S.
Azinas
ab,
F.
Bano
b,
I.
Torca
c,
D. H.
Bamford
d,
G. A.
Schwartz
e,
J.
Esnaola
c,
H. M.
Oksanen
d,
R. P.
Richter
*bf and
N. G.
Abrescia
*ag
aMolecular recognition and host–pathogen interactions programme, CIC bioGUNE, CIBERehd, Derio, Spain
bBiosurfaces Lab, CIC biomaGUNE, San Sebastian, Spain
cMechanical and Industrial Production Department, Mondragon University, Arrasate-Mondragón, Spain
dMolecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, Viikki Biocenter, University of Helsinki, Finland
eCentro de Física de Materiales, (CSIC-UPV/EHU) & Donostia International Physics Center, San Sebastian, Spain
fSchool of Biomedical Sciences, Faculty of Biological Sciences, School of Physics and Astronomy, Faculty of Mathematics and Physical Sciences, and Astbury Centre for Structural Molecular Biology University of Leeds, Leeds, UK. E-mail: R.Richter@leeds.ac.uk; Tel: +44 113 3431969
gIKERBASQUE, Basque Foundation for Science, Bilbao, Spain. E-mail: nabrescia@cicbiogune.es; Fax: +34 946572502; Tel: +34 946572523
First published on 16th April 2018
The protection of the viral genome during extracellular transport is an absolute requirement for virus survival and replication. In addition to the almost universal proteinaceous capsids, certain viruses add a membrane layer that encloses their double-stranded (ds) DNA genome within the protein shell. Using the membrane-containing enterobacterial virus PRD1 as a prototype, and a combination of nanoindentation assays by atomic force microscopy and finite element modelling, we show that PRD1 provides a greater stability against mechanical stress than that achieved by the majority of dsDNA icosahedral viruses that lack a membrane. We propose that the combination of a stiff and brittle proteinaceous shell coupled with a soft and compliant membrane vesicle yields a tough composite nanomaterial well-suited to protect the viral DNA during extracellular transport.
Fig. 1 (a) Schematic of PRD1 highlighting the main structural features of the virus particle. (b) Protein composition of wt PRD1 and subviral particles analysed by SDS-PAGE and Coomassie staining. Left: Molecular masses (M; kDa). Right: Positions of the most abundant proteins; the vertical black line refers collectively to membrane proteins (MPs, including P14, P16, P18, P20, P22, P31 and P32). Cementing protein P30 identified in the P3-shell by mass spectrometry is indicated by a black arrowhead. (c–f) Atomic force micrographs of wt PRD1 and subviral particles with scale bar (100 nm) and z range (75 nm) indicated in (f). (c) Wt PRD1 featuring different particle orientations. Inset: Crystal structure of wt PRD1.7 Yellow, green, cyan and blue denote the P3 pseudo-hexameric capsomers composing the icosahedral asymmetric unit; white lines delineate a virus facet; white pentagons, triangles, and ovals indicate icosahedral 5-, 3- and 2-fold symmetry axes, respectively; (d) Sus1 procapsids (no DNA within). Arrowheads indicate the visible depression due to the missing packaging portal in the unique vertex. Inset: Schematic of Sus1 procapsid; (e) P3-shell. Arrowheads highlight the depressions visible at all vertices due to the absence of the peripentonal capsomers and vertex complexes. Right inset: The star-shaped vertex depression at higher resolution (z range: 15 nm). Left inset: Schematic of P3-shell. Arrowheads indicate some of the de-capped vertices. (f) DNA-filled vesicle. Inset: Schematic of vesicle represented as an icosahedron for clarity and consistency with the wt PRD1 schematic representation – this shape, however, might not be retained in the purified membrane-vesicle. |
Guided by the available genetic, biochemical, and structural information, we chose to utilize (i) wild type PRD1 (wt PRD1), (ii) a PRD1 mutant that forms procapsids devoid of DNA (Sus1 procapsid), (iii) the icosahedral P3-shell composed of MCP P3 and the minor capsid protein P30 (Fig. 1b and Table S1†), but lacking the pentons and peripentonal capsomers (P3-shell), and (iv) proteo-lipidic membrane vesicles enclosing a complete genome (vesicle; Fig. 1b). We used AFM to examine the mechanical responses of these particles in an aqueous environment by assessing their stiffness and yield behaviour under an applied force and used finite element modelling, where possible, to aid the analysis.
For the production of wt and mutant phage particles, DS88 cells were infected using a multiplicity of infection of 8–10. For mutant particle production, infected cells were collected 15 min after infection (Sorvall rotor F12, 5000 rpm, 10 min, 22 °C) and transferred to a fresh pre-warmed medium. Virus particles were purified by polyethylene glycol–NaCl precipitation and rate zonal ultracentrifugation in sucrose (Sorvall rotor AH629), as previously described.19 For AFM, wt PRD1 and Sus1 procapsids were further purified by equilibrium ultracentrifugation in sucrose (Sorvall rotor AH629). The particles were concentrated by differential centrifugation (Sorvall rotor T647.5, 32000 rpm, 2 h, 5 °C). A buffer containing 20 mM potassium phosphate pH 7.2 and 1 mM MgCl2 was used for purification and resuspension.
For P3-shell preparation, the rate zonal purified Sus1 mutant particles (2 mg ml−1 in 20 mM Tris-HCl, pH 7.2, 1 mM MgCl2) were treated with 1% (w/v) sodium dodecyl sulfate (SDS) for 15 min at 25 °C.20 P3 shells were isolated by rate zonal centrifugation in a linear 5–20% (w/v) sucrose gradient using the Tris buffer (Sorvall rotor AH629, 24000 rpm, 1 h 45 min, 20 °C). The particles were concentrated by centrifugation (Sorvall rotor T865, 34000 rpm, 4 h, 5 °C) and resuspended in Tris buffer.
For membrane vesicle preparation, the rate zonal purified Sus607 particles devoid of the major membrane protein P11 (1 mg ml−1 in 20 mM Tris-HCl, pH 7.2) were treated with 2.5 M GuHCl21 and membrane vesicles were purified by equilibrium centrifugation in a linear 20–70% (w/v) sucrose gradient (Sorvall rotor TH641, 22000 rpm, 16–18 h, 20 °C). Protein concentrations were determined by Bradford assay.22 Particles were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis23 (SDS-PAGE; 16% acrylamide; Fig. 1b).
Virus particles were stored at 4 °C for no more than 4 weeks. During this time, more than 95% of the wt PRD1 particles remained intact and without the loss of their genome as visualized by cryo-electron microscopy (cryo-EM) 2D imaging [a JEM-2200FS (JEOL) transmission electron microscope equipped with an UltraScan 4000 SP 4k × 4k camera (GATAN)] (Fig. S2†). Sus1 procapsid, P3-shell particles and vesicles were similarly stored and also used within this time frame (Fig. S2†).
Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis was carried out on an EASY-nLC1000 (Thermo Fisher Scientific, Germany) connected to a Velos Pro-Orbitrap Elite hybrid mass spectrometer (Thermo Fisher Scientific) with a nano-electrospray ion source (Thermo Fisher Scientific). The LC-MS/MS samples were separated using a two-column setup consisting of a 2-cm C18-Pepmap trap column (Thermo Fisher Scientific), followed by a 15-cm C18-Pepmap analytical column (Thermo Fisher Scientific). The linear separation gradient consisted of 5% buffer B in 5 min, 35% buffer B in 60 min, 80% buffer B in 5 min and 100% buffer B in 10 min at a flow rate of 0.3 μl min−1 (buffer B: 0.1% TFA acid in 98% acetonitrile). Six microliters of sample were injected per LC-MS/MS run and analyzed. Full MS scan was acquired with a resolution of 60000 in the normal mass range in an orbitrap analyzer and followed with CID-MS2 top 20 most intense precursor ions within the ion trap (energy 35). Data were acquired using LTQ Tune software.
The acquired MS2 scans were searched against the enterobacteria phage PRD1 (NCBI) protein database using the Sequest search algorithms in Thermo Proteome Discoverer. The allowed mass error for the precursor ions was 15 ppm and for the fragment 0.8 Da. A static residue modification parameter was set for carbamidomethyl +57021 Da (C) of the cysteine residue. Methionine oxidation was set as dynamic modification +15995 Da (M). Only full-tryptic peptides were allowed for the maximum of one missed cleavage.
Nano-indentation measurements were performed at individually selected particles using the ‘point-and-shoot’ function within the NanoScope software. Briefly, the area of interest was first imaged to localize the virus particle; the ‘point-and-shoot’ function was then activated and force curves were taken at the particle centre. subsequently, the area was imaged once more to verify successful nano-indentation. The accuracy of localization of the particle centre was found to be limited by piezo drifts and estimated to be within 5 nm. Force vs. distance (F/z) curves were acquired at a constant approach velocity of 200 nm s−1. The approach and retract distances were 100 nm, corresponding to a total time of 1 s per complete approach and retract cycle. The maximal load was 4 nN, except for P3-shells, where the maximal load was lowered to 2 nN. F/z curves were analysed using the NanoScope software.
To avoid including particles that might have been displaced or changed orientation upon indentation, force curves where force levels dropped and remained below 500 pN over distances of 20 nm and more before hard-wall contact were also discarded from analysis. A representative force curve for each PRD1 particle type with AFM micrographs before and after indentation is shown in Fig. S3,† and additional force curves illustrating sample to sample variations are shown in Fig. S4.†
Fig. 3 Comparison of the average mechanical properties across the different PRD1-derived particles. In all panels: black bar, PRD1 wt; pink bar, Sus1 procapsid; blue bar, P3-shell; orange bar, vesicle. (a) Stiffness (k); (b) yield force (Fy); (c) yield strain (εy); (d) toughness (T). All panels report the mean values and standard errors of the mean that were derived from the data shown in Fig. 2 (for the vesicle, calculation of toughness is not possible because there is no defined yield point). |
The F/z curves typically exhibited a relatively small non-linear regime at the smallest indentation forces (F < 200 pN), which is likely to represent the Hertzian deformation of the capsid shell.27 This was followed by an extended linear regime, justifying the quantification of elastic properties in terms of the stiffness k. For quantitative stiffness analysis, we considered this linear regime for strains up to 10%, which was well below the yield point. For quasi-spherical shells such as the Sus1 procapsid and the P3-shell, this linear regime can be associated with shell bending.27
Wt PRD1 possessed the greatest stiffness (0.57 ± 0.03 N m−1, mean ± s.e.m.), followed by the Sus1 procapsid (0.39 ± 0.02 N m−1) and the P3-shell (0.22 ± 0.01 N m−1; Fig. 3a). Most likely, the enhanced resistance of wt PRD1 to elastic deformation arises from the pressure exerted by the DNA.8,30 PRD1 genome packaging produces a radial expansion of the internal membrane (e.g., the radius of the outer leaflet increases by ∼6%), which presses the vesicle closer to the capsid.12,28,30
The DNA-filled vesicle displayed the least stiffness (0.022 ± 0.002 N m−1; Fig. 3a), indicating that its direct effect on virion stiffness is marginal but that it contributes to virion stiffness by transmitting pressure from the DNA to the capsid. The volume inside the rigid capsid is reduced upon indentation accentuating the effect of DNA pressure on the stiffness of wt PRD1. In contrast, the soft membrane can stretch upon indentation, thus reducing any effect of DNA pressure on the vesicle stiffness.
Fig. 4 (a) Schematic of the spherical two-shell model (outer protein shell in blue, inner vesicle shell in yellow) for the finite-element modelling; three-quarters of the spheres are shown with the indenter apex represented as a grey sphere with the direction of the applied force as indicated by the black arrow; in the bottom right corner, the Cartesian coordinate system; (b) predicted curves of force vs. indentation for a protein shell (with E = 0.13 GPa, representing the P3-shell; black dots), a vesicle shell (with E = 0.021 GPa, representing the proteo-lipidic vesicle; red dots) and a composite of these protein and vesicle shells (blue dots). In matching colours: lines are linear fits through the origin, and texts the stiffness values corresponding to the slopes. The stiffness values of protein and vesicle shells match the experimental data for P3-shell and vesicle (Fig. 3a), respectively, confirming the correct choice of Young's modulus values; the stiffness of the composite is well approximated by the sum of the stiffness values of the constituent shells and thus only marginally larger than the stiffness of the protein shell alone. |
To extract material properties from the experimental data, we first considered the proteinaceous capsid individually. For the capsid, we estimated an outer radius R = 33.2 nm and a thickness d = 8 nm from the rotationally averaged electron density maps of the PRD1 virion.28,30 By treating the capsid as isotropic and linearly elastic, its properties can be described by two independent parameters: Young's modulus E and the Poisson ratio ν. By neglecting a non-linear regime at very small strains (ε < 3%), the predicted relationship between force and particle indentation δ was approximately linear and scaled with the Young's modulus E for indentations up to 2d (ε up to 24%; Fig. S6a†). Our results were rather insensitive to the Poisson ratio (Fig. S7a–c†), and we thus fixed ν = 0.4.
These features are consistent with previous computations by others and with the predictions of thin-shell theory,4,31,32 and thus validate the numerical model. The linear relationship between force and indentation is also consistent with our experimental data (Fig. S3b, e, h, and S4a–d†), where we note that the strain regime of ε < 10% used for the analysis of experimental data lies well within the range over which theory predicts a linear response. This implies that stiffness analysis is far away from any buckling transition.33 It justifies the use of linear elasticity in our simple theoretical model, and also the use of stiffness k = F/δ to characterize the shell's elastic properties. With F/E ∼ δ and k = F/δ, it is also clear that k ∼ E, and a linear fit to the data in Fig. S6a† gives k ≈ 1.65 nm × E. With this equation, we estimate E = 0.13 GPa for the P3-shell from the experimentally determined mean stiffness value for this particle (k = 0.22 N m−1; Fig. 3a).
A recent computational modelling study on a smaller non-membrane-containing virus has shown that capsid proteins can dynamically re-structure upon capsid indentation with appreciable effects on capsid elasticity compared to an idealized homogeneous shell.34 Our experimental data do not allow deconvoluting these effects. However, the extended linear regime in the F/z curves observed experimentally for the P3-shell and the Sus1 procapsid (Fig. S4c and d†) indicates that the bending elasticity of the PRD1 capsid shell remains appropriately described by a Young's modulus (where this would effectively include the possible re-structuration effects).
The elasticity of the proteolipidic membrane was estimated analogous to that of the proteinaceous capsid, assuming an outer radius identical to the inner radius of the capsid (R = 25.2 nm) for direct contact of the two shells, and a membrane thickness d = 5.5 nm from a rotationally averaged electron density map.30 Fig. S6b† shows the dependence of F/E on vesicle deformation for these geometrical parameters, from which k ≈ 1.07 nm × E can be derived. Assuming to a first approximation that the stiffness of the membrane shell is similar to the experimentally accessible value for the genome-containing vesicle (k = 0.022 N m−1; Fig. 3a), we can estimate E = 0.021 GPa.
To predict the elastic behaviour of the composite capsid-membrane system, we modelled a system of two concentric shells with the inner shell adopting the geometry and Young's modulus of the membrane and the outer shell adopting the geometry and Young's modulus of the P3-shell (Fig. 4a). The stiffness of this composite system was k = 0.24 N m−1, that is the presence of the vesicle enhanced the stiffness only marginally, by about 10%, compared to the P3-shell alone. More generally, the stiffness values shown in Fig. 4b exemplify that the stiffness of the composite (0.24 N m−1) is well approximated by the sum of the stiffness values of the constituent shells (0.22 N m−1 + 0.02 N m−1). The small enhancement in stiffness was virtually independent of the Poisson ratios of both the capsid and membrane (Fig. S7d†).
The above modelling exercise provides reasonable predictions about the trends that can be expected based on the experimental AFM-derived shell elastic mechanical properties. Here, we have operated with the P3-shell as a reference system because experimental data for this single-shell system were readily available. Using the above-identified stiffness relationship, we can now also estimate the Young's modulus of the complete capsid shell from the experimentally determined stiffness values of the Sus1 procapsid and the membrane. The closure of 11 of 12 vertices by additional proteins enhances the elasticity of the Sus1 procapsid shell compared to the P3-shell, whilst a further enhancement of the capsid elasticity by the unique vertex – missing in the Sus1 procapsid – is likely to be marginal. kcapsid ≈ kcapsid+membrane − kmembrane ≈ 0.39 N m−1 − 0.02 N m−1 = 0.37 N m−1 (Fig. 3a) and E ≈ k/1.65 nm (Fig. S5a†) give E ≈ 0.22 GPa, a value comparable to that for some non-enveloped and enveloped viral capsids.35
Indeed, the two-shell modelling confirmed that the stiffness is only marginally affected, by less than 10%, by an internal soft membrane contacting the capsid (Fig. 4b). The reduced stiffness of the P3-shell (E = 0.13 GPa) is likely due to the absence of the stabilizing pentons and peripentonal capsomers.
The mechanical failure of the PRD1 particles upon indentation was frequently accompanied by the loss of capsomers from the capsid shell (Fig. S3a–c,† insets). In addition, F/z curves of wt PRD1, the Sus1 procapsid, and the P3-shell revealed slip events coincident with the occurrence of micro-fractures during force loading likely reflecting the local displacements of MCPs (Fig. S5†). While all three particle populations presented a similar density of micro-fractures (average 1.3 per nN of applied compressive force), wt PRD1 and the procapsid withstood more of these fractures before yielding.
Previous studies on binary component viral systems – genome encapsulated by a protein shell – have highlighted the role of DNA or RNA in contributing to the capsid stiffness where the mechanical reinforcement is achieved by the genome anchoring the protein shell from the interior.35 In other cases, such as the herpes simplex virus type 1 (HSV-1) nucleocapsid, the stiffness and yield force remain practically the same whether the particle is fully packaged or devoid of the genome, and stabilizing viral proteins appear to be responsible for this assembly type.37,38 In PRD1, the Sus1 procapsid and the mature particle display similar yield forces whereas the relative increase in the stiffness of the wt PRD1 can be attributed to the presence of DNA. The packaging of the genome leads to an expansion of the membrane-vesicle increasing the membrane's interactions with the capsid proteins.12,28–30
Altogether, these comparisons highlight that the layered complex of the capsid and membrane vesicle relieves the genome from a stabilizing role and endows PRD1 with remarkably high mechanical stability.
To explore how the PRD1 vesicle and capsid together influence toughness, we compared the toughness of the PRD1-derived particles: the Sus1 procapsid, the P3-shell, and the vesicle. The P3-shell (T = 0.54 × 105 J m−3) was more susceptible to mechanical failure than the Sus1 procapsid (T = 2.1 × 105 J m−3; Fig. 3d). The fact that the P3-shell's toughness was ∼4-fold less but its stiffness was only 2-fold less revealed its rather stiff, brittle nature (Fig. 3a). The P3-shell lattice is held together by the (C-I type and C-II type) interactions established by the GON within each facet and along the facets via the MCPs C-termini and by the P30 proteins7 (Fig. S1a†). The relative ease of breakage of this lattice might facilitate morphological corrections as the capsomers assemble on the vesicle mould during the procapsid formation. Closing the icosahedral vertices with the peripentonal capsomers and penton proteins (Fig. S1a†) and plugging the unique vertex with the portal complex produce a stable procapsid that can withstand dsDNA translocation powered by the packaging ATPase P9.12
More generally, the brittle nature of the capsid is not only manifested in the small yield force of the P3-shell but also in the above-mentioned microfractures. The DNA-filled vesicles, on the other hand, did not show a clear yield point and typically recovered their original shape even after strains exceeding 60%, which indicated that they were ductile and very soft (Fig. 3a).
Thus, comparative analysis with other dsDNA viruses indicates that the toughness of PRD1 is superior to most other dsDNA viruses with comparable capsid organization but lacking the membrane, and is only rivalled by adenovirus – a non-membrane-containing virus – which is exceptionally rich in cementing proteins stabilizing the capsid (Fig. 5).
Fig. 5 PRD1's mechanical properties compared to other dsDNA viruses. Key for the viruses and particle types shown in the other panels (in the case of HSV-1 virion, we refer to its nucleocapsid). Comparison across viruses via Ashby plots of toughness (y-axis in log scale) vs. stiffness, yield force and yield strain (x-axis), respectively (see the Table S2† for references). |
Fig. 6 (a) Multilayered structure of an avian eggshell; (b) cryo-electron density of wt PRD1 with an octant removed displayed in Chimera45 to show (inset) the layered/composite structure of the virion with the proteinaceous shell in blue, the membrane-vesicle in yellow-lime (OL: outer leaflet; IL: inner leaflet) and with the horizontal black lines marking the distinct layers and the interacting matrix region between the capsid and the membrane. As cartoon representation in red fitted in density the P3 MCPs with N-terminal α-helices interacting with the OL and the P16 transmembrane protein crossing the membrane vesicle. The dsDNA has been removed from within the membrane vesicle for clarity; (c) schematic of a composite sandwich material to which (a) and (b) recapitulate. |
Altogether, these layers constitute a tough composite material that protects the progeny against mechanical stress while carrying out other essential functions. Analogously, the PRD1 capsid and vesicle are bonded to protect the integrity of the virion and its genome (Fig. 6b). Specifically, the connection between the capsid and membrane vesicle is made of polypeptide stretches: the capsid shell – composed of 240 copies of interacting trimers of the MCP P3 and glued at the icosahedral edges by the cementing protein P30 – anchors the membrane through several N-termini of MCPs P3; this connectivity is further augmented by the anchoring N-terminal transmembrane helix of protein P16 located at the base of the peripentonal MCPs P37 (inset in Fig. 6b).
Moreover, it is conceivable that during force loading onto the capsid, these connectors will act as nano-staples increasing the capability of the system of absorbing energy before mechanical failure (an additional energetic cost would be necessary to disrupt protein–membrane interactions44).
In summary, we here presented the nanomechanical characterization of a virus that features a membrane inside its capsid. The combination of a stiff, yet brittle, proteinaceous capsid with a soft proteolipidic vesicle in PRD1 facilitates multiple stages of the virus life cycle, including virus assembly, mechanical protection for the extracellular virion, and the vesicle-to-tube transformation during DNA ejection.13 From a broader perspective, it appears that the evolution of membrane-containing viruses has yielded, at the nanometer scale, composite properties comparable to those known for macroscopic natural materials, where the capsid and vesicle are bonded into a tough composite that protects the integrity of the virus and its genome.
Our findings provide both foundational quantitative information and inspiration that can encourage the engineering of tough nanoscale devices and particles capable of protecting fragile cargos.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8nr00196k |
This journal is © The Royal Society of Chemistry 2018 |