Hepatitis B Virus Genotype G forms core-like particles with unique structural properties

Abstract

We have determined the structure of the core capsid of an unusual variant of hepatitis B virus, genotype G (HBV/G) at 14Å resolution, using cryo-electron microscopy. The structure reveals surface features not present in the prototype HBV/A genotype. HBV/G is novel in that it has a unique 36-bp insertion downstream of the core gene start codon. This results in a twelve amino acid insertion at the N-terminal end of the core protein, and two stop codons in the precore region that prevent the expression of HBeAg. HBV/G replication in patients is associated with co-infection with another genotype of HBV, suggesting that HBV/G may have reduced replication efficiency in vivo. We localized the N-terminal insertion in HBV/G and show that it forms two additional masses on the core surface adjacent to each of the dimer-spikes and have modelled the structure of the additional residues within this density. We show that the position of the insertion would not interfere with translocation of nucleic acids through the pores to the core interior compartment. However, the insertion may partially obscure several residues on the core surface that are known to play a role in envelopment and secretion of virions, or that could affect structural rearrangements that may trigger envelopment after DNA second-strand synthesis.

Fig. 1

(a) Cryo-electron microscopy of HBV/G core-like particles. T = 3 particles (white arrows) and T = 4 particles (black arrows) are shown. Panels (b) and (c) illustrate the sorting of the T = 3 capsids from T = 4 capsids, respectively. The rotational average (top left in panels b and c) was used as a reference for sorting. Several representative sorted particles are presented. Characteristic class averages generated by projection matching for the T = 4 reconstruction are shown in (d).

Fig. 2

Surface shaded representation of the HBV/A capsid structure solved by X-ray crystallography (yellow), and HBV/G capsid structure solved by cryoEM (green) are shown in (a) and (b), respectively. Structures are Fourier filtered to 14 Å resolution for comparison. A difference map (c, shown in blue) was created by subtracting the HBV/A capsid structure from the HBV/G core structure is presented with a mass threshold set to account for the 12 extra residues from the HBV/G cryo-EM map. Panel (d) shows the difference map (blue) superimposed over the HBV/A core crystal structure (yellow). In all four panels, a red oval has been superposed over the reconstruction to highlight a single dimer spike in the structures at the base of the spike.

Fig. 3

(a) and (b). Stereo images of the HBV/A core protein structure. The red ribbon is the HBV/A core crystal structure, PDB accession 1QGT, and the yellow density is the structure derived from the HBV/A core X-ray crystallography data displayed at 14 Å resolution, to match resolution of the difference map shown in blue. The additional mass found on HBV/G (blue density) was determined by subtracting the HBV/A structure from the HBV/G structure. (a) The mass from the extra 12 amino acids on the N-terminal end of HBV/G is near to the N-terminal end of the HBV/A core (the portion of the ribbon coloured green). (b) Top view looking down one of the spike 2-fold axes. Locations of the amino acids critical for the encapsidation of the mature HBV particle, and that interact with the cytoplasmic domains of the HBsAg, are shown as purple spheres. The extra mass from the 12 amino acid insert may stearically interfere with access to some of these residues (notably S17, F18, L60, L95). (c) Side and top views of HBV/G core protein model (red ribbon) with the12 residue N-terminal insertion (yellow ribbon) modelled into the difference map density (transparent blue). Only one copy of the core molecule within the dimer is shown for clarity.