The Hepatitis B Virus Core Protein Intradimer Interface Modulates Capsid Assembly and Stability

During the hepatitis B virus (HBV) life cycle, capsid assembly and disassembly must ensure correct packaging and release of the viral genome. Here we show that changes in the dynamics of the core protein play an important role in regulating these processes. The HBV capsid assembles from 120 copies of the core protein homodimer. Each monomer contains a conserved cysteine at position 61 that can form an intradimer disulfide that we use as a marker for dimer conformational states. We show that dimers in the context of capsids form intradimer disulfides relatively rapidly. Surprisingly, compared to reduced dimers, fully oxidized dimers assembled slower and into capsids that were morphologically similar but less stable. We hypothesize that oxidized protein adopts a geometry (or constellation of geometries) that is unfavorable for capsid assembly, resulting in weaker dimer–dimer interactions as well as slower assembly kinetics. Our results suggest that structural flexibility at the core protein intradimer interface is essential for regulating capsid assembly and stability. We further suggest that capsid destabilization by the C61–C61 disulfide has a regulatory function to support capsid disassembly and release of the viral genome

The phosphorylation-mimic EEE mutation alters CTD structure.

<p>Viewed from the capsid exterior, along a fivefold axis, the Cp149 atomic model (PDB entry 1QGT, gray) fits into cryo-EM density of (A, red) Cp183_e-SSS and (B, green) Cp183_e-EEE. The last visible residue of the crystal structure (T142) is close to the CTD difference density. When the CTD density of both Cp183 forms are overlaid (C, shows the superimposition side views of A and B), the movement of the peptide and the increased degree of interaction in Cp183_e-EEE is immediately obvious, implying that the EEE mutation modulates a subunit-subunit interaction.</p

Cryo-EM 3D reconstructions of empty and pgRNA-filled Cp183 capsids.

<p>Surface shaded exterior maps of T = 4 (A) Cp183_e-SSS, (B) Cp183_e-EEE, (C) Cp183_RNA-SSS, (D) Cp183_RNA-EEE and their related central sections (E–H). Insets show enlarged views of the twofold (i.e. quasi-sixfold) vertex. All four maps have a similar external appearance with 120 spikes decorating a fenestrated capsid surface; the outer layer extends from a radius of 125 to 170 Å. In (A) Cp183_e-SSS and (B) Cp183_e-EEE, a thin layer of electron density partially occludes the central opening pore at the twofold axis (A, B, E, F, black arrows). This density is unique to the empty capsids. The pgRNA-filled capsids (C, D, G, H), on the other hand, lack the density across the twofold pore but display a substantial internal layer of density at the radii between 100–120 Å. In central sections (G, H), this density, corresponding to the co-assembled pgRNA, is clearly inhomogeneous indicating that the pgRNA has adopted a preferred conformation or constellation of conformations evident even though it has been icosahedrally averaged in these reconstructions. White arrows indicate the CTD tails tethered from the capsid inner surface. Oval, triangle, and pentagon indicate locations of twofold, threefold and fivefold axes, respectively.</p

Structural Organization of Pregenomic RNA and the Carboxy-Terminal Domain of the Capsid Protein of Hepatitis B Virus

<div><p>The Hepatitis B Virus (HBV) double-stranded DNA genome is reverse transcribed from its RNA pregenome (pgRNA) within the virus core (or capsid). Phosphorylation of the arginine-rich carboxy-terminal domain (CTD) of the HBV capsid protein (Cp183) is essential for pgRNA encapsidation and reverse transcription. However, the structure of the CTD remains poorly defined. Here we report sub-nanometer resolution cryo-EM structures of <em>in vitro</em> assembled empty and pgRNA-filled Cp183 capsids in unphosphorylated and phosphorylation-mimic states. In empty capsids, we found unexpected evidence of surface accessible CTD density partially occluding pores in the capsid surface. We also observed that CTD organization changed substantively as a function of phosphorylation. In RNA-filled capsids, unphosphorylated CTDs favored thick ropes of RNA, while the phosphorylation-mimic favored a mesh of thin, high-density strands suggestive of single stranded RNA. These results demonstrate that the CTD can regulate nucleic acid structure, supporting the hypothesis that the HBV capsid has a functional role as a nucleic acid chaperone.</p> </div

Structural organization of pgRNA.

<p>The difference maps of the pgRNA from (A) Cp183_RNA-SSS (blue) and (B) Cp183_RNA-EEE (gold) superimposed on cutaway of their respective empty capsids. By subtracting the empty Cp183 capsids from their respective pgRNA-filled capsids, the CTD-RNA interaction is evident as a gap, particularly evident on the fivefold of the EEE mutant (B). The pgRNA density in the unphosphorylated Cp183_RNA-SSS forms an icosahedral cage, similar to the organization of rcDNA observed in the native virion. The pgRNA in the phosphorylation-mimic Cp183-EEE capsid shows a continuous mesh-like network density that appears to be composed of short segments of density.</p

Cryo-micrographs of frozen-hydrated HBV capsids.

<p>(A) Cp183_e-SSS (e for empty), (B) Cp183_e-EEE, (C) Cp183_RNA-SSS, and (D) Cp183_RNA-EEE particles are shown, frozen hydrated in vitreous ice. These particles show the typical morphology of HBV capsids with characteristic spikes. These samples all have a minor population of smaller, T = 3 particles (black arrow). Inserts show translationally averaged images. Empty capsids (A, B) show a single ring corresponding to the protein shell; pgRNA-filled capsids (C, D) show two concentric rings, indicating the presence of an layer of nucleic acid. Note that the RNA ring in Cp183_RNA-EEE is thicker than in Cp183_RNA-SSS.</p

An assembly schema for HBV Cp183 capsids.

<p>Assembly of Cp183 dimers in the absence of RNA results in empty capsids with CTDs transiently exposed through the pores on the icosahedral twofold axes. Co-assembly of Cp183 dimers with pgRNA results in RNA-filled capsids where the RNA structure is responsive to the phosphorylation states of the CTD.</p

Spatial organization of the CTDs.

<p>Difference maps of CTD density were calculated by subtracting the low-pass filtered atomic model of Cp149 from the (A) Cp183_e-SSS and (B) Cp183_e-EEE. The resulting CTD density (red and green, respectively) was superimposed on the corresponding region of the interior of the Cp149 capsid. The bottom panels shows the enlarged views at the (C,E) threefold and (D,F) fivefold axes. The overall distributions of the CTDs in Cp183_e-SSS and Cp183_e-EEE are very similar except that the CTD density in Cp183_e-SSS is more scattered whereas the CTD density in Cp183_e-EEE forms a funnel-like shape under the fivefold vertex.</p

Interaction between the HBV capsid and pgRNA.

<p>A radially color-coded isosurface rendering of AB dimers and related pgRNA of (A) Cp183_RNA-SSS and (B) Cp183_RNA-EEE viewed from a 90° rotation of the region identified by an arrow in the rightmost inset. The Cp183_RNA-EEE forms a massive pentagonal density under the fivefold vertex and correlating with a thicker layer of pgRNA than seen with the Cp183_RNA-SSS reconstruction. Panels of radially cued densities of (C) Cp183_RNA-SSS and (D) Cp183_RNA-EEE, viewed along an icosahedral twofold axis at radii of 167, 153, 144, 126, 113 Å. In these images, the protein is presented in white with the high-to-low densities indicated by the gray scale. The density distribution patterns corresponding to capsid are very similar (three leftmost elements). CTDs are expected to be dominant features through radii of 117 to at 128 Å. At 126 Å the high-density features at CD dimer in Cp183_RNA-SSS are tilted toward to the threefold axis, but the related density in Cp183_RNA-EEE remains at the dimer position. Notably, the CTD density correlating with the A subunit is much weaker in the Cp183_RNA-SSS, whereas Cp183_RNA-EEE shows a strong propeller of density along the fivefold axes. At lower radius, pgRNA density shows distinct distributions. In the Cp183_RNA-SSS, the density is strongest along the twofold edge connecting fivefold axis, which forms an icosahedral cage. In the Cp183_RNA-EEE the density, while thinner than in Cp183_RNA-SSS, forms a more evenly distributed sphere.</p

Monitoring Assembly of Virus Capsids with Nanofluidic Devices

Virus assembly is a coordinated process in which typically hundreds of subunits react to form complex, symmetric particles. We use resistive-pulse sensing to characterize the assembly of hepatitis B virus core protein dimers into <i>T</i> = 3 and <i>T</i> = 4 icosahedral capsids. This technique counts and sizes intermediates and capsids in real time, with single-particle sensitivity, and at biologically relevant concentrations. Other methods are not able to produce comparable real-time, single-particle observations of assembly reactions below, near, and above the pseudocritical dimer concentration, at which the dimer and capsid concentrations are approximately equal. Assembly reactions across a range of dimer concentrations reveal three distinct patterns. At dimer concentrations as low as 50 nM, well below the pseudocritical dimer concentration of 0.5 μM, we observe a switch in the ratio of <i>T</i> = 3 to <i>T</i> = 4 capsids, which increases with decreasing dimer concentration. Far above the pseudocritical dimer concentration, kinetically trapped, incomplete <i>T</i> = 4 particles assemble rapidly, then slowly anneal into <i>T</i> = 4 capsids. At all dimer concentrations tested, <i>T</i> = 3 capsids form more rapidly than <i>T</i> = 4 capsids, suggesting distinct pathways for the two forms