Global structural changes in hepatitis B virus capsids induced by the assembly effector HAP1

Abstract

Hepatitis B virus (HBV) is a leading cause of liver disease and hepatocellular carcinoma; over 400 million people are chronically infected with HBV. Specific anti-HBV treatments, like most antivirals, target enzymes that are similar to host proteins. Virus capsid protein has no human homolog, making its assembly a promising but undeveloped therapeutic target. HAP1 [methyl 4-(2-chloro-4-fluorophenyl)-6-methyl-2-(pyridin-2-yl)-1,4-dihydropyrimidine-5-carboxylate], a heteroaryldihydropyrimidine, is a potent HBV capsid assembly activator and misdirector. Knowledge of the structural basis for this activity would directly benefit the development of capsid-targeting therapeutic strategies. This report details the crystal structures of icosahedral HBV capsids with and without HAP1. We show that HAP1 leads to global structural changes by movements of subunits as connected rigid bodies. The observed movements cause the fivefold vertices to protrude from the liganded capsid, the threefold vertices to open, and the quasi-sixfold vertices to flatten, explaining the effects of HAP1 on assembled capsids and on the assembly process. We have identified a likely HAP1-binding site that bridges elements of secondary structure within a capsid-bound monomer, offering explanation for assembly activation. This site also interferes with interactions between capsid proteins, leading to quaternary changes and presumably assembly misdirection. These results demonstrate the plasticity of HBV capsids and the molecular basis for a tenable antiviral strategy.

FIG. 1.

Structural effects of HAP1 are visible in the electron densities of the capsid structures. The +HAP1 density is shown as a magenta mesh, the −HAP1 density as a cyan mesh. Both are contoured at 1σ. Chain identities are indicated, as are the icosahedral fivefold, twofold, and threefold vertices. (A) Density superposed at a quasi-sixfold (icosahedral twofold) vertex reveals striations indicating movement of the CD dimers largely on the plane of the capsid surface. (B) “Side” view of the AB dimer, with the capsid surface indicated by a dashed white line, highlights the hinge at the B subunit and the upward radial movement of the A subunit.

FIG. 2.

HAP1 causes global structural changes. A surface rendering of the −HAP1 structure (A) and the +HAP1 structure (B), viewed looking down an icosahedral twofold (quasi-sixfold) axis; inset shows a fivefold vertex. Twofold and threefold symmetry axes are indicated. Note the changes in the shapes of the openings at all symmetry axes in +HAP1. A solvent radius of 3 Å was used in the calculation to produce a smoother surface. (C) A superposition of dimers from the −HAP1 (gray) and +HAP1 (colored) capsid structures. Magenta, chain A; blue, chain B; yellow, chain C; purple, chain D. Red spheres indicate the putative binding site of HAP1 on chain C. Note how the +HAP1 structures are pushed apart at this site. Residue Tyr132 is denoted for the A chains by a stick model; all other chains have Tyr132 in a quasi-equivalent position. A schematic of dimer arrangements surrounding the quasi-sixfold and threefold vertices is inset. The threefold and twofold (quasi-sixfold) symmetry operators are identified. (D) A CD dimer showing the putative HAP1 density from the averaged F_obs − F_calc density in red (15σ). The schematic is rotated 90° on the plane of the page relative to that in panel C, and the color coding is the same.

FIG. 3.

The putative HAP1-binding site is located in a hydrophobic pocket that appears central to major global movements. Only the pocket of the C subunit appears to have high occupancy (for other chains, see Fig. S6 and S7 in the supplemental material); wall-eyed stereo pairs for the −HAP1 C chain (A) and +HAP1 C chain (B) are shown, with the final averaged B-sharpened electron density map contoured at 2σ (magenta); averaged F_obs − F_calc density is also shown, contoured at 15σ in cyan. Ribbons indicate secondary structure; selected residues surrounding the pocket are labeled and represented as sticks. The view is roughly perpendicular to that shown in Fig. 2C.

FIG. 4.

A schematic representation of the global structural changes induced by HAP1. (A) A schematic of a capsid showing the global structural movement of icosahedral asymmetric units (triangles composed of an AB + CD dimer). Blue, −HAP1; red, +HAP1. Movements have been exaggerated (approximately 3×) for clarity; a yellow sphere at each C chain indicates the putative HAP1 site; the protrusion of fivefold vertices is particularly evident. (B) A close-up of an icosahedral facet shows how HAP1 changes the relationship between the icosahedral asymmetric units; symmetry operators are identified.