16

DO - 10.1128/JVI.00394-16

M3 - Article

C2 - 27076641

AN - SCOPUS:84971394435

VL - 90

SP - 5830

EP - 5844

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 12

ER -