Entropic forces drive clustering and spatial localization of influenza A M2 during viral budding.

The influenza A matrix 2 (M2) transmembrane protein facilitates virion release from the infected host cell. In particular, M2 plays a role in the induction of membrane curvature and/or in the scission process whereby the envelope is cut upon virion release. Here we show using coarse-grained computer simulations that various M2 assembly geometries emerge due to an entropic driving force, resulting in compact clusters or linearly extended aggregates as a direct consequence of the lateral membrane stresses. Conditions under which these protein assemblies will cause the lipid membrane to curve are explored, and we predict that a critical cluster size is required for this to happen. We go on to demonstrate that under the stress conditions taking place in the cellular membrane as it undergoes large-scale membrane remodeling, the M2 protein will, in principle, be able to both contribute to curvature induction and sense curvature to line up in manifolds where local membrane line tension is high. M2 is found to exhibit linactant behavior in liquid-disordered-liquid-ordered phase-separated lipid mixtures and to be excluded from the liquid-ordered phase, in near-quantitative agreement with experimental observations. Our findings support a role for M2 in membrane remodeling during influenza viral budding both as an inducer and a sensor of membrane curvature, and they suggest a mechanism by which localization of M2 can occur as the virion assembles and releases from the host cell, independent of how the membrane curvature is produced.

Immature HIV-1 lattice assembly dynamics are regulated by scaffolding from nucleic acid and the plasma membrane.

The packaging and budding of Gag polyprotein and viral RNA is a critical step in the HIV-1 life cycle. High-resolution structures of the Gag polyprotein have revealed that the capsid (CA) and spacer peptide 1 (SP1) domains contain important interfaces for Gag self-assembly. However, the molecular details of the multimerization process, especially in the presence of RNA and the cell membrane, have remained unclear. In this work, we investigate the mechanisms that work in concert between the polyproteins, RNA, and membrane to promote immature lattice growth. We develop a coarse-grained (CG) computational model that is derived from subnanometer resolution structural data. Our simulations recapitulate contiguous and hexameric lattice assembly driven only by weak anisotropic attractions at the helical CA-SP1 junction. Importantly, analysis from CG and single-particle tracking photoactivated localization (spt-PALM) trajectories indicates that viral RNA and the membrane are critical constituents that actively promote Gag multimerization through scaffolding, while overexpression of short competitor RNA can suppress assembly. We also find that the CA amino-terminal domain imparts intrinsic curvature to the Gag lattice. As a consequence, immature lattice growth appears to be coupled to the dynamics of spontaneous membrane deformation. Our findings elucidate a simple network of interactions that regulate the early stages of HIV-1 assembly and budding.

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

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.

Early stages of the HIV-1 capsid protein lattice formation.

The early stages in the formation of the HIV-1 capsid (CA) protein lattice are investigated. The underlying coarse-grained (CG) model is parameterized directly from experimental data and examined under various native contact interaction strengths, CA dimer interfacial configurations, and local surface curvatures. The mechanism of early contiguous mature-style CA p6 lattice formation is explored, and a trimer-of-dimers structure is found to be crucial for CA lattice production. Quasi-equivalent generation of both the pentamer and hexamer components of the HIV-1 viral CA is also demonstrated, and the formation of pentamers is shown to be highly sensitive to local curvature, supporting the view that such inclusions in high-curvature regions allow closure of the viral CA surface. The complicated behavior of CA lattice self-assembly is shown to be reducible to a relatively simple function of the trimer-of-dimers behavior.

Quantitative visualization of passive transport across bilayer lipid membranes.

The ability to predict and interpret membrane permeation coefficients is of critical importance, particularly because passive transport is crucial for the effective delivery of many pharmaceutical agents to intracellular targets. We present a method for the quantitative measurement of the permeation coefficients of protonophores by using laser confocal scanning microscopy coupled to microelectrochemistry, which is amenable to precise modeling with the finite element method. The technique delivers well defined and high mass transport rates and allows rapid visualization of the entire pH distribution on both the cis and trans side of model bilayer lipid membranes (BLMs). A homologous series of carboxylic acids was investigated as probe molecules for BLMs composed of soybean phosphatidylcholine. Significantly, the permeation coefficient decreased with acyl tail length contrary to previous work and to Overton's rule. The reasons for this difference are considered, and we suggest that the applicability of Overton's rule requires re-evaluation.

Interaction between charged surfaces mediated by rodlike counterions: the influence of discrete charge distribution in the solution and on the surfaces.

The interaction between two charged surfaces, with discrete or uniform charge distributions, embedded in a solution of rodlike counterions has been studied. Monte Carlo simulations and density functional theory have been applied to study the concentration profiles of counterions and the force between the charged surfaces. We show that for low surface charge densities the repulsive force between like-charged surfaces is observed regardless of the rod length. Where high surface charge densities are present, attractive forces at surface separations related to the rod length are observed.

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

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.

Highly Scalable and Memory Efficient Ultra-Coarse-Grained Molecular Dynamics Simulations.

The use of coarse-grained (CG) models can significantly increase the time and length scales accessible to computational molecular dynamics (MD) simulations. To address very large-scale phenomena, however, requires a careful consideration of memory requirements and parallel MD load balancing in order to make efficient use of current supercomputers. In this work, a CG-MD code is introduced which is specifically designed for very large, highly parallel simulations of systems with markedly non-uniform particle distributions, such as those found in highly CG models having an implicit solvent. The CG-MD code uses an unorthodox combination of sparse data representations with a Hilbert space-filling curve (SFC) to provide dynamic topological descriptions, reduced memory overhead, and advanced load-balancing characteristics. The results of representative large-scale simulations indicate that our approach can offer significant advantages over conventional MD techniques, and should enable new classes of CG-MD systems to be investigated.

Effects of Discrete Charge Clustering in Simulations of Charged Interfaces.

A system of counterions between charged surfaces is investigated, with the surfaces represented by uniform charged planes and three different arrangements of discrete surface charges - an equispaced grid and two different clustered arrangements. The behaviors of a series of systems with identical net surface charge density are examined, with particular emphasis placed on the long ranged corrections via the method of "charged slabs" and the effects of the simulation cell size. Marked differences are observed in counterion distributions and the osmotic pressure dependent on the particular representation of the charged surfaces; the uniformly charged surfaces and equispaced grids of discrete charge behave in a broadly similar manner, but the clustered systems display a pronounced decrease in osmotic pressure as the simulation size is increased. The influence of the long ranged correction is shown to be minimal for all but the very smallest of system sizes.

Decreased osmotic pressure via interfacial charge clustering.

Behaviors of a model interfacial system featuring the clustering of discrete, mobile wall charges and a counterion solution are investigated. The results demonstrate that even very small localized charge clusters produce significant effects for the osmotic pressure, effects that are not adequately represented in common colloidal models. We observe a pronounced decrease in osmotic pressure where a certain level of clustering is attained, with potentially significant implications for theories of colloidal stability and biochemical processes at microscopic length scales. The stable level of wall charge clustering, and thus the osmotic pressure, is found to be variable in the addition of an attractive potential, as measured via the excess free energy of clustering.