Coarse-grained simulation reveals key features of HIV-1 capsid self-assembly

Abstract

The maturation of HIV-1 viral particles is essential for viral infectivity. During maturation, many copies of the capsid protein (CA) self-assemble into a capsid shell to enclose the viral RNA. The mechanistic details of the initiation and early stages of capsid assembly remain to be delineated. We present coarse-grained simulations of capsid assembly under various conditions, considering not only capsid lattice self-assembly but also the potential disassembly of capsid upon delivery to the cytoplasm of a target cell. The effects of CA concentration, molecular crowding, and the conformational variability of CA are described, with results indicating that capsid nucleation and growth is a multi-stage process requiring well-defined metastable intermediates. Generation of the mature capsid lattice is sensitive to local conditions, with relatively subtle changes in CA concentration and molecular crowding influencing self-assembly and the ensemble of structural morphologies.

Figure 1. The fullerene cone model of the HIV-1 capsid is assembled from ‘mature-style' capsid lattice with hexameric (green) and pentameric (red) building blocks.

(a) CA dimer structure with both CG (tube) and all-atom (ribbon) monomer representations (NTDs green and CTDs grey). CTD dimer interface marked by dashed line; scale bar, 5 nm. (b) Quasi-equivalent pentamer and hexamer capsid inclusions (NTDs distinguished by colour, all CTDs grey), with schematic of adjacent packing in capsid lattice. (c) Mature capsid structure after Pornillos et al.. NTDs of hexamer-associated CA shown in green, with NTDs of pentamer-associated CA shown in red (all CTDs grey); scale bar, 20 nm.

Figure 2. Dimers of the CA protein are the units of self-assembly of HIV-1 capsid-like CG structures.

(a) Simulation snapshot of a population of metastable trimer-of-dimers. CA NTDs are blue, with CTDs grey (non-aggregated CA shown transparent for clarity). (b) Detail of a CG trimer-of-dimers, where ‘edges' are CA dimers, with two NTDs per triangle ‘vertex'. NTDs coloured by dimer for clarity, scale bar 10 nm. Final simulation snapshots for ρ_CR=200 mg ml⁻¹ and [CA]=2 mM (c), 3 mM (d) and 4 mM (e) are presented with NTDs coloured by monomer presence in a trimer (blue), pentamer (red) or hexamer (green). All CTDs grey, scale bar 20 nm. (f) Multiple aggregated capsid structures as revealed by electron cryotomography, with capsid structures highlighted in orange (g). Scale bar, 20 nm; f and g adapted from ref. .

Figure 3. Only a narrow range (indicated in green) of CA concentrations results in the nucleation and growth of a single-lattice region.

CG self-assembly as a function of active CA concentration [CA]₊ under fixed crowding conditions. Colour in the concentration bar indicates formation of only trimer-of-dimers structures (blue) and the nucleation and growth of single (green) or multiple (red) lattice regions. Example final structures are shown for [CA]₊=1, 2 and 4 mM (arrows). CA colour scheme as in Fig. 2, lamellar regions highlighted by ovals. Final structure for [CA]₊=2 mM formed via two lattice regions fusing. Grey panel shows cross-sectional slices (not to scale) to illustrate lamellar lattice in structure for [CA]₊=2 mM (top, cross-sectional plane indicated by dashed white line) and an example lamellar CA lattice inside a virion from electron cryotomography (bottom, CA lattice indicated by white arrow). Final structures for [CA]₊=4 mM wrap around periodic boundaries. Scale bar, 20 nm.

Figure 4. Putative assembly from 12 CA dimers that nucleates CG capsid lattice growth.

(a) Reversible addition of CA dimers (CA monomers depicted as black triangles, CTD/CTD interface as a dashed line) onto existing aggregates produces trimers with shared edges, eventually generating a central hexamer stabilized by trimer ‘skirt'. (b) Front and side view of example structure from CG simulation, with mild innate curvature visible. Hexamer-associated NTDs are green, NTDs in trimer skirt blue, CTDs grey. Scale bar, 20 nm.

Figure 5. Example CG simulation data under fixed molecular crowding.

(a) ‘Available' assembly-competent CA in solution for initial [CA]₊=0.5 mM (resulting in no nucleation and growth of mature lattice) and initial [CA]₊=1.0 mM (producing nucleation and growth of a single-lattice region, see the main text and Table 2). Lattice growth continues below the level of available [CA]₊ required for nucleation on the same timescale. (b) Number of separate lattice regions containing key structural motifs for [CA]₊=1 mM. The number of trimers in solution (blue curve) reduces significantly as a single region of lattice grows.

Figure 6. Steps in the assembly of the HIV-1 capsid by polymerization of CA dimers for [CA]_%=10% and conformational switching interval of 5 × 10⁵ time steps (see the main text).

Simulation snapshots at 120 × 10⁶ (a), 240 × 10⁶ (b), 440 × 10⁶ (c), 460 × 10⁶ (d), 600 × 10⁶ (e) and 1,700 × 10⁶ MD time steps (f) are shown with views perpendicular and parallel to the major structural axis. Colour scheme as in Fig. 2. Scale bar, 20 nm.

Figure 7. Disassembly of self-assembled CG CA lattice under simulated rapid dilution with constant molecular crowding.

Conformational switching intervals of 1 × 10⁴ time steps (a), 1 × 10⁵ time steps (b) and 5 × 10⁵ time steps (c) are shown. Simulation snapshots (inset) correspond to the systems at ≈175 × 10⁶ (a), ≈550 × 10⁶ (b) and ≈350 × 10⁶ (c) CG MD time steps. Colour scheme as in Fig. 2.