Structural Dynamics of Nonenveloped Virus Disassembly Intermediates

Disassembly or uncoating of an icosahedral capsid is a crucial step during infection by nonenveloped viruses. However, the dynamic and transient nature of the disassembly process makes it challenging to isolate intermediates in a temporal, stepwise manner for structural characterization. Using controlled, incremental heating, we isolated two disassembly intermediates: “eluted particles” and “puffed particles” of an insect nodavirus, Flock House virus (FHV). Cryo-electron microscopy and three-dimensional reconstruction of the FHV disassembly intermediates indicated that disassembly-related conformational alterations are minimally global and largely local, leading to asymmetry in the particle and eventual genome release without complete disintegration of the icosahedron.

ABSTRACT

The stability of icosahedral viruses is crucial for protecting the viral genome during transit; however, successful infection requires eventual disassembly of the capsid. A comprehensive understanding of how stable, uniform icosahedrons disassemble remains elusive, mainly due to the complexities involved in isolating transient intermediates. We utilized incremental heating to systematically characterize the disassembly pathway of a model nonenveloped virus and identified an intriguing link between virus maturation and disassembly. Further, we isolated and characterized two intermediates by cryo-electron microscopy and three-dimensional reconstruction, without imposing icosahedral symmetry. The first intermediate displayed a series of major, asymmetric alterations, whereas the second showed that the act of genome release, through the 2-fold axis, is actually confined to a small section on the capsid. Our study thus presents a comprehensive structural analysis of nonenveloped virus disassembly and emphasizes the asymmetric nature of programmed conformational changes.

IMPORTANCE Disassembly or uncoating of an icosahedral capsid is a crucial step during infection by nonenveloped viruses. However, the dynamic and transient nature of the disassembly process makes it challenging to isolate intermediates in a temporal, stepwise manner for structural characterization. Using controlled, incremental heating, we isolated two disassembly intermediates: “eluted particles” and “puffed particles” of an insect nodavirus, Flock House virus (FHV). Cryo-electron microscopy and three-dimensional reconstruction of the FHV disassembly intermediates indicated that disassembly-related conformational alterations are minimally global and largely local, leading to asymmetry in the particle and eventual genome release without complete disintegration of the icosahedron.

INTRODUCTION

Virus capsids with icosahedral geometry are exceptionally stable, symmetric structures that store, protect, and transport infectious genomes. Upon coming in contact with host cells, however, they disintegrate under relatively mild conditions to release the viral genome for downstream replication and translation. The process of disassembly or uncoating is crucial to establish an infection and requires major conformational changes in the capsid. A mature, infectious virus particle is primed for disassembly and responds to cues from the host cells leading to disintegration. Common cues that trigger conformational changes in capsids include receptor binding, exposure to low pH, low concentrations of divalent cations, and a reducing environment in endosomal compartments and interaction with cellular proteases (1). In some cases, sequential uncoating cues may be required for stepwise shedding of layers of capsid proteins. This complex, multistep process is not well established for most viruses; however, the structural alterations involved are not expected to be a straightforward and precise reversal of assembly-related associations (2).

Among viruses with outer icosahedral shells, the uncoating processes of picornaviruses, polyomaviruses, adenoviruses, and reoviruses have been studied in some detail (3, 4). Several members of the picornavirus family produce two disassembly intermediates under in vitro conditions: (i) 135S or A particle, a stain-permeable, slightly expanded form, and (ii) 80S or B particle, an empty capsid with icosahedral features (5, 6). Exceptions in this pathway occur in case of Equine rhinitis A virus and Triatoma virus, where the capsid quickly dissociates into smaller structural units like pentamers (7, 8). The picornavirus disassembly intermediates are defined by overall thinning of the capsid shell, radial outward movement of structural proteins, and expansion in capsid diameter, as well as by exposure or loss of internal capsid components involved in cellular membrane penetration. Disassembly intermediates of other viruses, such as reoviruses, rotaviruses, and adenoviruses, also appear to have lost capsid components involved in membrane penetration or have shed layers of capsid proteins (9–12). The association of poliovirus and rhinovirus intermediates with artificial membranes has indicated the structural basis of membrane interaction and genome transfer. Structural studies have also suggested that the site for genome release is likely the viral 2-fold axis of symmetry (13, 14). Whether this is a common fracture site for icosahedral capsids or whether genome release is localized to a single specific 2-fold axis or occurs from multiple axes on the capsid surface is not yet known. Compaction of the genome, as seen in human rhinovirus particles on the brink of uncoating, is another curious, relatively unexplored feature that potentially suggests the necessity of conformational changes in packaged genome prior to release.

The existing reports on a limited number of nonenveloped viruses do not provide a detailed map of sequential disassembly-associated conformational changes in icosahedral capsids. A primary reason for this gap involves difficulties involved in generating disassembly intermediates in vitro or isolating them from infected cells. The transient nature of the process makes it challenging to generate all conformational states in the pathway in a comprehensive, sequential manner. However, since infectious virus capsids are metastable particles programmed to disassemble within cells, it is possible that an incremental supply of energy could provide adequate impetus to compel an icosahedral capsid to transition toward disassembly in vitro.

We chose a model nonenveloped virus, Flock House virus (FHV) from the Nodaviridae family, for studying disassembly in vitro. FHV is an insect virus that has a bipartite, positive-strand RNA genome encapsidated in a symmetric, icosahedral capsid (T = 3) with a diameter of ∼34 nm (15, 16). The cognate genome consists of RNA1 (3.1 kb) and RNA2 (1.4 kb), which encode an RNA-dependent RNA polymerase and capsid protein, respectively. The capsid is initially assembled in association with nucleic acid, as a noninfectious provirion composed of 180 copies of a single coat protein, α (44 kDa), organized into 60 icosahedral asymmetric units (iASUs) (15). After assembly, α undergoes an autoproteolytic maturation cleavage, which generates the major coat protein β (residues 1 to 363) and a small, noncovalently capsid-associated, hydrophobic peptide, γ (residues 364 to 407) (17). γ, a membrane-lytic peptide, is exposed from the capsid interior during virus entry and facilitates endosomal membrane rupture (18). Mutations in α, which individually or together prevent maturation cleavage (D75N/D75V/D75T/N363T), also render the particle noninfectious (19). In the present study, incremental heat-induced structural alterations in FHV particles were examined with a combination of biophysical assays, electron microscopy, and single-particle three-dimensional (3D) reconstruction.

The heat-induced disassembly pattern of the infectious virus differed distinctly from its noninfectious, uncleaved counterpart, which established an important link between capsid maturation and disassembly. Two structurally altered forms of infectious FHV, one that closely matched a disassembly intermediate isolated earlier from infected cells and a distinctive particle caught in the process of releasing its genome, were isolated. Since icosahedral symmetry-based reconstruction usually causes averaging of features throughout the structure, asymmetric reconstruction was primarily utilized to identify global and local conformational changes in FHV. The intermediate particles displayed significant local differences in secondary and tertiary structure content, as well as in differential opening of the 2-fold symmetry axis, which appeared to be the site for genome exit, in addition to global loss of γ peptides throughout the capsid. This comprehensive structural analysis of the sequence of events during FHV disassembly may provide important insight into the principles of nonenveloped virus disassembly.

RESULTS

Incremental heating of wild-type FHV produces two successive, structurally distinct, disassembly intermediates.

Differential scanning calorimetry (DSC), a thermoanalytical technique that monitors physical transformations of a sample as a function of temperature, was used to identify endothermic transitions in wild-type and maturation-defective FHV (D75N/N363T). Controlled incremental heating of wild-type FHV from 50 to 100°C produced two prominent peaks, representing endothermic transitions, at 69.5 and 81.3°C and a dip at 77.6°C (Fig. 1A). In contrast, maturation-defective FHV displayed a notably different pattern of transformation akin to virus-like particles of FHV (20), with a single peak at ∼80°C concurrent with the second major peak generated by wild-type virus (Fig. 1A). These remarkable differences in thermally induced physical transition of different FHV particles, in spite of their close physical similarities (16), suggest the importance of cognate genome and covalent dissociation of gamma peptides in the conformational stability of particles.

FIG 1.

Structural transitions in FHV capsid upon incremental heating. (A) Differential scanning calorimetry of wild-type (solid line) and maturation-defective (dashed line) FHV from 50 to 100°C. (B) Electron micrographs of negatively stained wild-type (top panel) and maturation-defective (bottom panel) FHV, unheated or incrementally heated to 70, 75, and 80°C. White boxes mark the particles being displayed in insets, and the diameters of particles are highlighted with black lines. (C) Bar graph representing the number of different particle populations—native, eluted, puffed, and disintegrated—in wild-type (wt) and maturation-defective (mut) FHV samples under different heating conditions. A total of 100 particles were counted for each sample (in triplicate). (D) Electron micrographs of negatively stained wild-type FHV particles heated to 75°C without (upper image) and with RNase A treatment (lower image). White arrows point to puffed particles in the micrographs. Scale bar, 50 nm.

The conformational changes in particles heated to 70, 75, and 80°C for 30 min were visually evaluated by negative-staining electron microscopy. Unheated particles were spherical, ca. 34 nm in diameter, as expected (16). However, ∼93.2% of wild-type particles heated to 70°C appeared to have become stain permeable, as implied by a densely stained core, and were smaller in diameter (∼30 nm) (Fig. 1B, top panel, and Fig. 1C). Stain-permeable FHV particles were reported previously (21) as “eluted particles,” which were infection intermediates isolated from infected Drosophila cells. The close structural similarity of our heat-induced, in vitro-generated particles with eluted particles suggested that the former likely represent a distinct conformational variant in the disassembly pathway of FHV.

A second structurally distinct population, generated at 75°C, appeared to have a major protrusion “puffing” out from the viral surface (Fig. 1B, top panel, and Fig. 1C). Treatment of this population with RNase resulted in the disappearance of the altered particles (Fig. 1D), indicating that the puffs were probably comprised of extruding viral genome. The visual appearance of “puffed” particles suggests that these represent a more advanced stage in the disassembly pathway compared to eluted particles. Eventually, after heating at 80°C, a complete disruption of the particle structure was detected (Fig. 1B, top panel).

Maturation-defective FHV does not undergo distinct structural transitions like the wild type.

Incremental heating of the maturation-defective particles to 70 and 75°C did not produce any major structural alterations. These particles remained spherical and, unlike wild-type FHV, they did not show any internal staining or decrease in size. Heating to 80°C disrupted the overall particle structure (Fig. 1B, bottom panel, and Fig. 1C). This suggests that these noninfectious particles do not follow the complex, multistep, ordered pathway for dismantling of the capsid and that the formation of structurally distinct disassembly intermediates requires covalently cleaved mature virus.

FHV capsid protein shows secondary and tertiary structural changes upon incremental heating.

Structural alterations in the protein component of wild-type and maturation-defective particles during incremental heating were analyzed using circular dichroism, tryptophan fluorescence spectroscopy, and limited protease digestion (Fig. 2A to C). A sudden, abrupt decrease in negative ellipticity indicating the loss of secondary structural elements (Fig. 2A), a red shift in the emission maxima (λ_max) of the intrinsic tryptophan fluorescence (Fig. 2B), and substantial alterations in the pattern of trypsin cleavage (Fig. 2C) were noted upon heating wild-type particles to 70 and 75°C, indicating major conformational shifts at these temperatures. In contrast, the corresponding structural elements in maturation-defective particles appeared to be less sensitive to heat-induced denaturation effects, since the particles showed major alterations only after being heated to 80°C (Fig. 2).

FIG 2.

Secondary and tertiary structural changes in FHV capsid induced upon incremental heating. (A) Far-UV CD spectra of wild-type and maturation-defective FHV, at temperatures ranging from 25 to 90°C. (B) Intrinsic tryptophan fluorescence spectra of wild-type and maturation-defective FHV at temperatures ranging from 4 to 90°C. (C) Limited trypsin proteolysis of wild-type and maturation-defective FHV, unheated and heated to temperatures ranging from 25 to 90°C. Lanes are numbered at the bottom of the gels. The positions of α, β, and γ and that of trypsin and its autolytic degradation products are indicated with black arrows.

Incremental heating externalizes gamma peptides.

To check for externalization of γ peptides during disassembly, capsids were immunolabeled with an anti-γ polyclonal antibody and a gold-conjugated secondary antibody. While unheated wild-type particles did not show any gold particle binding, heated particles were decorated with dense gold dots, indicating increased accessibility of γ (Fig. 3A, top panel). The heated particles also appeared somewhat disintegrated, probably due to acceleration of disassembly due to antibody binding. The number of gold labels associated with each particle was far less than 180 (Fig. 3A, top panel), which was probably due to steric hindrances or because of the loss of the majority of γ from the particles. The latter hypothesis is supported by previous data which show that eluted particles isolated from infected cells have lost a fraction of γ (21). Maturation-defective FHV, which lacks covalently dissociated γ, did not display any binding to anti-γ antibody upon being heated to 70 and 75°C. Immunogold labeling to these particles was detected after heating to 80°C and significant disruption of the spherical cage (Fig. 3A, bottom panel).

FIG 3.

Status of γ peptide and RNA genome in heat-treated FHV particles. (A) Electron micrographs of negatively stained, unheated and heated wild-type (top panel) and maturation-defective (bottom panel) FHV particles, immunolabeled with anti-γ polyclonal and gold-conjugated secondary antibodies. White boxes mark the particles being displayed in insets, and white arrows point to the dense gold dots attributed to gold-conjugated secondary antibody. Scale bar, 50 nm. (B) Gel electrophoresis of viral RNA. Lanes 1, 3, 5, and 7 contain viral RNA extracted from wild-type FHV particles (labeled as Wt), unheated and heated to 70, 75, and 80°C, respectively, while lanes 2, 4, 6, and 8 contain similarly treated samples of maturation-defective FHV (labeled as Mut). Lanes 9 and 11 contain RNA extracted from unheated wild-type FHV (labeled as Wt*) and subsequently heated to 70 and 75°C, respectively, and lanes 10 and 12 contain similarly treated samples of maturation-defective FHV (labeled as Mut*). (C) Viral RNAs extracted from unheated and heated, wild-type (lanes 1 to 7) and maturation-defective FHV (lanes 9 to 13) particles, sequentially treated with RNase A and proteinase K, are shown. Lanes 8 and 14 contain viral RNA extracted from unheated wild-type (Wt*) and maturation-defective (Mut*) FHV, respectively, followed by sequential RNase A and proteinase K treatment.

Genomic RNA undergoes conformational change in both wild-type and maturation-defective FHV.

In contrast to the bipartite RNA (RNA1 and RNA2) packaged in native virions, RNA extracted from wild-type or maturation-defective particles heated to 70 or 75°C migrated as a single species, which appeared to be a complex of the two strands (Fig. 3B), as reported previously (22). The similarity in genomic conformational alterations in these particles suggests that it is probably the mature capsid protein, rather than genome, that propels stepwise disassembly in the wild-type capsid. The RNA complex was found to have degraded upon heating the particles to 80°C (Fig. 3B). Complex formation could not be achieved by heating RNA1 and RNA2 extracted from unheated particles (Fig. 3B), indicating that heat-induced RNA association requires support from the protein component of the virion.

To confirm that RNA complex formation occurs inside the capsid, heated particles were subjected to sequential RNase A and proteinase K treatment, followed by RNA extraction. Although RNase treatment of unheated particles or particles heated to 70°C did not affect bipartite or complexed RNA, partial digestion of the complex was observed upon heating the particles to 75°C. This indicates that while there is no drastic effect on particle integrity at 70°C, there is limited exposure of genome at 75°C. RNase treatment of particles heated to 80°C caused complete digestion of the complex, indicating full exposure of genome from disrupted particles (Fig. 3C).

Low-resolution structures of FHV disassembly intermediates indicate a local loss in capsid symmetry.

The disassembly pathway was not precisely synchronized for all particles. Eluted particles constituted ∼93.2% of the total population of wild-type FHV heated to 70°C (Fig. 1B and C). In addition to the eluted particles, this sample also contained small percentages of native (4.9%) and puffed particles (1.9%). Likewise, upon being heated to 75°C, a large proportion (55.0%) of wild-type particles were converted to “puffed” particles. This sample also contained a fair proportion (29.7%) of particles that appeared to be disintegrated, in addition to native (4.7%) and eluted (10.6%) particles (Fig. 1B and C). Eluted and puffed particles were therefore manually picked from negatively stained micrographs for low-resolution, single-particle reconstruction (Fig. 4A to C and Table 1). While reconstruction of native particle clearly showed icosahedral symmetry with easily distinguishable 2-, 3-, and 5-fold symmetry axes (Fig. 4A, left panel), the density map for eluted particles displayed an overall smooth surface with minimal features, but with slight depressions at all the 2-fold symmetry axes (Fig. 4A, middle panel). Another reconstruction of eluted particle without imposing icosahedral symmetry generated a unique, asymmetric structure with local differences in surface features (Fig. 4B). The capsid surface appeared uneven, with the greater part displaying icosahedral features, while there was a clear loss of symmetry restricted to a specific region, which appeared to protrude slightly from the surface (Fig. 4B). An asymmetric reconstruction for puffed particles resulted in a density map with a considerable protrusion on one side of the capsid surface (Fig. 4A, right panel), probably representing the RNA “puff” on the verge of exiting the capsid. This phenomenon points toward the directional nature of genome exit from virions, without completely damaging particle integrity. Although the finer details of the capsid surface could not be clearly observed due to the very low resolution of these reconstructions, the asymmetric nature of the particles and loss of local symmetry were fairly obvious. This local asymmetry was also observed in medium-resolution cryo-electron microscopy (cryo-EM) reconstruction of the eluted particle compared to a similar asymmetric reconstruction of native FHV (Fig. 4C and D and Table 1).

FIG 4.

Low- and medium-resolution reconstructions of wild-type FHV native, eluted, and puffed particles. (A) Electron micrographs of negatively stained wild-type FHV heated at different temperatures, along with the corresponding density maps of native, eluted, and puffed particles computed by applying symmetry during reconstruction of native and eluted particles. Red circles on the micrographs highlight the population of particles picked for the corresponding 3D reconstruction. The density map of the native particle (left panel) is marked with blue pentagons, triangles, and rectangles to show icosahedral symmetry elements 5-, 3-, and 2-fold, respectively. Quasi-3-fold and -2-fold axes are marked with orange triangles and rectangles. The icosahedral asymmetric unit (iASU) is outlined by a dotted, blue triangle. A 3D density map of eluted particle (middle panel), reconstructed with imposed icosahedral symmetry, is highlighted with a dotted arrow pointing to the depression at the 2-fold symmetry axis. The right panel shows the density map of puffed particle, with no symmetry imposed during reconstruction. (B) Asymmetric reconstruction of negatively stained eluted particle, represented by radially colored density maps in three different orientations. A color key (right) indicates the radial distance (in Å) from the particle center. (C) FSC curves for the reconstructions shown in panels A, B, and D. The resolutions of different reconstructions estimated at FSC 0.143 (dashed line) are highlighted. (D) Radially colored, medium-resolution, asymmetric cryo-EM reconstructions of wild-type native and eluted particles.

TABLE 1.

Statistics for both low-resolution, negatively stained particle reconstructions and medium-resolution, cryo-EM reconstructions of wild-type FHV native, eluted, and puffed particles

Data collection or processing parameter	Negatively stained			Cryo-EM
	Native particle	Eluted particle	Puffed particle	Native particle	Eluted particle
Magnification	50,000×	50,000×	50,000×	50,000×	50,000×
Voltage (keV)	200	200	200	200	200
Defocus range (μm)	0.5–3.0	0.5–3.0	0.5–3.0	0.5–3.0	0.5–3.0
Pixel size (Å/pixel)	2.21	2.21	2.21	2.21	2.21
Symmetry imposed	I	I/C1	C1	C1	C1
Micrographs (no.)	302	606	652	1,211	1,391
Particles boxed (no.)	5,528	11,122	1,761	21,952	39,327
Particles used in reconstruction (no.)	5,528	11,118	1,760	17,882	32,145
Map resolution (Å)	14.8	17.6/31.4	33.2	10.1	11.4
FSC threshold	0.143	0.143	0.143	0.143	0.143

High-resolution asymmetric cryo-EM reconstruction of eluted particles reveals global and local conformational changes.

Cryo-EM was utilized to generate a density map of eluted particles to a resolution of 4.7 Å (Fig. 5 and Table 2). Eluted particles had considerably less dense centers in cryomicrographs (Fig. 5A), as well as a smaller measured diameter of ca. 28 to 30 nm, compared to ∼34 nm for native particles (Fig. 5B). This reduction in diameter under cryoimaging conditions overruled the possibility of particle contraction being an artifact of negative staining. The difference in size was the primary selection criterion for manual picking and segregation of eluted particles from noneluted particles using RELION (23) (Table 2).

FIG 5.

Asymmetric cryo-EM reconstructions of eluted particle. (A) Cryo-electron micrograph of frozen hydrated wild-type FHV particles heated to 70°C. Red arrows point to the particles with considerably less dense centers. Scale bar, 50 nm. (B) Central section of the 3D reconstruction of eluted particle. The density is shown in gray, with the particle diameter (red and light blue lines), RNA shell diameter (light green line), and capsid shell thickness (yellow and dark blue lines) marked. (C) FSC curve, highlighting the resolution of the reconstruction estimated at FSC 0.143 (dashed line). (D) Surface-rendered, radially colored cryo-EM density map of eluted particle in different orientations, with a color key (right) displaying the radial distance (in Å) from the particle center. In the middle panel, the fine and coarse parts of the particles are marked by light blue and black dotted lines, respectively. A dotted gray circle highlights the front view of the coarse part of eluted particle in the right panel. (E) Density map colored by local resolution, with a color key (right) displaying the local resolution values in Å. (F) Central section of the radially colored density map, with a dotted arrow pointing to the interior bulk RNA.

TABLE 2.

Cryo-EM data collection, refinement, and validation statistics

Parameter	Eluted particle, fine part (EMD-9730; PDB ID 6ITB)	Eluted particle, coarse part (EMD-9730; PDB ID 6ITF)	Puffed particle (EMD-9732)
Data collection and processing
Magnification	81,000×	81,000×	81,000×
Voltage (keV)	300	300	300
Electron exposure/frame (e–/Å2)	∼0.5–1.0	∼0.5–1.0	∼0.5–1.0
Defocus range (μm)	0.5–3.0	0.5–3.0	0.5–3.0
Pixel size (Å/pixel)	1.7407	1.7407	1.7407
Symmetry imposed	C1	C1	C1
Micrographs (no.)	2,628	2,628	766
Initial particle images (no.)	61,905	61,905	1,301
Final particle images (no.)	58,918	58,918	1,293
B factor (Å2)	–181.093	–181.093
Map resolution (Å)	4.7	4.7	26.2
FSC threshold	0.143	0.143	0.143
Refinement
Initial model (PDB code)	4FTB	4FTB
Model-to-map fit
Correlation coefficient, CC_mask	0.77	0.71
RMSD
Bond length (Å)	0.005	0.008
Bond angle (°)	0.970	1.418
Validation (MolProbity)
All-atom clashscore	8.35	13.79
Rotamer outliers (%)	0.29	1.15
Cbeta deviations	0.0	0.0
Ramachandran plot (%)
Favored	93.94	85.68
Allowed	5.81	14.32
Outliers	0.25	0.00

The imposition of icosahedral symmetry during reconstruction led to the smearing of local characteristics throughout capsid surface, giving rise to an almost flat, featureless structure (data not shown). Single-particle reconstruction without imposing icosahedral symmetry showed a striking preservation in overall capsid shape and surface topography (Fig. 5D), except for an unusually disturbed region which lacked a significant portion of density corresponding to the capsid shell, was less resolved compared to the overall map, and was thus associated with more conformational flexibility (Fig. 5E). The average thickness of this part was ∼2 nm (corresponding to ∼10.01 pixels), while that of the well-refined symmetric part was ∼4 nm (∼22.63 pixels) (Fig. 5B). In the capsid shell, this region was positioned directly opposite a well-refined symmetric area with a centered 2-fold symmetry axis (Fig. 5B) and was probably the origin of disassembly-associated structural changes. For ease of reference, this altered region will be designated the “coarse part” of the capsid shell, while the well-refined area directly across from this region will be referred to as the “fine part” of the capsid (Fig. 5D).

Local differences were also observed in the internal RNA densities. Although there was a distinct overall gap between the densities of the capsid and the outer RNA shell, signifying major reduction in RNA-protein interactions, the coarse part appeared to have lost more RNA-protein contacts in comparison to the fine part at the 2-fold symmetry axis (Fig. 5F). In the innermost part of the capsid, the sparse density corresponding to bulk RNA (16) appeared to extend outward, toward the coarse part of the capsid shell, indicating that the genome might shift toward this weak part, which could represent the site for genome exit.

The crystal structure of wild-type FHV (PDB ID 4FTB) fitted well into the eluted particle density map, with a correlation coefficient of 0.72 (Fig. 6A, left). Although most of the capsid proteins could be placed into the map, the interior densities contributed by γ and by the N and C termini of β (Fig. 6A, right) were missing. This global loss of density may also explain the reduction in RNA-protein interactions, since they are primarily mediated via highly basic terminal regions essential for RNA recognition and particle assembly (24, 25).

FIG 6.

Atomic model fitting and refinement of eluted particle. (A) FHV crystal structure (PDB ID 4FTB) fitted into the cryo-EM density map for eluted particle (yellow mesh). The inset on the left represents the zoomed-in view of the crystal structure fitted into the cryo-EM density. The right panel shows the central section of the fitted capsid crystal structure, depicted as a ribbon diagram with subunits A, B, and C in red, blue, and green, respectively. The γ peptides and RNA are shown in magenta and yellow. (B) Surface-rendered, radially colored density map generated from the FHV capsid crystal structure, along with a color key (left). The dotted triangle on the map marks one iASU, displayed in two different rotational views. (C) Radially colored density map of asymmetric eluted particle (center), along with the refined iASU atomic models, fitted into the corresponding densities segmented from the fine (left panel) and coarse (right panel) parts of the eluted particle map.

Atomic model refinement reveals details of local conformational changes in the capsid subunits of eluted particle.

The crystal structure of wild-type FHV was utilized as a model for real space fitting and refinement against densities corresponding to the icosahedral asymmetric unit (iASU; containing chains A, B, and C) segmented from both the coarse and fine regions of the eluted particle map (Fig. 6B and C and Table 2). The fine part of the eluted particle sustained its β-strand content (29.23%) (data not shown) and preserved the typical β-barrel folding core, whereas its α-helical content was significantly reduced (8.02%) compared to wild-type iASU. In contrast, the coarse part showed noticeable decrease in both α-helical (5.07%) and β-strand (14.33%) content (Fig. 6B and C). Apart from density corresponding to γ peptide which is missing in both parts, the coarse part also lacked densities that could be mapped majorly to the termini and surface-exposed loops of the capsid protein. Particularly, the loop extending from residues 205 to 209 was missing in subunits B and C, whereas the 263-267 loop was missing in subunit C (Fig. 6C). The missing densities and lack of secondary structural content are probably the reason for the reduced thickness of the capsid shell in the coarse region.

The 2-, 3-, and 5-fold symmetry axes generated from refined iASUs from the coarse and fine parts of the eluted particle (Fig. 7) were compared to those generated similarly from the crystal structure of wild-type FHV. Although the 5-fold axis of symmetry in wild-type FHV contained an open channel with a diameter of ∼8 Å (Fig. 7A, left panel), the channel appeared to be in a relatively closed conformation in the eluted particle, with a reduced diameter of ∼6 Å in both fine and coarse models (Fig. 7A, middle and right panels). The loops framing the 5-fold axes appeared to be somewhat twisted in the coarse region. A similar blocking of the 5-fold axes has earlier been observed during uncoating of coxsackievirus A7 (26). The 3-fold axes appeared roughly similar in both particles, although a slight twisting of surrounding loops was noted in the coarse region of the eluted particle (Fig. 7B). Structural differences were most prominent at the 2-fold axis of symmetry (Fig. 7C). The center of this axis is formed by two helices, corresponding to amino acids 147 to 153 and amino acids 327 to 333 of subunit C, together with their 2-fold related copies. The distance between the Cα atoms of Phe-332 (calculated to represent the gap between symmetry-related helices) was reduced to 5.35 Å in the fine part of the eluted particle compared to 7.99 Å in the wild-type crystal structure, while an almost negligible decrease (7.82 Å) was observed in the coarse part (Fig. 7C). The coarse part also appeared to lack density corresponding to helix 147-153. The disorder in this helix, together with the movement of helix 327-333 away from the axis, is probably the pivotal event for widening of the 2-fold axis in the coarse part, and a simultaneous narrowing of the corresponding axis in the fine part could explain the preferential release of genome from the opposing 2-fold axis.

FIG 7.

Alterations at the 5-, 3-, and 2-fold symmetry axes of the eluted particle. (A) Ribbon diagram of the 5-fold axis from FHV crystal structure (PDB ID 4FTB) (left panel), as well as the fine (middle panel) and coarse (right panel) parts of the eluted particle. Chains A, B, and C and the γ peptide are colored red, blue, green, and magenta, respectively. The black dotted boxes mark the axes, while the inset shows the zoomed-in view. The channels at the axes are marked with black dotted circles, and their diameters are indicated in Å. (B) Three-fold axes of symmetry from FHV crystal structure, as well as the fine and coarse parts of the eluted particle, as in panel A. (C) Views of the 2-fold symmetry axis, with symmetry-related Phe-332 residues from the C-subunit marked in red and distances between their Cα (or CA) atoms indicated in Å in light blue.

Cα root mean square deviation (RMSD) calculations showed that all subunits in an iASU in the coarse part of the eluted particle showed significant movement compared to the wild type, with subunits A, B, and C showing RMSDs of 3.17, 4.82, and 3.60 Å, respectively (data not shown). In contrast, the Cα RMSD of the fine part iASU closely matched that of the wild type, with RMSDs of 0.84, 0.76, and 0.90 Å for subunits A, B, and C. The comparatively larger movements by subunits B and C in the coarse part may explain the opening at the 2-fold axis.

Asymmetric cryo-EM reconstruction of puffed particles captures genome exit from capsid and represents a novel structural intermediate.

Cryo-EM images of eluted particles also showed a small fraction (∼2%) of puffed particles (Fig. 1C and 8A). From a set of 766 selected cryo-EM images, 1,301 puffed particles were manually picked on the basis of prominent surface protrusions and processed, without imposition of icosahedral symmetry, to obtain a 3D reconstruction at a 26.2-Å resolution (Fig. 8A to E and Table 2). The structure of puffed particles was also notably asymmetric, with protruding density (probably representing RNA being released) localized to only a part of the map, while the other half appeared mostly symmetric with recognizable icosahedral surface features (2-, 3-, and 5-fold symmetry axes) (Fig. 8C and D). A 180° rotation of the particle about the central axis revealed that the region positioned directly opposite to the protruding density contained a centered 2-fold symmetry axis (Fig. 8E). This strongly indicates that the 2-fold symmetry axis is the site for RNA exit, which is a localized event and does not affect the icosahedral character of the opposite face on the capsid surface. The density for the interior RNA shell merged with the capsid density at the protruding site, while the densities were clearly separated at other parts of the map (Fig. 8D). Thus, cryo-EM-based reconstructions suggest that largely local conformational alterations are exploited for genome release during icosahedral particle disassembly.

FIG 8.

Asymmetric cryo-EM reconstruction of puffed particle. (A) Cryo-electron micrograph containing frozen hydrated FHV puffed particles (highlighted with red arrows). Scale bar, 50 nm. (B) FSC curve, highlighting the resolution of the reconstruction estimated at FSC 0.143 (dashed line). (C) Central section of the reconstructed puffed particle with density shown in gray and the particle diameter (light blue line) and RNA shell diameter (light green line) marked. (D) Central section of the radially colored cryo-EM density map of puffed particle with a dotted arrow pointing to the merging of interior RNA shell density with the capsid density at the protruding site. (E) Surface rendered, radially colored density map of puffed particle shown in different orientations with a color key (right) displaying the radial distance (in Å) from the particle center. A dotted arrow points to the 2-fold symmetry axis on the density map.

DISCUSSION

This study represents the first comprehensive, high-resolution structural study of FHV disassembly, which has revealed details of conformational changes in the icosahedral capsid leading to genome release. Although FHV is not a human pathogen, it is a convenient and minimal model system, reinforced by a wealth of biochemical and structural information and the availability of several variants (27). Studies related to the mechanism of capsid maturation and the positioning of membrane-penetrating peptides, though initially conducted on FHV, were found to be applicable to various other nonenveloped viruses (27). Thus, the structural changes mapped in this study might be applicable to nonenveloped virus disassembly in general. We utilized incrementally increasing temperature as an easily accessible and controllable parameter to trigger disassembly in vitro. So far, reports studying viral structural changes as a function of temperature have been scarce in the literature (28–30). Although in this study, two conformational states have been characterized, it is possible that several other transition states exist and can be isolated from judiciously chosen points in the DSC profile of FHV, as well as by the application of additional, host-cell specific parameters such as low pH or ionic strength, in conjunction with incrementally increasing temperature. FHV eluted particles have been isolated from infected cells before, but application of this in vitro method allowed isolation of particles in sufficient quantities for conducting biophysical and structural studies, underlining the potential applicability of incremental heat-induced disassembly for other viruses. A schematic outlining the link between FHV maturation and disassembly established by this biophysical and structural study is shown in Fig. 9.

FIG 9.

Schematic outlining the link between FHV maturation and disassembly. FHV maturation involves autoproteolytic cleavage of capsid protein α into β and γ. The trigger(s) for disassembly causes externalization and release of γ peptide from mature virus, which is followed by a decrease in particle diameter of ∼4 nm. The RNA genome condenses and extends toward one part of the capsid. This or previous events may induce asymmetry in the eluted particle. The RNA genome eventually exits from around a 2-fold symmetry axis, as seen in the puffed particle, leading to the disintegration of the capsid. Unlike the mature virus, maturation-defective FHV with uncleaved γ peptide does not undergo distinct structural alterations into eluted or puffed particles before disintegration.

Comparison of the structural changes observed here with the previously reported uncoating-related alterations in other nonenveloped virus capsids reveals intriguing similarities, as well as differences. First, in contrast to the radial expansion (∼4%) seen for the picornavirus 135S particle (31), a reduction of ∼4 nm in the diameters of eluted particles was observed. No further change in particle diameter was detected until complete disruption of the icosahedral capsid. Such particle contraction has never been reported for any nonenveloped virus disassembly, although Triatoma virus, a member of the insect virus family Dicistroviridae, also did not expand during disassembly (8). It is possible that insect viruses may not follow the typical expansion seen during uncoating of picornaviruses, although the lack of intermediate structures from a sufficient number of virus families makes such a conclusion ambiguous. Second, incremental heating of FHV did not generate a stable, intact but empty capsid. Instead, the icosahedral cage of FHV became largely disordered following RNA release. This is a fairly significant observation as presence of RNA (genomic or noncognate) is known to be crucial for FHV capsid assembly (32). This rationalizes that following RNA release the stability of FHV capsid, unlike that of picornaviruses, will drastically decrease, leading to its disintegration.

A few interesting disassembly-related conformational similarities were also observed between FHV and picornaviruses. Localized expansion of 2-fold axes due to the movement of helices lining the region suggests that exit of genome occurs through this axis. Our work thus supports the growing evidence for the 2-fold axis to be the weak link on icosahedral capsids for disassembly and genome release (13, 14). Similar pore opening due to the separation of α-helices lining the 2-fold axis has also been observed previously for several picornaviruses (26). The presence of ordered duplex RNA at the 2-fold axis in the FHV capsid provides a feasible explanation for this axis being the portal for genome release (15). A global loss of the γ peptide, analogous to that of VP4 during picornavirus uncoating (26), and localized loss of capsid protein components (33) also emerged as noticeable common conformational alterations. Differential movement of individual capsid protein subunits observed in this study has been noticed previously for CAV7 capsid proteins (VP1, VP2, and VP3) during progression from a native capsid to an empty one (26). These differential movements in the capsid subunits are transmitted throughout the particle, which results in the local changes seen in the FHV intermediate particles. Further, our work in conjunction with disassembly studies on HRV2 suggests that compaction of genome is a fascinating but unstudied step during disassembly. It is possible that the encapsidated genome, perhaps by virtue of its organization in the form of a rod-shaped structure, contributes actively toward its own release from the virus shell, instead of being a passive entity during disassembly. Our work also resulted in capturing a disassembling particle in the process of genome release, which has so far not been isolated in a relatively stable form.

A growing body of evidence suggests that local differences may persist in otherwise highly symmetric, icosahedral particles (34, 35). These differences, which are usually lost by imposing icosahedral symmetry on particles during reconstruction, may have biological relevance in discrete steps in viral life cycles, including assembly, trafficking, and genome release. Loss of stability during disassembly of the capsid may also intensify asymmetry in particles. Thus, asymmetric 3D reconstruction was primarily used to reconstruct FHV intermediate densities, which helped to unravel both global and local conformational changes involved in genome release. To reinforce our hypothesis on the relevance of asymmetry, a 3D reconstruction of eluted particle with icosahedral symmetry imposed resulted in a smooth, spherical structure with minimal recognizable features, probably due to the averaging of surface features. Asymmetric reconstruction appears to be a more meaningful approach for studying symmetry loss and related structural changes during icosahedral virus disassembly (36).

In conclusion, asymmetric 3D reconstructions of FHV disassembly intermediates have provided substantial insight into the mechanism of genome release and the structural complexities in transient intermediates, in addition to providing a unique snapshot of a particle in the midst of releasing packaged RNA. It is hoped that this study will advance our knowledge of disassembly-associated conformational alteration during nonenveloped virus uncoating and lead to the identification of common features in the unraveling of stable icosahedral structures.

MATERIALS AND METHODS

Cells and viruses.

Drosophila melanogaster cells (DL-1 or Schneider’s line 1) were propagated at 27°C in Schneider’s insect medium, supplemented with 15% heat-inactivated fetal bovine serum (Gibco) and antibiotics (Pen-Strep [100 U/ml penicillin and 100 μg/ml streptomycin]; Gibco) as described previously (37).

Wild-type or maturation-defective (D75N/N363T) virus was produced by transfection of DL-1 cells with FHV RNA1 and RNA2 with TransFectin (Bio-Rad) using established protocols (19). RNA1 was generated as described previously (24), while RNA2 was in vitro transcribed using either a wild-type cDNA sequence corresponding to the capsid protein, or one containing the D75N/N363T mutations, as the template. Wild-type or maturation-defective FHV particles were purified from transfected DL-1 cells as previously described (24). Purified virus was dialyzed, concentrated, and stored at 4°C in 50 mM HEPES (pH 7.0; MP Biomedicals) buffer. Virus particles were quantified by using the optical density at 260 nm (20).

DSC.

Differential scanning calorimetry (DSC) measurements of wild-type and maturation-defective particles, in 50 mM HEPES (pH 7.0) and at a concentration range of 0.5 to 1.25 mg/ml, were carried out in a VP-DSC MicroCalorimeter (MicroCal), with a cell volume 0.5 ml, operating at a scan rate of 1.5°C/min. Samples were degassed before loading, and 50 mM HEPES (pH 7.0) buffer was used as a reference. Temperature scans for all samples were carried out from 50 to 100°C, in order to determine the temperature of endothermic transitions.

Incremental heating of virus particles.

For each experiment, aliquots of wild-type or maturation-defective, purified particles, stored at 4°C in 50 mM HEPES (pH 7.0), were heated to a specified temperature for 30 min each, using a thermal cycler (Bio-Rad C1000) operating at a ramp rate of 0.1°C/s. After incubation at the indicated temperature, the samples were cooled immediately on ice for 2 min and used directly for further analysis.

CD spectroscopy.

A circular dichroism (CD) spectrometer (model 420SF; Aviv Biomedical, Lakewood, NJ) was used to measure the far-UV spectra of untreated or heat-treated particles, in a wavelength range of 207 to 260 nm, using a quartz cuvette with a path length of 1 mm and a bandwidth of 1 nm. Wild-type or maturation-defective particles, at a concentration of ∼0.2 mg/ml in 50 mM HEPES (pH 7.0) buffer, were used. Each CD spectrum was an average of three scans, and every scan was corrected for contribution from buffer.

Fluorescence spectroscopy.

Intrinsic tryptophan fluorescence spectra of both wild-type and maturation-defective FHV, in 50 mM HEPES (pH 7.0) and at concentrations ranging from 0.2 to 0.4 mg/ml, either unheated or heated to different temperatures ranging from 25 to 90°C, were recorded using a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies) using a quartz cuvette with a 10-mm path length (Perkin-Elmer). Samples were excited at 295 nm, and emission spectra were collected at between 300 and 450 nm, with excitation and emission slit widths set at 5 nm. Three spectra were averaged for each sample, and every spectrum was corrected for contribution from buffer.

Trypsin digestion assay.

The cleavage sites of the enzyme trypsin within the capsid protein sequences of wild-type and maturation-defective FHV were predicted using PeptideCutter (ExPASy server [38]). TPCK-treated trypsin was added to the particles at a protease/virus ratio of 1:10 (wt/wt), and the reaction was allowed to proceed for 1 h at 37°C. Wild-type or maturation-defective FHV particles, in 50 mM HEPES (pH 7.0) and at a concentration of 0.5 to 0.7 mg/ml, either unheated or heated to temperatures ranging from 25 to 90°C, were subjected to trypsin digestion, and the resultant cleavage products were analyzed by SDS-PAGE on a 18% tricine gel, followed by silver staining.

Transmission electron microscopy.

Next, 5-μl portions of unheated or heat-treated, wild-type or maturation-defective particles, at a concentration of 0.5 mg/ml, were allowed to adsorb onto the surface of a glow-discharged, 300-mesh, carbon-coated copper grid (Electron Microscopy Sciences) for 2 min. Excess solution was removed with filter paper (Whatman), and grids were stained with 2% (wt/vol) uranyl acetate for 2 min, followed by blotting and air drying. Grids were viewed in a FEI Tecnai TF20 microscope operating at 200 keV at a magnification of 50,000×.

Immunogold labeling of virus particles.

Then, 5-μl portions of unheated or heat-treated, wild-type or maturation-defective particles were adsorbed onto a glow-discharged, carbon-coated copper grid (300 mesh) for 2 min. The grid was then blotted to remove excess solution, followed by incubation with blocking buffer (1% [wt/vol] bovine serum albumin in 1× Tris-buffered saline [TBS]) for 15 min to prevent nonspecific interactions. Blocking was followed by incubation of the grid with primary antibody (rabbit anti-FHV gamma antiserum; 1:100 in TBS) for 60 min at room temperature. After a washing with TBS and incubation in blocking buffer for 5 min, the grids were treated with a goat anti-rabbit IgG (whole molecule)-gold (Sigma-Aldrich) secondary antibody for 60 min at room temperature. The grids were washed with TBS and finally stained with a 2% (wt/vol) uranyl acetate solution for 2 min, air dried, and viewed in an FEI Tecnai TF20 microscope.

RNase treatment of puffed particles.

A 5-μl portion of RNase A (0.2 mg/ml; Qiagen) was added to 5 μl of wild-type virus (0.5 mg/ml) and heated to 75°C, and the reaction mixture was incubated at 37°C for 15 min with periodic, gentle mixing. Postincubation, the mixture was immediately placed on ice for 1 min and adsorbed onto a glow-discharged, carbon-coated copper grid (300 mesh) for 2 min. The grids were stained with a 2% (wt/vol) uranyl acetate solution for 2 min, air dried, and viewed in an FEI Tecnai TF20 microscope.

Viral RNA extraction.

Viral RNA was extracted from ∼0.2 mg/ml of untreated or heat-treated, wild-type or maturation-defective FHV particles, using an RNeasy minikit (Qiagen) according to the manufacturer’s instructions. RNA was eluted in 40 μl of RNase-free water and stored at –80°C. Extracted viral RNA was analyzed on a nondenaturing 0.8% agarose gel, stained with 0.5 μg/ml ethidium bromide, and visualized under UV light.

RNase protection assay.

Unheated or heat-treated, wild-type or maturation-defective FHV particles at a concentration of 0.4 mg/ml were incubated with 10 μl of RNase A (1 mg/ml; Qiagen) at room temperature for 15 min. Subsequently, the samples were treated with 10 μl of proteinase K (1 mg/ml; Qiagen) at 37°C for 30 min to inactivate the RNase, followed by RNA extraction and analysis on a nondenaturing 0.8% agarose gel as described above.

3D reconstruction from negatively stained images.

Heated FHV particles were stained with uranyl acetate as described above, and the grids were viewed on an FEI Tecnai TF20 (200 keV) at a nominal magnification of 50,000×, corresponding to a pixel size of 2.21 Å per pixel. Images were acquired using a 4k×4k Eagle charge-coupled device camera with a defocus range of 0.5 to 3 μm. In total, 5,528, 11,122, and 1,761 particles were manually picked, extracted, and processed from 302, 606, and 652 contrast transfer function (CTF)-corrected micrographs (using CTFFIND4 [39]) of wild-type FHV, eluted particles, and puffed particles, respectively, using RELION-2.0 (23). The particles were sorted by 2D, as well as 3D, classification and selected for further 3D refinement. RELION-2.1 was used to generate a 3D initial model de novo from 2D class averages with no symmetry imposed, which was used as a reference map for 3D classification (with no symmetry imposed), as well as 3D refinement (with or without icosahedral symmetry imposed). After 3D autorefinement, the maps were sharpened using the postprocessing procedure in RELION. The global resolution of the 3D maps for each sample was estimated using a Fourier shell correlation (FSC) of 0.143 (the gold standard) in RELION, while the local resolution variability of the maps was determined in ResMap (40). Final density maps were visualized and analyzed using UCSF Chimera (41).

Cryo-EM data collection and image processing.

Portions (4 μl) of purified wild-type FHV particles at a concentration of 1.5 mg/ml were heated to 70°C and applied to glow-discharged 400-mesh Quantifoil R2/2 holey carbon grids. The sample was blotted for 3 s at 100% humidity, followed by plunge freezing of the grid in liquid ethane cooled by liquid nitrogen using a Vitrobot Mark IV (FEI). The frozen grids were imaged using a 300-keV FEI Titan Krios cryo-electron microscope (FEI) equipped with a K2 Summit direct electron detector (Gatan) and a Quantum LS imaging filter (Gatan). Movies (20 and 40 frames) were collected at a nominal magnification of 81,000× in counting mode, corresponding to a pixel size of 1.7407 Å per pixel and with a dose per frame of ∼0.5 to 1.0 e^–/Ų. Images were collected with a defocus range of 0.5 to 3 μm using the automated image acquisition software EPU (FEI).

MotionCor2 was used to correct the electron beam-induced sample motion in the movies, and both dose-weighted summed images for cryo-EM reconstructions and unweighted summed images for CTF estimation (42) were generated. All subsequent data processing steps were carried out using RELION-2.1. The contrast transfer function (CTF) was determined with CTFFIND4 (39) using the unweighted summed images. For eluted particle reconstruction, 61,905 particles were manually picked from 2,628 selected micrographs, while for puffed particle reconstruction 1,301 particles were manually picked from 766 selected micrographs from a total of 2,835 micrographs and sorted into 2D class averages. The selected good class averages (comprising of 58,918 eluted particles and 1,293 puffed particles) were utilized to generate the 3D initial models de novo, which were used as reference maps for 3D classification and autorefinement. The resulting density maps were first masked appropriately and then sharpened using postprocessing in RELION. All resolution values are based on an FSC 0.143 cutoff criterion. Local resolutions were estimated using ResMap.

Atomic model refinement.

Segmentation of the density corresponding to iASUs from fine and coarse parts of the reconstructed cryo-EM density map of eluted particle was performed using Segger (43) in Chimera. The crystal structure of the wild-type FHV iASU (PDB ID 4FTB) was used as a starting model and fitted manually to the segmented densities, followed by rigid-body fitting in Chimera. Subsequently, the fitted model structure was manually corrected using Coot (44), followed by real-space refinement in PHENIX (45). The quality of the refined structures was assessed with the MolProbity (46) tool in the PHENIX software package. Refinement statistics are listed in Table 2.

Data availability.

Cryo-EM density maps of FHV eluted particle and puffed particle were deposited into the Electron Microscopy Data Bank under accession codes EMD-9730 and EMD-9732, respectively. The atomic coordinates of the iASU of the fine and coarse parts of FHV eluted particle were deposited into the Protein Data Bank under PDB accession codes 6ITB and 6ITF, respectively.