Weak Protein−Protein Interactions Are Sufficient To Drive Assembly of Hepatitis B Virus Capsids

Abstract

Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.

High-resolution mass spectrometry of viral assemblies: Molecular composition and stability of dimorphic hepatitis B virus capsids

“…The capsid has protruding spikes at the dimer interfaces that display most of the antigenic epitopes and holes at the symmetry axes that allow infusion of nucleotides for reverse transcription (7,15). Although HBV capsid structure has been closely characterized by cryo-electron microscopy (EM) and x-ray crystallography (5), its biophysical properties and the assembly process are less well understood (16,17). Here, we present biophysical data from macromolecular (tandem) mass spectrometry (18,19) and atomic-force microscopy (AFM) that illuminate the molecular compositions and physical stability of HBV capsids.…”

Controlling viral capsid assembly with templating

“…For example, protein-protein interactions, and hence the critical subunit concentration (CSC), can be controlled in virus-like particle experiments by varying the salt concentration or pH [12,29,47]. Capsid proteins denature, however, if these parameters are changed too far from physiological conditions.…”

“…Thus, in addition to their biomedical and technological applications, studying viral capsids has revealed fundamental principles of assembly. Although specific assembly mechanisms are poorly understood for most viruses, a general mechanism has emerged for the spontaneous assembly of empty capsids [10][11][12][12][13][14][15]21,26,28,[47][48][49][50][51][52][53]. Assembly occurs through a sequential addition process in which individual subunits or larger intermediates [26,52] bind to a growing capsid.…”

Thermodynamic origins of protein folding, allostery, and capsid formation in the human hepatitis B virus core protein

“…1B), whereas α 1 , α 2 , and α 5 pack together and around the base of the four-helix bundle to create the hydrophobic core of "contact" domains (19). Weak interdimer interactions between contact domains stabilize HBV capsids (19,24) (Fig. 1B).…”

“…capsid assembly have inferred the existence of assembly-active (HBc Ass ) and assembly-incompetent (HBc Inc ) HBc conformations (12,13,21,24,25). However, there are few detailed insights on the thermodynamic origins of structure, allostery, and dynamics for the dimeric HBc 1-149 protomer, where structural plasticity must originate.…”