Assembly and Release of Hepatitis B Virus

Abstract

The hepatitis B virus (HBV) core protein is a dynamic and versatile protein that directs many viral processes. During capsid assembly, core protein allosteric changes ensure efficient formation of a stable capsid that assembles while packaging viral RNA-polymerase complex. Reverse transcription of the RNA genome as well as transport of the capsid to multiple cellular compartments are directed by dynamic phosphorylation and structural changes of core protein. Subsequently, interactions of the capsid with the surface proteins and/or host proteins trigger envelopment and release of the viral capsids or the transport to the nucleus. Held together by many weak protein-protein interactions, the viral capsid is an extraordinary metastable machine that is stable enough to persist in the cellular and extracellular environment but dissociates to allow release of the viral genome at the right time during infection.

Figure 1.

Morphology of the small hepatitis B surface antigen S-HBsAg (S), the medium M-HBsAg (M), and the large L-HBsAg (L) (adapted from Schädler and Hildt 2009). The large envelope protein has three domains called PreS1, PreS2, and S, whereas M-HBsAg contains PreS2 and S, and S-HBsAg comprises the S domain. Envelope proteins are translocated through the endoplasmic reticulum (ER), anchoring the transmembrane (TM)1/2 and TM3/4 domains into the membrane. L-HBsAg displays dual topology. In i-preS1, preS1 remains in the cytosol, whereas in e-preS1, preS1 is translocated through the membrane into the ER-lumen. N-glycosylation (N-glyc) of the S domain can occur at residue Asn146 (Peterson et al. 1982). M-HBsAg harbors another glycosylation site on Asn4 and an O-glycosylation (O-glyc) site on Thr37 (Stibbe and Gerlich 1983; Werr and Prange 1998; Schmitt et al. 2004). Capsid interaction sites include residues 96–116 of preS1 and part of a cytosolic loop in the S domain (in red).

Figure 2.

Virions and subviral particles (SVPs) use two different pathways for secretion (adapted from Prange 2012). Capsids are released through budding into multivesicular bodies (MVBs) using endosomal sorting complex required for transport (ESCRT)-0, -I, -II, -III, and Vsp4 protein complexes (Katzmann et al. 2002; Hanson and Cashikar 2012). Envelope proteins facilitate budding of the viral capsids at the MVB membrane; however, it is unclear how envelope proteins are transported from the pre-Golgi/endoplasmic reticulum (ER) membrane to the late endosome. Nonenveloped particles are hypothesized to be released in an ESCRT-independent pathway that uses the apoptosis-linked gene 2 (ALG2)-interacting protein X (Alix), also implicated in the secretion of enveloped particles (Watanabe et al. 2007; Bardens et al. 2011). It is not clear how capsid particles distinguish between pathways of enveloped and nonenveloped release. ERGIC, ER-Golgi intermediate compartment.

Figure 3.

Structures of hepatitis B core antigen (HBcAg) and hepatitis B e antigen (HBeAg) dimers. HBeAg harbors a 10-amino-acid-long amino-terminal extension (propeptide). The structure of the monomer structure shows modest conformational changes between free HBcAg, HBcAg in capsid, and HBeAg. The largest structural changes are found in helix α3 and α4, located at the intradimer interface (orange) (adapted from Zlotnick et al. 2013). Cysteine-7, located on the amino-terminal extension of HBeAg (3V6Z) (magenta spheres) can form a disulfide with cysteine 61 (yellow spheres). To accommodate this peptide, the monomers are rotated ∼140° about the intradimer interface from their orientation in HBcAg. In HBcAg, the monomers are parallel, and an intradimer Cys61-Cys61 disulfide can form. Structural differences in the spike region as well as the dimer–dimer interface can be observed between free HBcAg dimer (3KXS) and HBcAg from capsid (1QGT) (assembly-inactive and assembled states, respectively).

Figure 4.

The hepatitis B virus (HBV) virion. (A) Cryoelectron micrograph (cryo-EM) of virions isolated from chronically infected patients showing virions (Dane particles) containing spherical envelopes and virions with elongated appendages. Also shown are spherical and filamentous subviral particles (provided by Dr. Stefan Seitz). (B) Composite cryoelectron microscopy model of an HBV virion comprising the viral lipid envelope with hepatitis B surface antigen (HBsAg) protrusions (yellow), the icosahedral capsid (blue), and the enclosed double-stranded DNA (dsDNA) (red) (Dryden et al. 2006). The capsid displays icosahedral symmetry with protrusions representing the 4-helix bundles of the hepatitis B core antigen (HBcAg) dimers. Protrusions from the envelope are dimers of predominantly the small (S) form of the envelope protein, S-HBsAg. The viral envelope interacts with the spike tips of the capsid but is not arranged with icosahedral symmetry. Scale bar, 10 nm

Figure 5.

Predicted behavior for capsid assembly. (A) Capsid assembly at equilibrium. Very little capsid is observed until the total dimer concentration reaches a pseudocritical concentration of assembly, referred to as K_Dapp, the apparent dissociation constant. Above K_Dapp, almost all additional free dimer assemble into capsid (red), leaving a nearly constant concentration of free dimer (green); a slope of almost zero indicates that assembly is very close to equilibrium (Zlotnick 2007; Katen and Zlotnick 2009). (B) Assembly of large populations of particles displays sigmoidal kinetics (Endres and Zlotnick 2002; Hagan and Chandler 2006; Katen and Zlotnick 2009). The observed lag phase of assembly is proportional to the time to establish a steady state of intermediates. Following the lag phase, capsid particles accumulate (Hagan and Chandler 2006; Katen and Zlotnick 2009). As more free subunits are consumed to form capsids, the reaction slowly approaches equilibrium, plateauing when the pseudocritical concentration is reached (Katen and Zlotnick 2009). Nucleation and intermediate formation occurs throughout the entire assembly reaction.