Allosteric conformational changes of human HBV core protein transform its assembly

Abstract

Hepatitis B Virus core protein (HBc) has multiple roles in the viral lifecycle: viral assembly, compartment for reverse transcription, intracellular trafficking, and nuclear functions. HBc displays assembly polymorphism - it can assemble into icosahedral capsid and aberrant non-capsid structures. It has been hypothesized that the assembly polymorphism is due to allosteric conformational changes of HBc dimer, the smallest assembly unit, however, the mechanism governing the polymorphic assembly of the HBc dimer is still elusive. By using the experimental antiviral drug BAY 41-4109, we successfully transformed the HBc assembly from icosahedral capsid to helical tube. Structural analyses of HBc dimers from helical tubes, T = 4 icosahedral capsid, and sheet-like HBc ensemble revealed differences within the inter-dimer interface. Disruption of the HBc inter-dimer interface may likely promote the various assembly forms of HBc. Our work provides new structural insights into the HBV assembly mechanism and strategic guide for anti-HBV drug design.

Figure 1

Electron microscopy and 3-D reconstruction of HBc tubular ensembles. (a) Electron micrograph of negatively-stained HBc tubular ensembles. (b) A representative cryo-EM image of the HBc tubular ensembles and its Fourier transform. The visible diffraction layer lines extend to 12.1 Å. The reconstructed HBc tubular ensemble viewed from its top (c), edge (d), and side (e). In all images, scale bars represent 100 Å.

Figure 2

EM density map of HBc tubular ensemble and structural model of HBc dimer in tubular ensemble. (a) EM density map of reconstructed HBc tubular ensemble with helical lattice indexes and symmetry elements (quasi 3- and 6-fold axes) marked; the unit cell containing trimer of dimers is framed; the inset is an enlarged version of the trimer of dimer in the unit cell, composed of AB, CD, and EF dimers. (b) EM map of segmented AB dimer (transparent grey) with structural model (coloured ribbon) fitted in top (left) and side (right) views. (c) Structural model of the AB dimer built by MDFF in top (left) and side (right) views; the structural elements are colour-coded and the structural elements critical for HBc assembly are marked according to the scheme of Packianathan et al..

Figure 3

Structural variance of HBc dimers in tubular ensemble. Overlay of structural models of HBc dimers AB (red/brown), CD (light green/green), and EF (blue/cyan) from HBc tubular ensemble in side (upper panel) and top (lower panel) views. Significant conformational differences in helix α5 and helix α1 are indicated by arrows and stars respectively.

Figure 4

Structural comparison of HBc dimers in tube, capsid and sheet-like ensemble. (a,b) Overlays of the HBc AB dimer in tube with that in capsid (PDB ID 1QGT) and sheet-like ensemble (PDB ID 3KXS). (c,d) Overlays of HBc CD dimer in tube with that in capsid (PDB ID 1QGT) and sheet-like ensemble (PDB ID 3KXS). (e) Overlay of HBc EF dimer in tube with that in sheet-like ensemble (PDB ID 3KXS). The significant conformational differences are marked with the helix shift angles labelled.

Figure 5

Structural features of HBc dimers in tube, capsid and sheet-like ensemble. (a) Structural models of dimers in tube (left), capsid (middle) and sheet-like ensemble (right), displayed as space-filled models with hydrophilic and hydrophobic residues rendered blue and red respectively, or (b) depictured as grey ribbon models with hydrophobic residues in helix α5 and helix α1 displayed as red sticks. (c) The trimers of dimers in the unit cell/asymmetric unit of HBc tube, capsid and sheet-like ensemble are displayed as grey ribbon models with hydrophobic residues in helix α5 and helix α1 displayed as red sticks. The three inter-dimer distances within the trimer of dimers of tube, capsid and sheet-like ensemble are labelled.

Figure 6

Schematic model of HBc assembly controlled by allosteric conformational changes. In wild-type, the HBc dimer is in the assembly-active (HBc^Act) conformation and the HBc dimers first assemble into trimer of dimers, in which the interactions between dimers are weak, hence the trimer is thermodynamically unstable therefore the assembly proceeds further to a large, closed ensemble, the capsid (upper panel). BAY41-4109 induces a conformational change of HBc dimer and renders it into an aberrant conformation (HBc^Abb), the HBc dimers first assemble into trimer of dimers, in which the interactions between dimers are even weaker, hence the trimer is thermodynamically unstable therefore the assembly proceeds further to an even large ensemble, the tube (lower panel). The binding of BAY41-4109 to the preformed capsid can also change the conformation of HBc dimer from HBc^Act to HBc^Abb and destabilize the capsid, which may render the capsid to dissociate into trimers of dimers or dimers; the trimers of dimers or dimers then re-associate to assemble into a tube.