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. 2024 Jan 12;10(2):eadi7606.
doi: 10.1126/sciadv.adi7606. Epub 2024 Jan 10.

Structural basis for nuclear import of hepatitis B virus (HBV) nucleocapsid core

Ruoyu Yang  1 , Ying-Hui Ko  2 , Fenglin Li  1 , Ravi K Lokareddy  2 , Chun-Feng David Hou  1 , Christine Kim  3 , Shelby Klein  4 , Santiago Antolínez  5 , Juan F Marín  5 , Carolina Pérez-Segura  5 , Martin F Jarrold  4 , Adam Zlotnick  3 , Jodi A Hadden-Perilla  5 , Gino Cingolani  2
Affiliations

Structural basis for nuclear import of hepatitis B virus (HBV) nucleocapsid core

Ruoyu Yang et al. Sci Adv. .

Abstract

Nuclear import of the hepatitis B virus (HBV) nucleocapsid is essential for replication that occurs in the nucleus. The ~360-angstrom HBV capsid translocates to the nuclear pore complex (NPC) as an intact particle, hijacking human importins in a reaction stimulated by host kinases. This paper describes the mechanisms of HBV capsid recognition by importins. We found that importin α1 binds a nuclear localization signal (NLS) at the far end of the HBV coat protein Cp183 carboxyl-terminal domain (CTD). This NLS is exposed to the capsid surface through a pore at the icosahedral quasi-sixfold vertex. Phosphorylation at serine-155, serine-162, and serine-170 promotes CTD compaction but does not affect the affinity for importin α1. The binding of 30 importin α1/β1 augments HBV capsid diameter to ~620 angstroms, close to the maximum size trafficable through the NPC. We propose that phosphorylation favors CTD externalization and prompts its compaction at the capsid surface, exposing the NLS to importins.

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Figures

Fig. 1.
Fig. 1.. Binding of purified importins to HBV EC by native agarose gel electrophoresis.
Purified EC was incubated with an excess of the importin α1/β1 heterodimer (A) or individually purified ΔIBB–Imp α1 (B) and importin β1 (C).
Fig. 2.
Fig. 2.. Binding stoichiometry between EC and the importin α/β1 heterodimer determined by CD-MS.
(A) EC peaked at 5 MDa, while (B) the EC:importin α1/β1 complex was detected at 10.15 MDa, which is consistent with one capsid bound to 30 copies of the ~160-kDa importin α1/β1 heterodimer. The charge distribution for ions (right column) is consistent with a compact structure for EC where the ion distribution is close to the Rayleigh limit (red line) and a much less compact structure for importin-decorated capsids.
Fig. 3.
Fig. 3.. Crystal structure of HBV-NLS bound to ΔIBB–Imp α1.
(A) A ribbon diagram of dimeric Cp183 showing the amino acid sequence for the unresolved CTD. Four arginine-rich boxes are underlined with arginines and phosphoacceptors colored in black and red, respectively. (B) The crystal structure of ΔIBB–Imp α1 (shown as a yellow solvent surface) bound to the HBV-NLS (in green sticks) refined at 2.0-Å resolution. A magnified view of the HBV-NLS overlaid to the experimental Fo-Fc electron density contoured at 2σ shows that the guanidinium group of R173 at the P2 site exists in two conformations. (C) Schematic diagram of all major contacts between HBV-NLS and ΔIBB–Imp α1 observed at the major NLS binding site. Hydrogen bonds and salt bridges are shown as dashed black and green lines. A red frame highlights R173 at the P2 position.
Fig. 4.
Fig. 4.. Cryo-EM analysis of HBV EC bound to human importins.
Icosahedral reconstructions of HBV EC (A), EC bound to a molar excess of ΔIBB–Imp α1 (B), and EC bound to a molar excess of importin α1/β1 (C). The three reconstructions were determined at 3.0-, 3.8-, and 3.4-Å FSC resolution, respectively, and are contoured at 2.2 σ. HBV EC is colored gray, while density features ascribed to importins are colored yellow.
Fig. 5.
Fig. 5.. Importin α1 binds to the HBV-NLS exposed at the quasi-sixfold channel.
(A) A global view of 30 importin α1/β1 blobs visible around the quasi-sixfold channels of HBV EC. The zoom-in panel shows the arched electron density for importin α1/β1 (yellow) obtained after focused refinement. (B) A model of importin α1 (yellow ribbon) docked into the density observed at the quasi-sixfold bound to the HBV-NLS (green stick). Phospho-S155, -S162, and -S170 are colored in red. Importin β1 is schematized as a blue circle connected to importin α1 IBB by a flexible linker (dashed black line).
Fig. 6.
Fig. 6.. Cryo-EM single particle analysis of the purified importin α1/β1 heterodimer.
(A) SDS–polyacrylamide gel electrophoresis (PAGE) analysis of the purified importin α1/β1 heterodimer and (B) relative 2D class averages obtained using cryoSPARC. (C) Quality of the 3.95-Å cryo-EM density (gray) contoured at 2.2 σ and overlaid with the IBB (yellow). (D) Refined atomic models of importin β1 (cyan) bound to the IBB domain (yellow). (E) Cartoon model of the importin α1/β1 complex. The flexible linker in importin α1 (residues 53 to 77) is colored in black. Magnified view of WT importin α1/β1 (F) and the engineered importin Δ53–76–α1/β1 (G) bound to a quasi-sixfold pore. The capsid is colored gray, while the importin is colored yellow. The small cartoon models on the top right illustrate the proposed arrangement of importin α1 (yellow) and β1 (cyan).
Fig. 7.
Fig. 7.. Role of phosphorylation in CTD.
(A) Model of CTD residues 150 to 183 emerging at the quasi-sixfold pore assuming (left) an extended conformation that lacks intramolecular bonding; (right) the conformation modeled in complex with importin α1/β1 (Fig. 5B). (B) CTD peptide end-to-end distance versus radius of gyration (Rg) and (C) NLS SASA distributions for unphosphorylated (orange) and S155, S162, and S170 phosphorylated (blue) states. The minimum NLS SASA required for binding of importin α1/β1 was estimated on the basis of the structure of ΔIBB–Imp α1:HBV-NLS complex by subtracting the SASA of the bound NLS from that of the unbound NLS in its crystallographic conformation.
Fig. 8.
Fig. 8.. A model for HBV capsid nuclear import through the NPC.
The diameter of the HBV capsid decorated by 30 importin α1/β1 is ~62 nm. The dilated NPC is estimated to be ~69 nm (64). (i to vi) Proposed steps leading to HBV capsid nuclear import. The illustration was created using BioRender.
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