Effect of capsid confinement on the chromatin organization of the SV40 minichromosome

Abstract

Using small-angle X-ray scattering, we determined the three-dimensional packing architecture of the minichromosome confined within the SV40 virus. In solution, the minichromosome, composed of closed circular dsDNA complexed in nucleosomes, was shown to be structurally similar to cellular chromatin. In contrast, we find a unique organization of the nanometrically encapsidated chromatin, whereby minichromosomal density is somewhat higher at the center of the capsid and decreases towards the walls. This organization is in excellent agreement with a coarse-grained computer model, accounting for tethered nucleosomal interactions under viral capsid confinement. With analogy to confined liquid crystals, but contrary to the solenoid structure of cellular chromatin, our simulations indicate that the nucleosomes within the capsid lack orientational order. Nucleosomes in the layer adjacent to the capsid wall, however, align with the boundary, thereby inducing a 'molten droplet' state of the chromatin. These findings indicate that nucleosomal interactions suffice to predict the genome organization in polyomavirus capsids and underscore the adaptable nature of the eukaryotic chromatin architecture to nanoscale confinement.

Figure 1.

Radially integrated solution SAXS intensities (symbols) versus the magnitude of the momentum transfer vector q of (a) wt SV40, (b) empty capsid (c) VLPs containing dsDNA (DNA-VLPs) (d) wt SV40 T = 7 model. For (a–c), the solid red curves are the best fitted form-factor models of multiple spherical shells with a smoothly varying radial ED profiles, represented by hyperbolic tangent functions (Equation 1) and shown as insets to each plot. For (d), the solid red curve is the best fitted T = 7 form-factor model (Equation 2). The average radial ED is shown as insets. The dashed blue curves show the assumed power-law background functions (see text and Tables 1 and 2). The best fitted parameters of the three models (a–c) are presented in Table 1 and for T = 7 form-factor model (d) in Table 2. The R² values for the models of wt SV40, empty capsid, DNA-VLPs and wt SV40 with the T = 7 model are 0.997, 0.979, 0.999, 0.996, respectively. (e) Representation of the fitted geometrical T = 7 model for wtSV40 (Equation 2). The red core represents the ED of the confined minichromosome, and the blue spheres represent the 72 VP1 pentamers building the virus capsid. The transparent spheres enable to view the capsid interior. Doted spheres mark the pentamer vertices of the icosahedron. The blue, yellow, red and green bars correspond to inter-pentamer distances of 9.0, 10.3, 9.7 and 8.5 nm, respectively, as the best fitted parameters establish. The SAXS data were measured at the ID02 beamline at the ESRF synchrotron (Grenoble). The experimental q range is q = 0.07556–4.42314 nm⁻¹.

Figure 2.

Chromatin local density d(r) in simulations as would be observed along the radius of the capsid. (a) For three models at λ = 1.75: Blue curve, FM; Green, in the absence of tethering (NT); Red in the absence of tethering and lacking attractive forces to the boundary (NT-NAB). Also shown are the best model fit for the SAXS data for SV40 (taken from the inset to Figure 1d), normalized to the FM density at low r (orange dashed line); the spherically averaged density profile of SV40 measured by Cryo-EM adapted from Shen et al. (48) and normalized to FM density at low r (magenta dashed line). (b) d(r) at λ = 1.75 for green, NT model; Black, strong tethering well (minimal sliding, TM-1 nm); Red, weaker tethering (less sliding, TM-10 nm); Navy, weak tethering (maximal sliding, TM-35 nm); Blue, the FM. See text for additional details.

Figure 3.

Nucleosomal order in simulations. (a) Order parameter S versus λ (associated with the concentration of salt) at a constant density of ρ_Nuc ≈ 0.6. Navy is for PBC N = 100; Black, PBC N = 20; Red, NT-NAB model; Green, NT model; Blue, FM. Top right insets show snapshots of the system from two different perspectives for the FM at λ = 0.25, taken from simulations conditions marked in plots by blue square. The highlighted area marks the range 1.5 < λ < 1.75, corresponding to the range of physiological salt concentrations. Dashed lines mark the average S for 100 or 20 randomly oriented ellipsoids. Black and orange asterisks mark the critical point for two consecutive transitions (isotropic-like to nematic-like and nematic-like to columnar-like, respectively), calculated via the derivative of S. (b) Snapshots of 100 ellipsoids under bulk-like conditions (i.e. under PBC) at constant density, ρ_Nuc ≈ 0.6. Left, snapshot of the system for λ = 0.1, taken from simulations conditions marked in plots by navy square. Right, snapshot of the system for λ = 1 with additional snapshot of a slice through the center in the xy plane. All snapshots are oriented so that the eigenvector of the highest order parameter is defined as .

Figure 4.

Nucleosome orientational order along the capsid radius from simulations. Full dotted lines follow the radial order parameter Σ(r) versus distance from the boundary, for the FM. Short straight lines indicate the average order parameter (S) for all the ellipsoids that are located away from the boundary (r < 1.5σ), shown for λ = 1.75 (black) and λ = 0.75 (red). Inset shows snapshots for the FM at λ = 1.75 (left) and λ = 0.75 (right). Snapshots are oriented with the eigenvector of the highest order parameter defined as .