Molecular Dynamics of Viral Capsid Assembly and Stability

Introduction

Viral capsids are protein shells that encapsulate the viral genome, playing essential roles in genome protection, host cell recognition, and entry [1, 2]. The assembly of a capsid from individual capsid protein (CP) subunits into a highly ordered, often icosahedral or helical lattice is a complex process governed by weak non-covalent interactions [3, 4]. Understanding the molecular dynamics underlying capsid assembly and stability is critical for developing antiviral strategies and designing virus-like particles (VLPs) for vaccine and therapeutic applications [5, 6]. Computational virology, particularly molecular dynamics (MD) simulations, has become indispensable for probing these processes at atomic and near-atomic resolution [7, 8].

This article reviews the principles and applications of MD simulations in the study of viral capsid assembly and stability, with emphasis on veterinary-relevant viruses. We discuss coarse-grained (CG) and all-atom (AA) modeling approaches, the simulation of capsid maturation, mechanical stability analyses, and the use of MD in drug discovery targeting capsid assembly. The discussion is grounded in the biophysical and structural literature, drawing on studies of hepatitis B virus (HBV), human immunodeficiency virus (HIV), satellite tobacco mosaic virus (STMV), picornaviruses, and others.

Coarse-Grained Simulations of Capsid Assembly

The sheer size of a viral capsid (often 20–100 nm in diameter) precludes routine all-atom simulation of the entire assembly process. Coarse-grained (CG) models reduce computational cost by grouping several atoms into single interaction sites, enabling simulations of assembly pathways and stability at length scales up to entire virions [3, 9]. The CG approach has been applied to study the self-assembly of icosahedral capsids, including the roles of capsid protein pentamers and hexamers in forming the closed shell [10, 11]. For example, CG models of HBV core protein dimers have elucidated how assembly-promoting antivirals (APAs) alter the free energy landscape of capsid formation [4, 12]. Similarly, CG simulations of the HIV-1 capsid have revealed how protein-protein interactions at the hexamer-hexamer and hexamer-pentamer interfaces dictate the fullerene cone morphology [3, 13]. A key advantage of CG models is the ability to simulate large assemblies (e.g., virus-like particles) on desktop computers using efficient solvent representations such as supra-CG solvation [9].

Table 1 summarizes representative CG and AA simulation studies of viral capsids.

Table 1. Representative molecular dynamics studies of viral capsid assembly and stability.

All-Atom Simulations and Capsid Maturation

All-atom MD simulations provide the highest level of chemical detail, capturing hydrogen bonding, side chain packing, and water-mediated interactions that govern capsid stability [17, 13]. The advent of massively parallel computing and optimized MD codes (e.g., NAMD, GROMACS) has enabled simulations of entire capsids (e.g., HIV-1 core, STMV) at atomic resolution for microsecond timescales [7, 13]. All-atom simulations have been instrumental in characterizing capsid maturation – the structural rearrangement from a non-infectious precursor to the mature, infectious particle. For HIV-1, maturation involves cleavage of the Gag polyprotein by the viral protease, leading to disassembly of the immature hexagonal lattice and reassembly into the mature fullerene cone [5, 18]. MD simulations of the immature HIV-1 Gag lattice, including the CA-SP1 junction, have revealed that the maturation inhibitor bevirimat (BVM) stabilizes the SP1 six-helix bundle, thereby blocking the final CA-SP1 cleavage [18]. The host factor inositol hexakisphosphate (IP6) also facilitates SP1 bundle formation, and simulation studies have shown that BVM and IP6 binding are non-cooperative [18].

All-atom simulations have also been used to explore the effects of mutations on capsid stability. For example, the HIV-1 E45A mutation stabilizes the capsid via unexpected inter-hexamer NTD-NTD interactions, while P38A destabilizes the hexamer by loosening protomer interactions [5]. Second-site mutations such as R132T and T216I rescue infectivity by restoring lattice flexibility and electrostatic balance [5]. Similarly, MD studies of the HIV-1 CTD dimerization domain have shown that mutations in the major homology region (MHR) allosterically destabilize the dimeric interface, even though the MHR is distant from the contact surface [14]. These findings underscore the importance of long-range allosteric communication within the capsid lattice.

Mechanical Stability and Force-Induced Uncoating

The mechanical properties of viral capsids are critical for surviving environmental stresses and for the controlled release of the genome during infection [19, 20]. Steered MD (SMD) simulations and atomic force microscopy (AFM) experiments have been combined to probe the directional mechanical stability of capsid components. A notable example is the bacteriophage φ29 motor’s three-way junction (3WJ) pRNA, which must withstand enormous strain during DNA packaging. SMD simulations revealed that the 3WJ exhibits extraordinary rigidity along the portal axis due to two Mg²⁺ clamps, while being flexible in transverse directions [19]. This anisotropic mechanical stability is essential for its biological function.

For viral capsids, MD simulations have been used to simulate nanoindentation and evaluate the Young’s modulus and failure modes. Such studies have been performed on HBV capsids, picornaviruses, and plant viruses, providing insights into how capsid protein interactions and lattice geometry contribute to overall virion robustness [17, 21, 20]. The directional stability of capsids is also relevant to disinfection and inactivation. For coxsackievirus B5, amino acid substitutions at the C-terminus of VP1 were found to reduce heat sensitivity by improving molecular packing, as revealed by cryo-electron microscopy and MD simulations [6]. These findings have implications for understanding the thermal stability of vaccine formulations and for predicting the persistence of viral pathogens in the environment [22, 23].

Computational Drug Targeting of Capsid Assembly

Given the essential role of capsid assembly in the viral life cycle, many antiviral compounds have been designed to modulate this process. MD simulations serve as a powerful tool for rational drug design by revealing the binding modes of small molecules and their effects on capsid dynamics [1, 17, 24]. For HBV, capsid assembly modulators (CAMs) bind to a hydrophobic pocket at the dimer-dimer interface, altering the kinetics of assembly and resulting in either empty capsids or aberrant structures [1, 25]. All-atom MD simulations of the piperazine-thioureidobenzamide derivative 35a showed that it retains critical hydrogen bonds with Trp-102, Thr-128, and Leu-140 while forming new contacts with Ser-106, Thr-142, and Asn-136 [1]. These interactions stabilize a conformation that promotes mis-assembly.

Similarly, molecular docking and MD simulations have been used to screen compounds against the HBV core protein, including benzoylated emodin derivatives [10] and novel inhibitors identified by high-throughput virtual screening [24]. For HIV-1, the capsid protein is a validated target for inhibitors such as PF74 and CPSF6, which bind to the NTD and disrupt interactions with host factors like Nup153 and CPSF6 [5, 26]. MD simulations of the HIV-1 capsid lattice have shown that drug binding can allosterically modulate pore size, ion permeability, and electrostatic potential [5]. More recently, the role of the central pore of HIV-1 capsomers in maintaining capsid stability has been investigated using combined experimental and computational approaches [27, 28, 29].

Workflow for Simulating Capsid Assembly and Stability

The following Mermaid diagram illustrates a typical computational workflow for studying capsid assembly and stability using multiscale modeling.

flowchart TD
    A[Structural Data: Cryo-EM, X-ray, NMR], > B[Model Building: All-atom or Coarse-grained]
    B, > C{Scale}
    C, >|Coarse-grained| D[CG Simulation of Assembly Pathways]
    C, >|All-atom| E[AA Simulation of Capsid Maturation]
    D, > F[Analysis of Free Energy Landscapes]
    E, > G[Mechanical Stability: SMD, Nanoindentation]
    F, > H[Validation with Mutagenesis & AFM]
    G, > H
    H, > I[Drug Design: Virtual Screening & MD Refinement]
    I, > J[Lead Compound Optimization]

Case Studies in Veterinary Virology

Although many MD studies have focused on human pathogens, the principles are directly transferable to veterinary viruses. The infectious bursal disease virus (IBDV) VP3 protein is a multifunctional scaffold that stabilizes the capsid. MD simulations combined with SAXS and X-ray crystallography revealed that the N-terminal domain (D1) of VP3 drives dimerization, and its deletion severely impairs capsid assembly and viral infectivity in chickens [15]. This work highlights the utility of MD in uncovering critical assembly determinants in avian pathogens.

Porcine circovirus type 2 (PCV2) VLPs are used as vaccines, but stability issues limit their biotechnological applications. CG and all-atom simulations have been employed to assess the effects of pH and temperature on PCV2 VLP integrity, guiding formulation improvements [16]. Similarly, MD studies of dengue virus and other flaviviruses have provided insights into the conformational changes of the capsid protein during RNA encapsulation, which could inform the design of assembly inhibitors for veterinary use [30].

The use of ankyrin repeat proteins (DARPins) as intracellular antivirals against HIV-1 has also been explored computationally. MD simulations of the Ank1D4-NTD CA complex showed that the S45Y mutation in the DARPin improves binding free energy, providing a template for designing antiviral proteins against retroviruses that affect animals, such as the Jembrana disease virus or equine infectious anemia virus [31].

Conclusion

Molecular dynamics simulations have become an essential tool for dissecting the assembly, maturation, and stability of viral capsids at the molecular level. Coarse-grained models enable the study of large-scale assembly pathways, while all-atom simulations provide the chemical detail required to understand drug binding and mutation effects. The integration of MD with structural biology and biophysical assays has led to significant insights into capsid mechanics and allosteric regulation. These approaches are increasingly applied to veterinary viruses, with the potential to accelerate the development of vaccines, antivirals, and stable VLPs for animal health.

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