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Hepatitis B virus (HBV) contains 3 types of particles, i.e., 22-nm-diameter spherical and tubular subviral particles (SVPs) and 44-nm-diameter Dane particles. The SVPs are non-infectious and present strong immunogenicity, while Dane particles are infectious. In this study, we isolated spherical SVPs from HBV carriers’ sera and determined their 3D structure at the resolution of ∼30 A by cryo-electron microscopy (cryo-EM) single-particle reconstruction. Our cryo-EM structure suggests that the native HBV spherical SVP is irregularly organized, where spike-like features are arranged in a crystalline-like pattern on the surface. Strikingly, the hepatitis B surface antigen (HBsAg) in the native spherical SVPs folds as protrusions on the surface, as those on the native tubular SVPs and Dane particles, but is largely different from that in the recombinant octahedral SVPs. These results suggest a universal folding shape of HBsAg on the native HBV viral and subviral particles.

Core protein of hepatitis B virus (HBV) with various C-terminal lengths (residue 154, 164, 167 and 183) can self-assemble into recombinant hepatitis B core antigen (HBcAg) particles. To understand the RNA encapsidation mechanism of HBV, the three-dimensional structures of these particles were reconstructed by cryo-electron microscopy (cryoEM). Detailed structural comparisons showed that their capsid structures are highly similar, while the RNA content is increased upon the retention of more amino acid residues at the C-terminus of core protein, suggesting the crucial role of the basic C-terminal tail on determining the genome size.

The brevidensovirus is one of the smallest viruses in the world and the capsid of Aedes albopictus C6/36 cell densovirus (C6/36DNV) is the simplest and most compact capsid in brevidensovirus. To understand the assembly mechanism of icosahedral-virus capsid from this simplest model, we tried to express various lengths of virus proteins (VPs) of C6/36DNV in Bac-to-Bac system and evaluate their self-assembly capacities in insect Spodoptera frugiperda 9 (Sf9) cells. The result showed that the N-terminal GGSG sequence (residue 23-26), highly conserved glycine-rich region in Parvoviridae, and C-terminal GTGGVVTCMP (residue 344-353) were essential for capsid assembly, while the N-terminal nuclear localization signal, GTKRKR sequence (residue 15-20), was nonessential for the virus-like particles (VLPs) assembly, but did effect the formation of crystalline arrays in infected Sf9 cells. These information provided clues for how icosahedral-virus capsids formed and showed the potential of C6/36DNV-VLPs becoming a powerful nanoparticle vector.