Cryo-electron microscopy of hepatitis B virions reveals variability in envelope capsid interactions

Abstract

Hepatitis B virus (HBV) is a major human pathogen causing about 750,000 deaths per year. The virion consists of a nucleocapsid and an envelope formed by lipids, and three integral membrane proteins. Although we have detailed structural insights into the organization of the HBV core, the arrangement of the envelope in virions and its interaction with the nucleocapsid is elusive. Here we show the ultrastructure of hepatitis B virions purified from patient serum. We identified two morphological phenotypes, which appear as compact and gapped particles with nucleocapsids in distinguishable conformations. The overall structures of these nucleocapsids resemble recombinant cores with two alpha-helical spikes per asymmetric unit. At the charged tips the spikes are contacted by defined protrusions of the envelope proteins, probably via electrostatic interactions. The HBV envelope in the two morphotypes is to some extent variable, but the surface proteins follow a general packing scheme with up to three surface protein dimers per asymmetric unit. The variability in the structure of the envelope indicates that the nucleocapsid does not firmly constrain the arrangement of the surface proteins, but provides a general template for the packing.

Figure 1

Micrograph and gallery of HBV embedded in vitrified buffer. (A) Part of a micrograph (inverted contrast) recorded with a Philips CM120 Biotwin. (B) Gallery of selected particles with compact morphology. (C) Gallery of selected particles with gapped morphology. (D) Gallery of particles with mixed morphology. Areas consistent with the gapped morphology are delineated in white. Particles appear bright against darker background. The length of the bar in panel D equals 20 nm.

Figure 2

Surface representations of compact particles (A) A-map and (B) B-map, and gapped particles (C). The A- and B-maps of the compact particles were derived from iterative alignment, supervised classification and reconstruction using two reference maps (see Supplementary data). The A-, B- and C-maps were reconstructed from 114, 106 and 26 particles, respectively. In the upper row a third of the envelope is cut away to reveal the structure of the nucleocapsid inside. For all three maps the Fourier-Shell-correlation dropped to 0.5 at 1/22 Å⁻¹ (see Supplementary Figure 3). For representation the maps were low-pass-filtered to this spatial frequency. The view is centered on a local three-fold axes. In panel C the positions of a strict two-fold, three-fold and five-fold axes are labeled. The lower row shows a view onto the inner surface of the envelope centered at a local three-fold axis. The protrusions around the three-fold (short arrows) and five-fold axes (long arrows) that contact the spikes of the nucleocapsid are marked. The length of the scale bars equals 10 nm. The threshold for calculating the surface representation of the envelope of the gapped particles was adjusted to compensate for the reduced density in this area (see density plots in Supplementary Figure 1).

Figure 3

Spike organization and core envelope contacts. (A) A-map, (B) B-map and (C) C-map show side views of the two types of spikes (labeled as AB and CD spike) in contact to the envelope. The envelope is shown in yellow, the lower part of the nucleocapsid in blue. The radial assignment of color is the same in panels A, B and C. The two dimers in the asymmetric unit (AB-dimer, red, blue; CD-dimer, green, orange, 1QGT; Wynne et al, 1999) fitted into the densities of the A-map (D) and B-map (E) of compact virions and in the C-map (F) of gapped particles. Negatively charged amino acids at the tips of the spikes are shown in yellow. (G) Ribbon diagram of the two dimers in the asymmetric unit (1QGT). The observed contact areas are highlighted by arrows and circles. Contacts in the A-map are colored black, in the B map medium gray and in the C-map light gray. (H) shows a superposition of the nucleocapsid of the A map (blue) and B map (red). In the six spikes closest to the two-fold axis the AB- and CD-dimers are labeled. The change in surface color indicates a relative lateral displacement of the CD-spikes (approximately 4–6 Å). The position of the centers of the AB-spikes in the two maps is identical at the current resolution (less than 3 Å displacement). For spherical slices see Supplementary Figure 3.

Figure 4

Organization of HBs in the membrane. (A–D) Transmembrane region of compact particles (EMD-1399, EMD-1403, EMD-1404, EMD 1405); (E–G) transmembrane region of gapped particles (EMD-1406, EMD-1407, EMD-1408). Densities in the asymmetric unit are color-coded: blue, closest to the five-fold axes, red, closest to the two-fold axes and green close to the three-fold axes but weaker in some reconstructions. The common building principle is shown in (H). (I) Superposition of maps (A–D) (compact particles); (J) superposition of maps (E–G) (gapped particles). The superposition shows that without separating the particles according to their HBs-packing, an almost featureless envelope region is observed. (K, L) Envelope region of panel B, which had the lowest phase residuals in cross common lines of the compact maps after refinement. (K) Envelope seen from the outside and (L) envelope seen from the inside (see Supplementary Figure 6 for other envelopes of the other particle reconstructions).