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Article Dans Une Revue Journal of Molecular Biology Année : 2020

Hepatitis B Virus Core Protein Domains Essential for Viral Capsid Assembly in a Cellular Context

Résumé

Hepatitis B virus (HBV) core protein (HBc) is essential to the formation of the HBV capsid. HBc contains two domains the N-terminal domain corresponding to residues 1-140 essential to form the icosahedral shell and the C-terminal domain corresponding to a basic and phosphorylated peptide, and required for DNA replication. The role of these two domains for HBV capsid assembly was essentially studied in vitro with HBc purified from mammalian or non-mammalian cell lysates, but their respective role in living cells remains to be clarified. We therefore investigated the assembly of the HBV capsid in Huh7 cells by combining fluorescence lifetime imaging microscopy/Förster's resonance energy transfer, fluorescence correlation spectroscopy and transmission electron microscopy approaches. We found that wild-type HBc forms oligomers early after transfection and at a sub-micromolar concentration. These oligomers are homogeneously diffused throughout the cell. We quantified a stoichiometry ranging from ~170 to ~230 HBc proteins per oligomer, consistent with the visualization of eGFP-containingHBV capsid shaped as native capsid particles by transmission electron microscopy. In contrast, no assembly was observed when HBc-N-terminal domain was expressed. This highlights the essential role of the C-terminal domain to form capsid in mammalian cells. Deletion of either the third helix or of the 124-135 residues of HBc had a dramatic impact on the assembly of the HBV capsid, inducing the formation of mis-assembled oligomers and monomers, respectively. This study shows that our approach using fluorescent derivatives of HBc is an innovative method to investigate HBV capsid formation.

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Dates et versions

hal-02635147 , version 1 (28-05-2020)

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Virgile Rat, Xavier Pinson, Florian Seigneuret, Stéphanie Durand, Charline Herrscher, et al.. Hepatitis B Virus Core Protein Domains Essential for Viral Capsid Assembly in a Cellular Context. Journal of Molecular Biology, 2020, 432 (13), pp.3802-3819. ⟨10.1016/j.jmb.2020.04.026⟩. ⟨hal-02635147⟩
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