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. 2016 Nov;12(42):5862-5872.
doi: 10.1002/smll.201601712. Epub 2016 Sep 16.

Virus Matryoshka: A Bacteriophage Particle-Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus

Pooja Saxena  1 , Li He  2 , Andrey Malyutin  1 , Siddhartha A K Datta  3 , Alan Rein  3 , Kevin M Bond  1 , Martin F Jarrold  1 , Alessandro Spilotros  4 , Dmitri Svergun  4 , Trevor Douglas  1 , Bogdan Dragnea  1
Affiliations

Virus Matryoshka: A Bacteriophage Particle-Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus

Pooja Saxena et al. Small. 2016 Nov.

Abstract

Immature human immunodeficiency virus type 1 (HIV-1) is approximately spherical, but is constructed from a hexagonal lattice of the Gag protein. As a hexagonal lattice is necessarily flat, the local symmetry cannot be maintained throughout the structure. This geometrical frustration presumably results in bending stress. In natural particles, the stress is relieved by incorporation of packing defects, but the magnitude of this stress and its significance for the particles is not known. In order to control this stress, we have now assembled the Gag protein on a quasi-spherical template derived from bacteriophage P22. This template is monodisperse in size and electron-transparent, enabling the use of cryo-electron microscopy in structural studies. These templated assemblies are far less polydisperse than any previously described virus-like particles (and, while constructed according to the same lattice as natural particles, contain almost no packing defects). This system gives us the ability to study the relationship between packing defects, curvature and elastic energy, and thermodynamic stability. As Gag is bound to the P22 template by single-stranded DNA, treatment of the particles with DNase enabled us to determine the intrinsic radius of curvature of a Gag lattice, unconstrained by DNA or a template. We found that this intrinsic radius is far larger than that of a virion or P22-templated particle. We conclude that Gag is under elastic strain in a particle; this has important implications for the kinetics of shell growth, the stability of the shell, and the type of defects it will assume as it grows.

Keywords: HIV; self-assembly; strain; virus shells.

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Figures

Figure 1.
Figure 1.. Schematic representation of template-directed assembly of ΔMA-Gag.
(a) ΔMA-Gag molecules assembled on the surface of a P22 procapsid functionalized with strands of ssDNA (shown as yellow zig-zag lines). ΔMA-Gag domains have been drawn to scale using size estimates from domain structures published from cryo-EM studies of ΔMA-Gag (flexible spacer peptides not shown). Scale bar = 10 nm. (b) Schematic of ΔMA-Gag drawn to scale. Scale bar = 10 nm. (c) Schematic depicting the top view of a ΔMA-Gag hexagonal lattice.
Figure 2.
Figure 2.. SAXS analysis of P22-ΔMA-Gag.
(a) Scattering patterns of P22 VLPs (blue dots), a fit from its ab initio model (red curve) and the intensity calculated for the published atomic model (dashed line; PDB: 2XYY). The inset shows the two models superimposed: transparent beads represent the ab initio model and spacefilling colors represent the atomic model. (b) Scattering from P22-ΔMA-Gag VLPs (blue dots), a fit from the ab initio model (red curve) and the best-fit model for scattering computed for polydisperse hollow spheres (dashed line). The inset shows two orthogonal orientations of the ab initio model.
Figure 3.
Figure 3.. Cryo-EM of P22-ΔMA-Gag.
(a) VLPs imaged by cryo-EM at a magnification of 40000x. Scale bar = 100 nm. (b) A histogram of the size distribution of diameters measured from 55 P22-ΔMA-Gag particles.
Figure 4.
Figure 4.. Fourier Transform of Gag intensity in P22-ΔMA-Gag.
The average FFT wave (shown in red) of the intensity profiles obtained for the 10 randomly picked P22-ΔMA-Gag particles at a radius of 405 Å from the particle center. Inset: Representative micrographs, and rectangular strips generated for particles by converting the micrograph image into polar coordinates using Spider:PO. An inner radius of 2 pixels (6 Å) and an outer radius of 200 pixels (600 Å) from the center (0,0) was selected.
Figure 5.
Figure 5.. Radial density profile of P22-ΔMA-Gag.
(a) An average image obtained by superposing cryo-electron micrographs of 260 P22-ΔMA-Gag particles using EMAN1. Scale bar = 100 nm. (b) Radial profile for P22-ΔMA-Gag (red) obtained from averaging the gray intensity profiles of 10 individual P22-ΔMA-Gag particles. The horizontal axis shows distance from the particle center in Å.
Figure 6.
Figure 6.. Effect of ssDNA polarity on assembly of P22-ΔMA-Gag.
Negatively-stained images of representative particles from samples of P22-ΔMA-Gag assembled on templates with (a) inward ssDNA strands and (b) outward ssDNA strands. (c) Box plots of the diameters measured for >50 particles in each set.
Figure 7.
Figure 7.. Nuclease-treatment of P22-ΔMA-Gag.
Representative images of P22-ΔMA-Gag particles (a) before and (b) after digestion with nuclease. (c) Dots: Continuous arcs selected from the TEM image of digested P22-ΔMA-Gag (shown in (b)) fit using polynomial curves (red). Curvature was then calculated at each interpolation point based on polynomial fit coefficients. (d) Histogram of radii of curvature (K) of arcs selected from particles before (blue) and after (orange) nuclease digestion.
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