Off-Pathway Assembly: A Broad-Spectrum Mechanism of Action for Drugs That Undermine Controlled HIV-1 Viral Capsid Formation

Abstract

The early and late stages of human immunodeficiency virus (HIV) replication are orchestrated by the capsid (CA) protein, which self-assembles into a conical protein shell during viral maturation. Small molecule drugs known as capsid inhibitors (CIs) impede the highly regulated activity of CA. Intriguingly, a few CIs, such as PF-3450074 (PF74) and GS-CA1, exhibit effects at multiple stages of the viral lifecycle at effective concentrations in the pM to nM regimes, while the majority of CIs target a single stage of the viral lifecycle and are effective at nM to μM concentrations. In this work, we use coarse-grained molecular dynamics simulations to elucidate the molecular mechanisms that enable CIs to have such curious broad-spectrum activity. Our quantitatively analyzed findings show that CIs can have a profound impact on the hierarchical self-assembly of CA by perturbing populations of small CA oligomers. The self-assembly process is accelerated by the emergence of alternative assembly pathways that favor the rapid incorporation of CA pentamers, and leads to increased structural pleomorphism in mature capsids. Two relevant phenotypes are observed: (1) eccentric capsid formation that may fail to encase the viral genome and (2) rapid disassembly of the capsid, which express at late and early stages of infection, respectively. Finally, our study emphasizes the importance of adopting a dynamical perspective on inhibitory mechanisms and provides a basis for the design of future therapeutics that are effective at low stoichiometric ratios of drug to protein.

Figure 1

Schematic of the hierarchical process that is central to HIV-1 mature capsid assembly. Within each oligomeric state of N monomers (N-mer), a variety of configurations are possible. The canonical assembly pathway relies on constant self-correction across N-mer states, which is contingent on a dynamic and broad population of small N-mer intermediates. In this work, we effectively introduce the presence of capsid inhibitor (CI) drugs using a small but fixed population of trimers of dimers, a 6-mer with up to three bound CIs (one at each dimer–dimer interface), thereby perturbing the natural dynamics of the assembly process. Snapshots of the final structures from 12 CG-MD simulations are depicted, from which a majority population of eccentric or malformed capsids can be seen. Here, eccentric (canonical) end-points refer to structures with regions of densely accumulated pentamers and defective hexamers (broadly distributed pentamers) while malformed assemblies are non-enclosed and semi-amorphous structures.

Figure 2

Topology of the assembled CA lattice is assessed by the eccentricity (λ) of each vertex (i.e., each CA monomer) in a graph representation of the lattice. We show the distribution of λ (P(λ)) at select points during one trajectory of [CA⁺]_CI = 0.100 mM for demonstrative purposes: at (a) 1 × 10⁸, (b) 7 × 10⁸, and (c) 14 × 10⁸ MD timesteps. The same analysis is performed in (d) using half of the lattice from (c), i.e., after cleavage by a plane perpendicular to the long axis of the capsid. Representative lattices are depicted as insets in (a-d) with each monomer shown as a sphere colored by its λ from red (λ = 0.5) to blue (λ = 1.0). The topology of the lattice can be qualitatively characterized by the skew of the distribution of λ (γ(λ)) in which (a,d) zero skew is indicative of an open lattice with isotropic edges, (b) negative skew is indicative of an open anisotropic lattice, and (c) positive skew is indicative of a closed, non-spherical lattice.

Figure 3

Assembly time-series plots that depict (a) the number of assembled hexamers, (b) the number of assembled pentamers, (c) the number of incorporated inhibitor-bound TODs (TOD_CI), and the (d) skew (γ(λ)) and (e) kurtosis (κ(λ)) of the distribution of eccentricities (λ) throughout the assembled lattice as a function of CG MD time step (shifted with respect to the onset of lattice growth) for each system within the listed concentration of capsid inhibitors (CIs). We find accelerated assembly, especially with respect to pentamers, in the CI-present simulations, which appear to be commensurate with negative γ(λ) and κ(λ), i.e., a descriptor that indicates anisotropic edge growth in the protein lattice. The shaded region depicts the standard deviation around the mean from four trajectories within the listed range of concentrations while the solid line depicts a single trajectory. In (f), molecular snapshots of the protein lattice for the 0.100 mM case (green line in (a)–(e)) at the listed MD time step (τ [×10⁶]) are shown; green, red, and blue NTD domains in capsomers indicate hexamers, pentamers, and incomplete capsomers, respectively.

Figure 4

(a) Schematic of the canonical assembly pathway (top) through the dual association of CA dimers and accelerated assembly pathway (bottom) through the association of a trimer of dimers (TOD) that is stabilized by capsid inhibitors (CIs) and a CA dimer. The stars represent potential binding interfaces; yellow stars indicate the need for an adjacent CA dimer to associate nearly simultaneously, while red stars indicate sites that are energetically satisfied by a single CA dimer. The topological character of an assembling lattice over time is quantified by the skew of its eccentricities (γ(λ)) with distributions calculated from a swarm of 50 trajectories in the (b) absence and (c) presence of CIs. Here, γ(λ) ≈ 0 is suggestive of an open isotropic lattice, γ(λ) < 0 is suggestive of an open anisotropic lattice, and γ(λ) > 0 is suggestive of a uniform and oblong lattice (such as in closed mature capsids). These results suggest the preference of isotropic (emergence of anisotropic) lattice growth during canonical (CI-perturbed) assembly, which is highlighted in the region bound by the dashed red box. Each trajectory was discretized into 20 equal bins before histograms were computed (n = 375 per histogram).

Figure 5

(a) Disassembly time-series plot that depicts the fraction of remaining hexamers and pentamers (with respect to the initial size of the mature capsid core) for the five listed systems as a function of MD time step. All four of the eccentric cases exhibit spontaneous disassembly, which appear to be initiated at sites with large pentamer density. Molecular snapshots of the protein lattice for the 0.075 mM case (cyan line in (a)) at the indicated points are shown; green, red, and blue capsomers indicate hexamers, pentamers, and incomplete capsomers, respectively. (b) Molecular snapshots from each trajectory at the indicated MD time step (τ [×10⁶]) are shown. The inhibitor-bound TODs are represented as gray capsomers. This data suggests that capsid inhibitors promote the formation of “leaky” mature cores that spontaneously open at regions of high curvature (or high pentamer density), and represents one failure mechanism that suppresses infectivity.