Probe capsid structure stability and dynamics of adeno-associated virus as an important viral vector for gene therapy by hydrogen-deuterium exchange-mass spectrometry.

Adeno-associated virus (AAV), a widely used gene therapy vector, is a small, nonenveloped virus that contains a single-stranded DNA genome with a maximum length of 4.7 kb. Despite extensive biophysical and structural characterization, many aspects of AAV functions remain elusive. This knowledge gap is primarily due to a lack of structurally resolved dynamic information and the absence of structural coverage of functionally critical segments on the AAV capsid. Here, we developed a protocol to study AAV structural dynamics by hydrogen-deuterium exchange mass spectrometry (HDX-MS), a powerful method for monitoring protein structure stability and dynamics in solution. We performed HDX-MS measurements on AAVs without or with different DNA payloads of different sizes, and obtained detailed dynamic information on the entire AAV sequence including the two functionally important segments not previously structurally characterized. The unique N terminus of the capsid protein VP1 (VP1u) was found to adopt a highly dynamic and unstable conformation with low HDX protection across the entire region, whereas the presence of a DNA payload increased its protection. The VP1 and VP2 shared region (VP1/2) showed no measurable protection, with or without DNA. Differential HDX between empty and full capsid samples allowed us to identify potential new DNA-capsid interaction sites located primarily around the five-fold channel, which differ from the three-fold pocket binding site previously identified. Our HDX-MS method for characterizing AAV structural dynamics opens a new way for future efforts to understand AAV structure-function relationships and engineer next-generation AAV vectors with improved gene delivery properties.

An Online Native Mass Spectrometry Approach for Fast, Sensitive, and Quantitative Assessment of Adeno-Associated Virus Capsid Content Ratios.

Adeno-associated viruses (AAVs) have emerged as a leading platform for in vivo therapeutic gene delivery and offer tremendous potential in the treatment and prevention of human disease. The fast-paced development of this growing class of therapeutics, coupled with their intrinsic structural complexity, places a high demand on analytical methods capable of efficiently monitoring product quality to ensure safety and efficacy, as well as to support manufacturing and process optimization. Importantly, the presence and relative abundance of both empty and partially filled AAV capsid subpopulations are of principal concern, as these represent the most common product-related impurities in AAV manufacturing and have a direct impact on therapeutic potential. For this reason, the capsid content, or ratio of empty and partial capsids to those packaged with the full-length therapeutic genome, has been identified by regulatory agencies as a critical quality attribute (CQA) that must be carefully controlled to meet clinical specifications. Established analytical methods for the quantitation of capsid content ratios often suffer from long turnaround times, low throughput, and high sample demands that are not well-suited to the narrow timelines and limited sample availability typical of process development. In this study, we present an integrated online native mass spectrometry platform that aims to minimize sample handling and maximize throughput and robustness for rapid and sensitive quantitation of AAV capsid content ratios. The primary advantages of this platform for AAV analysis include the ability to perform online buffer exchange under low flow conditions to maintain sample stability with minimal sample dilution, as well as the ability to achieve online charge reduction via dopant-modified desolvation gas. By exploiting the latter, enhanced spectral resolution of signals arising from empty, partial, and full AAV capsids was accomplished in the m/z domain to facilitate improved spectral interpretation and quantitation that correlated well with the industry standard analytical ultracentrifugation (AUC) method for capsid content ratio determination. The utility of this approach was further demonstrated in several applications, including the rapid and universal screening of different AAV serotypes, evaluation of capsid content for in-process samples, and the monitoring of capsid stability when subjected to thermal stress conditions.

Separation of full, empty, and partial adeno-associated virus capsids via anion-exchange chromatography with continuous recycling and accumulation.

The field of recombinant adeno-associated virus (rAAV) gene therapy has attracted increasing attention over decades. Within the ongoing challenges of rAAV manufacturing, the co-production of impurities, such as empty and partial capsids containing no or truncated transgenes, poses a significant challenge. Due to their potential impact on drug efficacy and clinical safety, it is imperative to conduct comprehensive monitoring and characterization of these impurities prior to the release of the final gene therapy product. Nevertheless, existing analytical techniques encounter notable limitations, encompassing low throughput, long turnaround times, high sample consumption, and/or complicated data analysis. Chromatography-based analytical methods are recognized for their current Good Manufacturing Practice (cGMP) alignment, high repeatability, reproducibility, low limit of detection, and rapid turnaround times. Despite these advantages, current anion exchange high pressure liquid chromatography (AEX-HPLC) methods struggle with baseline separation of partial capsids from full and empty capsids, resulting in inaccurate full-to-empty capsid ratio, as partial capsids are obscured within peaks corresponding to empty and full capsids. In this study, we present a unique analytical AEX method designed to characterize not only empty and full capsids but also partial capsids. This method utilizes continuous N-Rich chromatography with recycling between two identical AEX columns for the accumulation and isolation of partial capsids. The development process is comprehensively discussed, covering the preparation of reference materials representing full (rAAV-LacZ), partial (rAAV-GFP), and empty (rAAV-empty) capsids, N-rich method development, fraction analysis, determination of fluorescence response factors between capsid variants, and validation through comparison with other comparative techniques.

Inside the capsid: Revealing viral genome organization through multiscale simulations.

In a recent issue of Nature, Coshic et al. employ a computational multiscale approach to package the complete HK97 viral genome into its capsid. They find both good agreement with experimental observations and shed new light on the heterogeneity of genome structures and the mechanism by which they package.

Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts.

The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking, widespread across viruses and within the Totiviridae, and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two flexible opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity within the cell extract. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.

Guanidine Hydrochloride-Induced Hepatitis B Virus Capsid Disassembly Hysteresis.

Hepatitis B virus (HBV) displays remarkable self-assembly capabilities that interest the scientific community and biotechnological industries as HBV is leading to an annual mortality of up to 1 million people worldwide (especially in Africa and Southeast Asia). When the ionic strength is increased, hepatitis B virus-like particles (VLPs) can assemble from dimers of the first 149 residues of the HBV capsid protein core assembly domain (Cp149). Using solution small-angle X-ray scattering, we investigated the disassembly of the VLPs by titrating guanidine hydrochloride (GuHCl). Measurements were performed with and without 1 M NaCl, added either before or after titrating GuHCl. Fitting the scattering curves to a linear combination of atomic models of Cp149 dimer (the subunit) and T = 3 and T = 4 icosahedral capsids revealed the mass fraction of the dimer in each structure in all the titration points. Based on the mass fractions, the variation in the dimer-dimer association standard free energy was calculated as a function of added GuHCl, showing a linear relation between the interaction strength and GuHCl concentration. Using the data, we estimated the energy barriers for assembly and disassembly and the critical nucleus size for all of the assembly reactions. Extrapolating the standard free energy to [GuHCl] = 0 showed an evident hysteresis in the assembly process, manifested by differences in the dimer-dimer association standard free energy obtained for the disassembly reactions compared with the equivalent assembly reactions. Similar hysteresis was observed in the energy barriers for assembly and disassembly and the critical nucleus size. The results suggest that above 1.5 M, GuHCl disassembled the capsids by attaching to the protein and adding steric repulsion, thereby weakening the hydrophobic attraction.

Methodological Validation of Sedimentation Velocity Analytical Ultracentrifugation Method for Adeno-Associated Virus and Collaborative Calibration of System Suitability Substance.

Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term in vivo gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio. This includes specificity, accuracy, precision, linearity, and parameter variables involving the sedimentation velocity analytical ultracentrifugation (SV-AUC) method. Specifically, SV-AUC differentiated among the empty, partial, full, and high sedimentation coefficient substance (HSCS) AAV particles while evaluating their sedimentation heterogeneity. The intermediate precision analysis of HE (high percentage of empty capsid) and HF (high percentage of full capsid) samples revealed that the specific species percentage, such as empty or full, was more significant than 50%. Moreover, the relative standard deviation (RSD) could be within 5%. Even for empty or partially less than 15%, the RSD could be within 10%. The accuracy recovery rates of empty capsid were between 103.9% and 108.7% across three different mixtures. When the measured percentage of specific species was more significant than 14%, the recovery rate was between 77.9% and 106.6%. Linearity analysis revealed an excellent linear correlation between the empty, partial, and full in the HE samples. The AAV samples with as low as 7.4 × 1011 cp/mL AAV could be accurately quantified with SV-AUC. The parameter variable analyses revealed that variations in cell alignment significantly affected the overall results. Still, the detection wavelength of 235 nm slightly influenced the empty, partial, and full percentages. Minor detection wavelength changes showed no impact on the sedimentation coefficient of these species. However, the temperature affected the measured sedimentation coefficient. These results validated the SV-AUC method to quantify AAV. This study provides solutions to AAV empty and full capsid ratio quantification challenges and the subsequent basis for calibrating the AAV empty capsid system suitability substance. Because of the AAV structure and potential variability complexity in detection, we jointly calibrated empty capsid system suitability substance with three laboratories to accurately detect the quantitative AAV empty and full capsid ratio. The empty capsid system suitability substance could be used as an external reference to measure the performance of the instrument. The results could be compared with multiple QC (quality control) laboratories based on the AAV vector and calibration accuracy. This is crucial for AUC to be used for QC release and promote gene therapy research worldwide.

Strategy toward In-Cell Self-Assembly of an Artificial Viral Capsid from a Fluorescent Protein-Modified ß-Annulus Peptide.

In-cell self-assembly of natural viral capsids is an event that can be visualized under transmission electron microscopy (TEM) observations. By mimicking the self-assembly of natural viral capsids, various artificial protein- and peptide-based nanocages were developed; however, few studies have reported the in-cell self-assembly of such nanocages. Our group developed a ß-Annulus peptide that can form a nanocage called artificial viral capsid in vitro, but in-cell self-assembly of the capsid has not been achieved. Here, we designed an artificial viral capsid decorated with a fluorescent protein, StayGold, to visualize in-cell self-assembly. Fluorescence anisotropy measurements and fluorescence resonance energy transfer imaging, in addition to TEM observations of the cells and super-resolution microscopy, revealed that StayGold-conjugated ß-Annulus peptides self-assembled into the StayGold-decorated artificial viral capsid in a cell. Using these techniques, we achieved the in-cell self-assembly of an artificial viral capsid.

Intrinsic disorder in flaviviral capsid proteins and its role in pathogenesis.

A high level of disorder in many viral proteins is a direct consequence of their small genomes, which makes interaction with multiple binding partners a necessity for infection and pathogenicity. A segment of the flaviviral capsid protein (C), also known as the molecular recognition feature (MoRF), undergoes a disorder-toorder transition upon binding to several protein partners. To understand their role in pathogenesis, MoRFs were identified and their occurrence across different flaviviral capsids were studied. Despite lack of sequence similarities, docking studies of Cs with the host proteins indicate conserved interactions involving MoRFs across members of phylogenetic subclades. Additionally, it was observed from the protein-protein networks that some MoRFs preferentially bind proteins that are involved in specialized functions such as ribosome biogenesis. The findings point to the importance of MoRFs in the flaviviral life cycle, with important consequences for disease progression and suppression of the host immune system. Potentially, they might have impacted the way flaviviruses evolved to infect varied hosts using multiple vectors.

Transformation of a Viral Capsid from Nanocages to Nanotubes and Then to Hydrogels: Redirected Self-Assembly and Effects on Immunogenicity.

The ability to manipulate the self-assembly of proteins is essential to understanding the mechanisms of life and beneficial to fabricating advanced nanomaterials. Here, we report the transformation of the MS2 phage capsid from nanocages to nanotubes and then to nanotube hydrogels through simple point mutations guided by interfacial interaction redesign. We demonstrate that site 70, which lies in the flexible FG loop of the capsid protein (CP), is a "magic" site that can largely dictate the final morphology of assemblies. By varying the amino acid at site 70, with the aid of a cysteine-to-alanine mutation at site 46, we achieved the assembly of double-helical or single-helical nanotubes in addition to nanocages. Furthermore, an additional cysteine substitution on the surface of nanotubes mediated their cross-linking to form hydrogels with reducing agent responsiveness. The hierarchical self-assembly system allowed for the investigation of morphology-related immunogenicity of MS2 CPs, which revealed dramatic differences among nanocages, nanotubes, and nanotube hydrogels in terms of immune response types, antibody levels and T cell functions. This study provides insights into the assembly manipulation of protein nanomaterials and the customized design of nanovaccines and drug delivery systems.

Point mutation in a virus-like capsid drives symmetry reduction to form tetrahedral cages.

Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of unique symmetry-reduced structures. Starting with the encapsulin from Myxococcus xanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple-point mutation to various amino acids and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent a unique example of tetrahedral geometry when surveying all characterized encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in the protein sequence.

Collision induced unfolding and molecular dynamics simulations of norovirus capsid dimers reveal strain-specific stability profiles.

Collision induced unfolding (CIU) is a method used with ion mobility mass spectrometry to examine protein structures and their stability. Such experiments yield information about higher order protein structures, yet are unable to provide details about the underlying processes. That information can however be provided using molecular dynamics simulations. Here, we investigate the gas-phase unfolding of norovirus capsid dimers from the Norwalk and Kawasaki strains by employing molecular dynamics simulations over a range of temperatures, representing different levels of activation, together with CIU experiments. The dimers have highly similar structures, but their CIU reveals different stability that can be explained by the different dynamics that arises in response to the activation seen in the simulations, including a part of the sequence with previously observed strain-specific dynamics in solution. Our findings show how similar protein variants can be examined using mass spectrometric techniques in conjunction with atomistic molecular dynamics simulations to reveal differences in stability as well as differences in how and where unfolding takes place upon activation.

Cryo-EM structure of flagellotropic bacteriophage Chi.

The flagellotropic bacteriophage χ (Chi) infects bacteria via the flagellar filament. Despite years of study, its structural architecture remains partly characterized. Through cryo-EM, we unveil χ's nearly complete structure, encompassing capsid, neck, tail, and tail tip. While the capsid and tail resemble phage YSD1, the neck and tail tip reveal new proteins and their arrangement. The neck shows a unique conformation of the tail tube protein, forming a socket-like structure for attachment to the neck. The tail tip comprises four proteins, including distal tail protein (DTP), two baseplate hub proteins (BH1P and BH2P), and tail tip assembly protein (TAP) exhibiting minimal organization compared to other siphophages. Deviating from the consensus in other siphophages, DTP in χ forms a trimeric assembly, reducing tail symmetry from 6-fold to 3-fold at the tip. These findings illuminate the previously unexplored structural organization of χ's neck and tail tip.

Virus-like particles derived from bacteriophage MS2 as antigen scaffolds and RNA protective shells.

The versatile potential of bacteriophage MS2-derived virus-like particles (VLPs) in medical biotechnology has been extensively studied during the last 30 years. Since the first reports showing that MS2 VLPs can be produced at high yield and relatively easily engineered, numerous applications have been proposed. Particular effort has been spent in developing MS2 VLPs as protective capsules and delivery platforms for diverse molecules, such as chemical compounds, proteins and nucleic acids. Among these, two are particularly noteworthy: as scaffolds displaying heterologous epitopes for vaccine development and as capsids for encapsulation of foreign RNA. In this review, we summarize the progress in developing MS2 VLPs for these two areas.

Hollow, nanosized protein particles have many potential uses. If they can be appropriately engineered, they may for example be able to carry therapeutic cargoes to diseased cells or be used as a vaccine where appropriate antigens are mounted on their external surface. Many viruses offer a ready-made protein particle, the capsid, which can be made hollow by exclusion of the viral genetic material. MS2 is a virus that targets bacteria ­ a bacteriophage ­ which is well characterized and has been developed over many years for a number of applications. It has particular promise for development as a vaccine and for RNA delivery, both of which are reviewed here.

Imaging of adeno-associated viral capsids for purposes of gene editing using CEST NMR/MRI.

PURPOSE: Gene therapy using adeno-associated virus (AAV) vector-mediated gene delivery has undergone substantial growth in recent years with promising results in both preclinical and clinical studies, as well as emerging regulatory approval. However, the inability to quantify the efficacy of gene therapy from cellular delivery of gene-editing technology to specific functional outcomes is an obstacle for efficient development of gene therapy treatments. Building on prior works that used the CEST reporter gene lysine rich protein, we hypothesized that AAV viral capsids may generate endogenous CEST contrast from an abundance of surface lysine residues. METHODS: NMR experiments were performed on isolated solutions of AAV serotypes 1-9 on a Bruker 800-MHz vertical scanner. In vitro experiments were performed for testing of CEST-NMR contrast of AAV2 capsids under varying pH, density, biological transduction stage, and across multiple serotypes and mixed biological media. Reverse transcriptase-polymerase chain reaction was used to quantify virus concentration. Subsequent experiments at 7 T optimized CEST saturation schemes for AAV contrast detection and detected AAV2 particles encapsulated in a biocompatible hydrogel administered in the hind limb of mice. RESULTS: CEST-NMR experiments revealed CEST contrast up to 52% for AAV2 viral capsids between 0.6 and 0.8 ppm. CEST contrast generated by AAV2 demonstrated high levels of CEST contrast across a variety of chemical environments, concentrations, and saturation schemes. AAV2 CEST contrast displayed significant positive correlations with capsid density (R2 > 0.99, p < 0.001), pH (R2 = 0.97, p = 0.01), and viral titer per cell count (R2 = 0.92, p < 0.001). Transition to a preclinical field strength yielded up to 11.8% CEST contrast following optimization of saturation parameters. In vivo detection revealed statistically significant molecular contrast between viral and empty hydrogels using both mean values (4.67 ± 0.75% AAV2 vs. 3.47 ± 0.87% empty hydrogel, p = 0.02) and quantile analysis. CONCLUSION: AAV2 viral capsids exhibit strong capacity as an endogenous CEST contrast agent and can potentially be used for monitoring and evaluation of AAV vector-mediated gene therapy protocols.

DNA Released by Adeno-Associated Virus Strongly Alters Capsid Aggregation Kinetics in a Physiological Solution.

While adeno-associated virus is a leading vector for gene therapy, significant gaps remain in understanding AAV degradation and stability. In this work, we study the degradation of an engineered AAV serotype at physiological pH and ionic strength. Viral particles of varying fractions of encapsulated DNA were incubated between 30 and 60 °C, with changes in molecular weight measured by changes in total light scattering intensity at 90° over time. Mostly full vectors demonstrated a rapid decrease in molecular weight corresponding to the release of capsid DNA, followed by slow aggregation. In contrast, empty vectors demonstrated immediate, rapid colloid-type aggregation. Mixtures of full and empty capsids showed a pronounced decrease in initial aggregation that cannot be explained by a linear superposition of empty and full degradation scattering signatures, indicating interactions between capsids and ejected DNA that influenced aggregation mechanisms. This demonstrates key interactions between AAV capsids and their cargo that influence capsid degradation, aggregation, and DNA release mechanisms in a physiological solution.

Higher-Order Structure of Adeno-Associated Virus Serotype 8 by Hydrogen/Deuterium Exchange Mass Spectrometry.

The higher-order structure (HOS) is a critical quality attribute of recombinant adeno-associated viruses (rAAVs). Evaluating the HOS of the entire rAAV capsid is challenging because of the flexibility and/or less folded nature of the VP1 unique (VP1u) and VP1/VP2 common regions, which are structural features essential for these regions to exert their functions following viral infection. In this study, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used for the structural analysis of full and empty rAAV8 capsids. We obtained 486 peptides representing 85% sequence coverage. Surprisingly, the VP1u region showed rapid deuterium uptake even though this region contains the phospholipase A2 domain composed primarily of α-helices. The comparison of deuterium uptake between full and empty capsids showed significant protection from hydrogen/deuterium exchange in the full capsid at the channel structure of the 5-fold symmetry axis. This corresponds to cryo-electron microscopy studies in which the extended densities were observed only in the full capsid. In addition, deuterium uptake was reduced in the VP1u region of the full capsid, suggesting the folding and/or interaction of this region with the encapsidated genome. This study demonstrated HDX-MS as a powerful method for probing the structure of the entire rAAV capsid.

The structure and physical properties of a packaged bacteriophage particle.

A string of nucleotides confined within a protein capsid contains all the instructions necessary to make a functional virus particle, a virion. Although the structure of the protein capsid is known for many virus species1,2, the three-dimensional organization of viral genomes has mostly eluded experimental probes3,4. Here we report all-atom structural models of an HK97 virion5, including its entire 39,732 base pair genome, obtained through multiresolution simulations. Mimicking the action of a packaging motor6, the genome was gradually loaded into the capsid. The structure of the packaged capsid was then refined through simulations of increasing resolution, which produced a 26 million atom model of the complete virion, including water and ions confined within the capsid. DNA packaging occurs through a loop extrusion mechanism7 that produces globally different configurations of the packaged genome and gives each viral particle individual traits. Multiple microsecond-long all-atom simulations characterized the effect of the packaged genome on capsid structure, internal pressure, electrostatics and diffusion of water, ions and DNA, and revealed the structural imprints of the capsid onto the genome. Our approach can be generalized to obtain complete all-atom structural models of other virus species, thereby potentially revealing new drug targets at the genome-capsid interface.

Identification of clickable HIV-1 capsid-targeting probes for viral replication inhibition.

Of the targets for HIV-1 therapeutics, the capsid core is a relatively unexploited but alluring drug target due to its indispensable roles throughout virus replication. Because of this, we aimed to identify "clickable" covalent modifiers of the HIV-1 capsid protein (CA) for future functionalization. We screened a library of fluorosulfate compounds that can undergo sulfur(VI) fluoride exchange (SuFEx) reactions, and five compounds were identified as hits. These molecules were further characterized for antiviral effects. Several compounds impacted in vitro capsid assembly. One compound, BBS-103, covalently bound CA via a SuFEx reaction to Tyr145 and had antiviral activity in cell-based assays by perturbing virus production, but not uncoating. The covalent binding of compounds that target the HIV-1 capsid could aid in the future design of antiretroviral drugs or chemical probes that will help study aspects of HIV-1 replication.

Virtual indentation of the empty capsid of the minute virus of mice using a minimal coarse-grained model.

A minimal coarse-grained model for T=1 viral capsids assembled from 20 protein rigid trimers has been designed by extending a previously proposed form of the interaction energy written as a sum of anisotropic pairwise interactions between the trimeric capsomers. The extension of the model has been performed to properly account for the coupling between two internal coordinates: the one that measures the intercapsomer distance and the other that gives the intercapsomer dihedral angle. The model has been able to fit with less than a 10% error the atomic force microscopy (AFM) indentation experimental data for the empty capsid of the minute virus of mice (MVM), providing in this way an admissible picture of the main mechanisms behind the capsid deformations. In this scenario, the bending of the intercapsomer dihedral angle is the angular internal coordinate that can support larger deformations away from its equilibrium values, determining important features of the AFM indentation experiments as the elastic constants along the three symmetry axes of the capsid and the critical indentations. From the value of one of the parameters of our model, we conclude that trimers in the MVM must be quite oblate tops, in excellent agreement with their known structure. The transition from the linear to the nonlinear regimes sampled in the indentation process appears to be an interesting topic for future research in physical virology.