Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids

Abstract

Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (<4 Å), we introduced a disulfide crosslink that rescued particle symmetry. We deduced that HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection.

Keywords: biochemistry; cryo-EM; direct acting antiviral; hepatitis B virus; image reconstruction; infectious disease; microbiology; self-assembly; virus.

Figure 1.. Synthesis of HAP-TAMRA.

HAP-TAMRA is a derivative of HAP13 (Bourne et al., 2008) with a tetramethylrodamine (TAMRA) moiety.

Figure 2.. HAP-TAMRA drives core protein assembly.

(a) A negative stain micrograph of a typical assembly reaction using purified core protein. (b) Like other HAPs, stoichiometric excess of HAP-TAMRA drives assembly of non-capsid polymers of core protein. (c) A representative size exclusion chromatograph, monitored at 555 nm to pick up TAMRA, shows that HAP-TAMRA co-elutes with capsid. (inset) Spectra of the capsid-bound HAP-TAMRA (blue) and free forms show an unusual chromatic shift, suggestive of π-stacking of TAMRA moieties in the capsid. (d) Using the absorbance shift as an indicator (either the value at 520 nm or the ratio of 520/555 nm), we find that preformed capsids saturate at a stoichiometry of about one small molecule per dimer. The x axis represents ‘effective’ probes per Cp dimer, considering that half the input probe is an inactive enantiomer. Each point is an average of three measurements with error bars showing the standard deviation. The scale bars in (a) and (b) are 100 nm.

Figure 3.. HAP-TAMRA absorbance and fluorescence change in response to binding capsid.

(a) HAP-TAMRA in solution for a matched pair of samples with 8 µM capsid (colored lines) and without (black lines). In the presence of capsid, the absorbance spectrum shifts towards one that resembles a π-stacked TAMRA dimer (Adachi et al., 2014). The change is not as dramatic as in Figure 2c, due to the presence of free dye in sample that have not been purified by SEC. (b) The change in absorbance at 520 nm can be used to observe kinetics of binding different HAP-TAMRA concentrations to 8 µM capsid, showing the time dependence of forming the samples in panel a. The kinetic traces are averages of three independent experiments. The dashed lines fit to a two-phase exponential association. The half-life for binding is between 5 and 10 min. (c) Further normalization of the kinetic curves by their maximum value gives a visual expression of the similarity of the half-lives, and reiterates that the kinetics appear to be similar across input concentrations (d) The time course in b was monitored for 350 min. Shown are the endpoint values of the change in 520 nm absorbance. The result is comparable to the one in Figure 2d, where the bound probe is isolated from the free probe. (e) Assuming the maximum amount of HAP-TAMRA binds to the protein in each condition, we can calculate the concentration of HAP-TAMRA bound. Plotting extinction coefficient for the ΔA520nm signal is constant across the titration, with a value of 11196 M⁻¹ ± 4%. (f) A demonstration that fluorescence is also quenched upon binding using 40 µM HAP-TAMRA, 8 µM Cp dimer. In the presence of capsid, we estimate that 75% of the HAP-TAMRA was unbound, accounting for most of the residual fluorescence.

Figure 4.. Cryo-microscopy shows that HAP-TAMRA distorts capsids.

(a) A cryo-micrograph shows a section of a field of Cp149 capsids treated with HAP-TAMRA. Particles appear asymmetric and have marked angles. None have the spherical cross-section typical of Cp149 capsids. (b–d) Cryo-micrographs of capsids were subject to 2D class averaging to identify their major characteristics. In each case, the classes are sorted by descending class distribution, (b) For apo-capsids of Cp149, lacking any HAP-TAMRA, all classes have a circular cross-section and a periphery of spikes. These seven classes encompass more than 80% of the Cp149 capsid particles sampled. (c) For micrographs of Cp149 particles soaked with HAP-TAMRA the most common class has an elliptical cross-section. Other classes have distinct and asymmetric faceting. The data in panels a and c are independent acquisitions of the same experimental conditions, demonstrating the repeatability of the effect. (d) The fourteen most populated classes from a micrograph of cysteine cross-linked Cp150 capsids treated with HAP-TAMRA resemble those of apo-Cp149 in panel b.

Figure 5.. Reconstruction of T = 4 and T = 3 particles.

(a) A T = 4 capsid. A single asymmetric unit is composed of two dimers, and by convention the component subunit chains are labeled with letters A,B and C,D. The two subunits that form the AB dimer (light green) and a CD dimer (dark green) are highlighted. The HAP-TAMRA molecules are highlighted (red), and bind around a quasi-sixfold (icosahedral twofold) symmetry axis. The remaining AB dimers are shown in darker grey than the CD dimers. The scale bar is 5 nm and also applies to panel (c). (b) A single quasi-sixfold cluster from a T = 4 capsid. The coloring scheme is that same as in (a) except that all HAP-TAMRA molecules at the quasi-sixfold are highlighted. (c) A T = 3 capsid with the asymmetric unit comprised of an AB dimer (light purple) and one half of a CC dimer (dark purple) highlighted. (d) As per (b) except a T = 3 quasi-sixfold has threefold symmetry and three bound HAP-TAMRA molecules. In panels (b) and (d) symmetry-related subunits are denoted by the prime or double prime symbols.

Figure 6.. Fourier shell correlation and 2D class averages of T = 4 and T = 3 particles.

(a) Fourier shell correlation for the T = 4 and T = 3 reconstruction in green and purple, respectively. The dashed line indicates a correlation of 0.143. (b) The most populated 2D class averages from the T = 4 dataset reconstructed in Figure 5a. (c) The most populated 2D class averages from the T = 3 dataset reconstructed in Figure 5c.

Figure 7.. HAP-TAMRA has a very similar structure in three different quasi-equivalent environments.

The HAP site consists of a pocket in one subunit that is capped by a neighboring subunit. The pockets of the T = 4 B and C subunits (panels a and b) and the T = 3 B subunit (c) each has unambiguous density for the HAP and linker moieties of HAP-TAMRA at a contour level of 3.6 σ. The B subunits of T = 3 and T = 4 show well defined density for the TAMRA moiety. The TAMRA density of the T = 4 C subunit is more ambiguous (b), suggesting multiple orientations. We show a fit consistent with available density, and note that the HAP and TAMRA regions become contiguous by 2.6σ contour. When the HAP pockets are compared, based on overlaying the respective protein monomers (d), we observe that the HAP and linker moieties have essentially the same conformation in all three sites. (e) The B site for the T = 4 structure, from a different perspective than shown in panel (a), showing density for protein and HAP-TAMRA (contoured a 3.6σ). Note that Y132, the lower left of this view, is in the peptide turn on the right side of panels a-d.

Figure 8.. The dimer-dimer interactions, including the HAP-pocket, are structurally conserved.

T = 3 (a,b) and T = 4 (c,d) capsids are color coded by resolution. Spike tips are the lowest resolution features at 4.5–5 Å resolution. The interdimer interfaces near the HAP pockets and hydrophobic cores (i.e. the dimer chassis subdomain) at the intradimer interfaces are the highest resolution regions of both capsids. Variation of resolution is analogous to differences in crystallographic B factor.

Figure 9.. The flexibility of interdimer geometry is demonstrated by comparison of T = 3 and T = 4 quasi-sixfold vertices.

Dimer–dimer interaction varies in response to quasi-equivalent interaction, T number, and the presence of CpAM. The difference in the geometry of interaction can be difficult to see. (a) Quasi-sixfold clusters of dimers from the T = 4 (white) and T = 3 (purple) structures are overlaid based on the leftmost dimer; in both cases this is an AB dimer. (b) The aligned dimers (left) are nearly identical. The dimer opposite shows a dramatic difference in 4° structure imposed by a series of relatively subtle differences. (c, d) Side views of T = 4 (c) and T = 3 quasi-sixfold show that their 4° structures are analogous to the boat and chair conformations, respectively, associated with 6-membered rings in organic chemistry (e.g. cyclohexane).

Figure 10.. Structural defects induced by HAP-TAMRA are concentrated at icosahedral fivefold.

(a, b) Differences between the HAP-TAMRA capsid and an apo capsid (1QGT) were calculated by overlaying the two capsids based on a common center. Residues are color coded based on the displacement of the α carbons. Though the HAP-TAMRA capsids are systematically larger than the apo capsids, the smallest displacement is at the base of the CD dimers surrounding icosahedral three-fold (triangles in panel b). The A subunits surrounding the fivefold vertices (pentagons in panel b) show the greatest displacement.

Figure 11.. A low resolution reconstruction of Cp149+HAP TAMRA shows that defects are concentrated at fivefold vertices.

(a) T = 4 reconstructions Cp149+HAP TAMRA show an absence of density on the fivefold. This suggests structural heterogeneity. Consistent with this, the reconstruction only achieved 22 Å resolution. (b) A density map based on the 1QGT molecular model down-sampled to 22 Å resolution shows the resolved features of a well-ordered fivefold. (c,d) Central slices through the experimental Cp149+HAP TAMRA map (c) and the 1QGT render (d). The effect of HAP-TAMRA was to angle the dimers surrounding the fivefold axis towards twofold axes. (e) 2D class averages of the particles collected for the reconstruction in panel a, sorted by descending class distribution demonstrate faceting and irregular morphology. (f) FSC for reconstruction of Cp149+HAP-TAMRA.