Engineering Recombinant Virus-like Nanoparticles from Plants for

Feb 15, 2017 - Understanding capsid assembly following recombinant expression of viral structural proteins is critical to the design and modification ...
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Engineering Recombinant Virus-like Nanoparticles from Plants for Cellular Delivery Lou Brillault,† Philippe V. Jutras,‡,# Noor Dashti,‡,# Eva C. Thuenemann,§ Garry Morgan,∥ George P. Lomonossoff,§ Michael J. Landsberg,†,⊥ and Frank Sainsbury*,‡ †

Institute for Molecular Bioscience, ‡Australian Institute for Bioengineering and Nanotechnology, ∥Centre for Microscopy and Microanalysis, and ⊥School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, Australia § Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney, Norfolk NR4 7UH, United Kingdom S Supporting Information *

ABSTRACT: Understanding capsid assembly following recombinant expression of viral structural proteins is critical to the design and modification of virus-like nanoparticles for biomedical and nanotechnology applications. Here, we use plant-based transient expression of the Bluetongue virus (BTV) structural proteins, VP3 and VP7, to obtain high yields of empty and green fluorescent protein (GFP)-encapsidating core-like particles (CLPs) from leaves. Single-particle cryo-electron microscopy of both types of particles revealed considerable differences in CLP structure compared to the crystal structure of infection-derived CLPs; in contrast, the two recombinant CLPs have an identical external structure. Using this insight, we exploited the unencumbered pore at the 5-fold axis of symmetry and the absence of encapsidated RNA to label the interior of empty CLPs with a fluorescent bioconjugate. CLPs containing 120 GFP molecules and those containing approximately 150 dye molecules were both shown to bind human integrin via a naturally occurring Arg-Gly-Asp motif found on an exposed loop of the VP7 trimeric spike. Furthermore, fluorescently labeled CLPs were shown to interact with a cell line overexpressing the surface receptor. Thus, BTV CLPs present themselves as a useful tool in targeted cargo delivery. These results highlight the importance of detailed structural analysis of VNPs in validating their molecular organization and the value of such analyses in aiding their design and further modification. KEYWORDS: virus-like nanoparticle, bluetongue virus, cryo-EM, core-like particle, guest protein encapsidation, bioconjugation, receptor targeting plants has emerged as an efficient and flexible platform permitting facile coexpression of multiple capsid proteins, the rapid assessment of capsid protein modifications, and high fidelity assembly of the capsid particles.11,12 Central to engineering the properties of VNPs and understanding their interactions with their environment is detailed knowledge of their molecular organization. Secondary, tertiary, and quaternary structure is often assumed based on knowledge of the cognate virus; however, detailed analysis of modified or recombinant VNPs can reveal critical structural details explaining the functional behavior of the particle. Seminal work in this field used X-ray crystallography to guide the design of peptides presented on the surface of Cowpea mosaic virus (CPMV) particles such that they would elicit

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irus-like nanoparticles (VNPs) are emerging as versatile tools in nanotechnology as a consequence of the highly evolved control over their composition and morphology. Based on the protein shell of viruses, these supramolecular self-assembled nanoparticles comprise either infection-derived virions or recombinant virus-like particles lacking the infectious genome. In many cases, the capsid structure is known to nearatomic resolution, permitting precise rational modification of their structures by genetic and/or chemical means.1−3 Proof-ofconcept studies aimed at turning virus capsids into drug delivery vehicles and molecular imaging reagents demonstrate their amenability to, and the potential of, chemical conjugation to VNPs. 4,5 Likewise, modification of capsid-encoding sequences and manipulation of the diverse mechanisms viruses use to encapsidate protein and nucleic acid have also demonstrated the potential of a number of VNP platforms to encapsidate guest proteins.6−10 Among expression systems for the production of recombinant VNPs, transient expression in © 2017 American Chemical Society

Received: November 17, 2016 Accepted: February 1, 2017 Published: February 15, 2017 3476

DOI: 10.1021/acsnano.6b07747 ACS Nano 2017, 11, 3476−3484

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ACS Nano antibodies that could bind to the native antigen and not just the denatured form.13,14 Single-particle cryo-electron microscopy (cryo-EM) is particularly well suited to investigating the structure of symmetrical protein assemblies, such as virus capsids,15 and has been used to confirm the presentation16,17 and encapsidation18 of proteins by recombinant virus capsids. Cryo-EM of transiently expressed empty CPMV virus-like particles showed that they were structurally extremely similar to native CPMV virions and confirmed that the recombinant particles are devoid of RNA.19 Bluetongue virus (BTV) core-like particles (CLPs) are double-layered icosahedral protein shells consisting of 120 molecules of the VP3 subcore protein arranged with pseudo-T = 2 icosahedral symmetry, superimposed with 260 trimers of VP7 protein arranged in a T = 13 lattice.20 The particles have an average diameter of 70 nm, and the atomic structure of the BTV CLP derived from infectious virions has been solved to 3.5 Å.20 BTV CLPs can be produced by overexpression of VP3 and VP7 in eukaryotic cells,21,22 resulting in particles with an internal cavity 46 nm in diameter. Fusion of GFP to the Nterminus of VP3 results in fluorescent CLPs when coexpressed with VP7 in insect cells. This fusion was used to elucidate the kinetics and localization of particle assembly in vivo, although the fusion was unstable and it is not clear whether GFP was contained within the internal cavity of the CLPs.23 Here, we present the recombinant CLP of BTV as a VNP with specific cell-receptor binding capability and demonstrate its amenability to loading with guest protein and small molecule bioconjugates. Using single-particle cryo-EM analysis to guide and validate the modification of BTV VNPs, we found substantial deviations from the infection-derived CLP structure but found that the encapsidation of a guest protein did not affect the self-assembly, surface charge, or capsid structure of recombinant CLPs. In addition to the encapsidation of green fluorescent protein (GFP), we demonstrate that BTV CLPs are amenable to chemical modification by labeling addressable lysines of the interior surface of VP3. Finally, we demonstrate the biomedical potential of this VNP platform, which is efficiently and easily produced in leaves, to deliver proteins and small molecules using a natural affinity for human integrin via the multivalent presentation of canonical Arg-Gly-Asp (RGD) motifs.

Figure 1. Plant-based expression and purification of BTV CLPs. (A) Molecular models based on PBD entry 2BTV showing how the recombinant CLP is assembled through the deposition of VP7 trimers onto the VP3 subcore. (B) Schematic representation of the expression constructs used to generate CLPs and GFP-CLPs. (C) Representative photographs of density gradients following ultracentrifugation to isolate CLPs and GFP-CLPs. (D) Reducing SDSPAGE of isolated particles show the presence of CLP proteins the 103 kDa VP3 and the 39 kDa VP7. In the case of GFP-CLPs, VP3 exists as a 130 kDa fusion to GFP.

by a single-step density gradient ultracentrifugation process, enables the facile production of highly pure BTV CLPs. Using this procedure, we regularly obtain 200−250 μg of purified CLP/g fresh weight leaf tissue. Guest Protein Encapsidation. Using the incircle radius of the approximate triangle given by the interior face of VP3, r = 2.75 nm, and assuming an average partial specific volume for globular proteins of 1.212 × 10−3 nm3/Da,27 we predict that fusion to VP3 termini should permit the encapsidation of 72 kDa proteins within the BTV CLP. Staying well below the theoretical limit in this instance, we fused GFP to the Nterminus of VP3 via a short Gly2Ser linker and coexpressed this with VP7. A greenish band can be observed at the same sedimentation position as unmodified CLPs on an iodixanol gradient (Figure 1C), which was shown to contain purified GFP-VP3 and VP7 (Figure 1D), as would be expected from the self-assembly of these proteins into GFP-containing CLPs (GFP-CLPs). TEM and SEC were used to demonstrate that empty and GFP-loaded CLPs are essentially indistinguishable in size and appearance from empty (unmodified) CLPs (Figure 2). DLS shows that both CLP (RH, 43.1 ± 0.3) and GFP-CLP

RESULTS AND DISCUSSION BTV CLP Expression and Purification. BTV CLPs selfassemble in vivo following the expression of two structural proteins, VP3 and VP7 (Figure 1A,B). Plasmid constructs for the expression of these proteins were created using the highyielding transient expression system based on hyper-translated CPMV RNA-2 regulatory regions.24,25 Co-infiltration of Nicotiana benthamiana leaves with agrobacterium cultures containing each plasmid is known to yield CLPs,22 which self-assemble in the cytosol.26 Density gradient ultracentrifugation of detergent-extracted and clarified leaf tissue expressing VP3 and VP7 yields a distinct band at a higher density than soluble plant proteins (Figure 1C). Reducing SDS-PAGE of the material in these bands shows that they contained the highly purified constituent proteins of BTV CLPs, the 103 kDa VP3 and 39 kDa VP7 (Figure 1D). Transmission electron microscopy (TEM), size exclusion chromatography (SEC), and native agarose gel electrophoresis (Figure 2) were used to confirm that these proteins are isolated as intact CLPs. Thus, the transient expression of BTV CLPs in plant leaves, followed 3477

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map are consistent with this, allowing us to resolve salient topographies of the secondary structure (Figure 3A) and unequivocally fit crystal structures previously determined for native BTV CLPs (Figure 3C). A striking difference to the virion structure is the absence of VP7 (P-)trimers at the 5-fold axis of symmetry, a feature that was also suggested by initial 2D and 3D classes (Supporting Information). Densitometric analysis of CLPs subjected to SDS-PAGE was used to confirm the presence of VP7 and VP3 at a ratio of 5:1, rather than the 13:2 ratio expected of native CLPs (Supporting Information). The phenomenon of missing P-trimers at the 5-fold axis has been noted previously in low-resolution cryo-EM reconstructions of insect cell-derived recombinant CLPs. This was thought to be related to either the stoichiometry of the available coat proteins or other parameters related to features of the recombinant expression system such as the absence of posttranslational processing and specific viral proteins that may be necessary for the binding of VP7.30 When expressed in plant leaves, full BTV virus-like particles possess a third protein layer consisting of VP2 and VP5 that relies on complete VP7 coverage, which indicates that there are no limitations on protein maturation in plant cells or requirements for additional BTV proteins.22 In a related Reovirus, P-trimers are believed to be the last VP7 trimers to be added to form the core particle,31 thus the missing P trimers in recombinant CLPs could simply be due to insufficient VP7 accumulation. Alternatively, the structure of recombinant CLPs may result from the loss of the P-trimer during purification. Indeed, the P-trimer interaction with the VP3 layer is mediated by the least favorable contacts among VP3-VP7 interactions.20 Nevertheless, it is clear that such deviation from the expected structure based on the infection-derived core particle has considerable implications for the modification and application of VNPs. Furthermore, the electron density map indicates that neither RNA nor host proteins interact with the interior face of VP3, which is an important consideration for internal modification of VNPs.32−34 Thus, the single particle Cryo-EM analysis reported here not only validates the structure of recombinant BTV CLPs but also suggests opportunities for their further engineering as VNPs. Cryo-EM and subsequent single-particle analysis of GFPCLPs to examine the internal location of GFP yielded a 3D reconstruction at a resolution of 21.1 Å as estimated by Gold Standard FSC (Supporting Information). As expected, the size and the symmetry of the GFP-CLP are similar to the empty CLP (Figure 4A). The inferior resolution of the GFP-CLP is consistent with the smaller size of the GFP-CLP data set (approximately 20-fold fewer particle images) and the inferior microscope used to image the sample. To facilitate a more accurate comparison of the GFP-CLP and CLP particles, a second CLP reconstruction was produced using a randomly sampled subset of the data to produce the structure shown in Figure 4B at a resolution of 22.9 Å (Supporting Information). The middle slice profiles of both the high- and low-resolution CLP reconstructions and the GFP-CLP particle reconstruction indicate that the fused GFPs are located at the 5-fold axis of symmetry (Figure 4B). Radial density analysis shows the presence of additional density around 50 Å wide at a radius of about 200 Å. The extra density is not seen in either of the CLP profiles, which only show density profiles corresponding to the double layer core−shell extending from VP3 located at a radius of 250 Å to the VP7 trimeric spikes from about 275 to 350 Å. Thus, despite the relatively low protein mass and expected

Figure 2. Physical characterization of recombinant BTV CLPs. (A) TEM images of negatively stained CLPs and GFP-CLPs; scale bar = 200 nm. (B) SEC analysis of CLP and GFP-CLP preparations using self-packed Sephacryl S-500 media showing protein detection by absorbance at 280 nm (A280) and GFP-specific detection by absorbance at 488 nm (A488). (C) Native agarose gel electrophoresis of CLPs and GFP-CLPs showing staining with Coomassie and fluorescence detection following UV excitation.

(RH, 42.8 ± 0.6) preparations were highly monodisperse with average percent polydispersity of 8.6 and 6.9, respectively. The two types of CLPs migrated to the same point on native agarose gels, which separate VNPs on the basis of size and surface charge rather than internal components,28,29 indicating that their external composition is identical. As expected of GFPencapsidating particles, GFP-CLPs are fluorescent on native gels (Figure 2). In contrast to GFP-VP3 produced in insect cells,23 the GFP-VP3 isolated by the density gradient from plant leaves remains a fully intact protein fusion (Figure 1). The absence of free VP3 on reducing SDS-PAGE demonstrates that there is a full complement of 120 GFP molecules per GFP-CLP (Figure 1), which highlights the excellent capacity of BTV CLPs for guest protein encapsidation and the ease with which they can be engineered to encapsulate guest protein when expressed in plants. Single Particle Cryo-EM. Single particle cryo-EM was used to examine the structure of the recombinant BTV CLPs. Following 2D and 3D classification, a refined 3D map was produced with icosahedral symmetry imposed (Supporting Information). The resolution of this map, as estimated by Fourier Shell Correlation (FSC) 0.143 criteria applied to the unfiltered map following gold standard refinement, is 9.1 Å; however, estimates of local resolution show that this is clearly an underestimattion, likely to be heavily skewed by the large empty cavity inside the capsid shell. The local resolution varies between 5.5 and 9.5 Å, and the majority of the capsid shell itself is resolved to 6.0 Å or better. Structural details visible in the 3478

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Figure 3. Single-particle cryo-EM analysis of recombinant BTV CLPs. (A) Micrograph of frozen hydrated BTV CLPs; scale bar = 200 nm. (B) 3D reconstruction of empty recombinant BTV CLPs refined to 9.1 Å showing the external and internal views. (C) Fitting of the crystal structure of the native BTV CLP structure into the cryo-EM 3D reconstruction with P-trimers of the crystal structure shown in red. (D) Internal and clipped (selection) views of the crystal structure fitting, showing the VP3 (yellow and purple) and the VP7 trimer (blue and red); the missing P-trimers in the cryo-EM reconstruction are colored red on the crystal structure. Magnified view of the boxed selection shows the accurate fitting of the crystal structure into the cryo-EM 3D reconstruction.

detected. The fact that it is located about the 5-fold axis of symmetry could be due to the proximity of the VP3 N-terminus to this location or the favorable biochemical environment in this region of the interior face of VP3. Small-Molecule Conjugation. The BTV CLP is porous to egressing RNA at the 5-fold axis of symmetry and is thought to selectively import nucleoside triphosphates through a pore between A and B monomers of VP3 along the 2-fold axis.35 To explore the scope of BTV CLP use in nanotechnology, we sought to determine whether we could address the interior surface of CLPs, taking advantage of the unencumbered space about the 5-fold axis of the VP3 layer; the absence of internal proteins, which are normally located at the 5-fold axis in virions;36 and the absence of RNA, which is normally associated with much of the interior surface of VP3.36 Chemical bioconjugation to the interior surface could open opportunities to conjugate drugs and imaging agents for the use of CLPs in therapy or biomolecular imaging.33 As a model compound that has application in molecular imaging, we used the far-red fluorescent dye Cy5. Labeling addressable Lys residues with a soluble Cy5 N-succinimidyl (NHS) ester results in specific labeling of VP3 (Figure 5C). According to SEC, the yield of intact particles following labeling is approximately 60% (Figure 5B). Although DLS from a single sample (RH, 37.6 ± 0.3; % polydispersity, 5.2) indicates that CLPs are stable and remain monodisperse during labeling, some aggregation was observed during an ultrafiltration concentration step prior to SEC. TEM and native agarose gel electrophoresis also confirm that the Cy5-CLPs remain intact and that the labeling protocol does not change the external

Figure 4. Single-particle cryo-EM analysis of GFP CLPs. (A) Middle slice view of BTV CLPs refined at 9.1 Å (CLP) as well as at 22.9 Å (CLP-low) and GFP-CLPs at 21.1 Å. An example of the extra density observed inside GFP-CLPs is indicated with the arrowhead. (B) Radial density profiles derived from the middle slices. Density corresponding to VP3, VP7, and presumably, GFP is indicated.

absence of ordered orientation of the guest protein, which is fused via a flexible linker, a putative GFP density can be 3479

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ferrin,46 and integrin.47 αvβ3 integrin is overexpressed on numerous cancers and has been identified as a promising target for the directed delivery of chemotherapeutics.48 The RGD tripeptide motif binds to αvβ3/β5 integrins with high affinity49,50 and has been used to selectively target imaging agents and drugs to cancer cells with diagnostic and therapeutic effects.48,51 An RGD motif found on exposed loops at the top of BTV VP7 (Figure 6A) has been shown to be accessible to antibodies and, through mutation analysis, involved in CLP uptake by an insect cell line.52 Here, we sought to determine whether the RGD motif presented by VP7 on recombinant CLPs does, indeed, bind human integrin for potential delivery of cargoloaded BTV CLPs. A solid-phase binding assay53 was employed to show that both GFP-CLPs and Cy5-CLPs bind human αvβ3 integrin against a background of bovine serum albumin (Figure 6B). The specificity of this interaction was confirmed by competition with the established integrin peptide ligand, cRGD.49 Reduced binding of Cy5-CLPs is observed in the presence of increasing concentrations of the ligand (Figure 6C). The Cy5 signal appears to be relatively insensitive to competitive binding, which could be due to the multivalency of the RGD motif present on the BTV CLP and, therefore, its strong avidity for high-density, immobilized receptor. As a preliminary demonstration of the ability of BTV-derived VNPs to deliver therapeutic cargos to target cells, we investigated the interaction of BTV CLPs with MCF-7 human breast adenocarcinoma cells, an established model for RGD-mediated targeting.48,54 Confocal microscopy of MCF-7 cells incubated with Cy5-CLPs shows the interaction and potential internalization of Cy5-CLPs (Figure 6D). Cy5 can be detected as both bright puncta, possibly corresponding to uptake via endocytosis and as diffuse signal around the nucleus of all cells, indicative of cytosolic localization. The specific binding of functionalized BTV VNPs to integrin and their interaction with MCF-7 cells highlights their potential as cell surface receptor-targeted delivery vehicles.

Figure 5. Bioconjugation to reactive amines on the interior surface of BTV CLPs. (A) Incubation of CLPs with excess sulfo-Cy5 NHS ester to label interior amines. (B) SEC of labeled CLPs. (C) SDSPAGE analysis of labeled (+) and unlabeled (−) CLPs showing staining with Coomassie Blue and fluorescence detection. (D) TEM images of labeled CLPs; scale bar = 200 nm. (E) Native agarose gel electrophoresis of labeled (+) and unlabeled (−) CLPs showing staining with Coomassie and fluorescence detection following green illumination.

CONCLUSIONS Assumptions are generally made regarding the molecular structure of VNPs based on what is known of the structure of the cognate virus. Recombinant production of VNPs may result in capsids which are highly similar to the virus from which structural proteins are derived,19 or as is the case with the BTV CLP, the recombinant capsid may differ considerably, resulting in unexpected biochemical properties and an altered landscape for rational modification. Single particle cryo-EM is particularly suited to the analysis of VNPs as modifications that would affect crystal formation for X-ray diffraction would not affect cryo-EM grid preparation as long as the VNP remains soluble. Our results underline the utility and importance of structural analysis of VNPs in both confirming their molecular organization and aiding their further modification. We used single particle cryo-EM to demonstrate that encapsidated protein is associated with the interior surface of BTV CLPs and to determine that the interior surface of empty CLPs was amenable to bioconjugation of functional molecules. The VNP presented here, derived from the BTV CLP, is easily produced in plants, from where it can be isolated with a single purification step. We show that the BTV CLP is able to carry cargos of encapsidated protein or bioconjugated small molecules. Their potential to deliver diverse cargos to target cells was demonstrated by exploiting their natural affinity to human

properties of the particles (Figure 5D). Using the extinction coefficient of sulfo-Cy5 (A646, 271,000 mol−1·cm−1) and the calculated protein mass extinction coefficient of 1.075 (mg/ mL)−1cm−1 for recombinant CLPs, we determined that there are approximately 150 Cy5 molecules per CLP, or 1.25 labeled Lys residues per VP3 protein. Due to the absence of exposed Lys residues on the exterior face of VP3 (Supporting Information), we suggest the labeling is specific to the interior surface of VP3. These results indicate that BTV CLPs are porous to small molecules, significantly expanding the possible uses of this VNP in nanotechnology. In particular, the use of VNPs as nanoreactors for controlling encapsidated enzymatic activities is an area of great potential and relies on efficient protein encapsidation and diffusion of substrates into the particle.37−39 Integrin Binding of Fluorescent Particles. Specifically targeting VNPs to cell-surface receptors is a key aspect in their development as drug and imaging agent delivery vehicles. Targeting moieties, including small molecules,40 peptides,41 polypeptide ligands,42,43 and antibodies,44 can either be conjugated to reactive surface residues of VNPs or genetically engineered for display by VNP coat proteins. Alternatively, some VNPs have been exploited for their inherent tropism for cells possessing certain receptors such as vimentin,45 trans3480

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integrins and by using encapsidated entities to report binding to these medically relevant proteins and interaction with cells that overexpress them.

METHODS Materials. EDTA-free COMPLETE protease inhibitor tablets were purchased from Roche (www.roche.com). OPTIPREP density gradient media (60% iodixanol solution), cyclic RGDFV peptide (cRGD), Fluoroshield, and Corning black 96-well plates were purchased from Sigma-Aldrich (www.sigmaaldrich.com). Polyclear ultracentrifuge tubes were purchased from Seton Scientific (www.setonscientific. com). Disposable PD-10 desalting columns and Sephacryl S500 were purchased from GE Healthcare (www.gehealthcare.com), and 10K MWCO SNAKESKIN dialysis tubing, DMEM media, and Hoechst stain were purchased from Thermo Scientific (www.thermofisher. com). The NHS ester of sulfonated Cyanine 5 (Cy5) was purchased from Lumiprobe (www.lumiprobe.com), and Alexa Fluor 488 phalloidin was purchased from Cell Signaling Technology (www. cellsignal.com). Miracloth and purified human integrin αvβ3 were purchased from EMD Millipore (www.merckmillipore.com), and electron microscopy grids were purchased from Electron Microscopy Sciences (www.emsdiasum.com). All enzymes for cloning were sourced from New England Biolabs (www.neb.com), and all other reagents were purchased from Sigma-Aldrich. Molecular Cloning. Expression vectors for codon-optimized versions of BTV-8 VP3 and VP7 have been described previously.22 To construct pEAQ-GFP:VP3HT, three fragments were ligated together: (1) The gf p gene of pEAQ-HT-GFP25 was amplified with the primers 5-acgttgtaaaacgacggccag-3 and 5-agctccggatttgtatagttcatccatgccatg-3 then digested with PacI and BspEI; (2) the codonoptimized BTV-8 VP3 gene of pEAQ-VP3HT22 was amplified with 5cctccggaggcatggctgctcaaaat-3 and 5- ctgaagggacgacctgctaaacaggag-3 and then digested with BspEI and AvrII; and (3) pEAQ-VP3HT was digested with AvrII and PacI. Reactions were performed according to the manufacturer’s protocol, and constructs were verified by Sanger sequencing. All expression constructs were transformed into the disarmed Agrobacterium tumefaciens strain LBA4404 by electroporation and propagated at 28 °C in Luria−Bertani media containing 50 μg/L of kanamycin, streptomycin, and rifampicin. Transient Expression and Purification. Overnight LBA4404 cultures were pelleted by centrifugation at 2000g for 20 min at ambient temperature and resuspended in 10 mM MES (pH 5.6) with 10 mM MgCl2 and 100 μM acetosyringone) to an OD600 of 0.4 and incubated for 3−4 h at ambient temperature. VP3 and VP7 suspensions were mixed at equal volumes and pressure-infiltrated with a needleless syringe into the underside of the leaves of 7-week-old N. benthamiana plants. Extraction for CLP purification was performed on 8 g of whole infiltrated leaves 8 days post infiltration. Tissue was disrupted using a Kinematica PT1200 POLYTRON at 25,000 rpm for 2 × 45 s in 3 volumes of cold extraction buffer (50 mM Bicine, pH 8.4 with 140 mM NaCl, 0.1% (w/v) N-lauroylsarcosine sodium salt, 1 mM dithiothreitol) containing protease inhibitor. Cell debris was removed by passing the lysate through two layers of Miracloth, and the filtered sample was then centrifuged at 18000g for 10 min at 4 °C. Stepwise gradients of 50, 40, 30, and 20% iodixanol in BTV buffer (20 mM Tris−HCl, pH 8.4 with 140 mM NaCl) were prepared in polyclear ultracentrifuge tubes using 3 mL of each step, overlaid with 23 mL of clarified lysates. Tubes were placed in a Sorvall SURESPIN 630/36 rotor and centrifuged at 25000 rpm for 3 h at 8 °C. CLPs were harvested by piercing the side of the tube with a 21 gauge needle and drawing out the visible band. CLPs were desalted using PD-10 disposable columns and extensively dialyzed against BTV buffer. For long-term storage BTV buffer was supplemented with 5% (v/v) ethylene glycol. Gel Electrophoresis. Constituent proteins of CLPs were analyzed by SDS-PAGE using Mini-PROTEAN TGX gels (www.Bio-rad.com) under reducing conditions. Gels for native agarose gel electrophoresis of CLPs comprised 0.70% Tris-borate-buffered agarose and were run at 6 V/cm for 70 min in 89 mM Tris-borate. Prior to loading, 10% (v/

Figure 6. Biochemical activity of BTV CLPs; human integrin binding. (A) Molecular model based on PDB entry 2BTV of the side and top view of a single VP7 trimer with VP3 for spatial reference showing the location of the RGD motif in yellow on the externally projecting face of VP7. (B) Binding of GFP-CLPs and Cy5-CLPs to human integrin in a solid-phase assay as measured by the presence of the fluorescence cargo. (C) Integrin binding competition of Cy5-CLPs with cyclic RGD as determined by the reduction of Cy5-CLP binding (fluorescence) increasing titrations of cyclic RGD (cRGD). (D) Confocal micrographs showing the interaction of Cy5-CLPs with MCF-7 cells; scale bar = 10 nm. Actin signal corresponds to Alexa Fluor 488 phalloidin (green), nuclei to Hoechst stain (blue), and CLPs to Cy5 (magenta). 3481

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ACS Nano v) glycerol and 0.05% (w/v) bromophenol blue were added to 15 μg of CLPs. Chemical Bioconjugation. Addressable amine groups were labeled using CLPs at 2.5 mg/mL and dialyzed into 200 mM sodium bicarbonate (pH 8.4) at ambient temperature overnight. The NHS ester of water-soluble Sulfo-Cy5 was added to CLPs at a 10000-fold molar excess and incubated for 4 h at ambient temperature with gentle agitation. Labeled CLPs were then dialyzed back into BTV buffer with 5% (v/v) ethylene glycol overnight at 4 °C. Size-Exclusion Chromatography. SEC was performed on CLPs using a Tricorn 20/5 mm column packed with Sephacryl S500 on an AKTA Explorer 100 system and run in BTV buffer with 5% (v/v) ethylene glycol. The flow rate was set to 0.1 mL/min. Integration of the 280 nm peaks was used to estimate intact CLP content. UV/vis Spectrophotometry. Absorbance values measured by the 1 cm UV detector of the AKTA Explorer 100 system were used to calculate concentrations. The molar extinction coefficient at 280 nm for BTV CLPs was calculated using the 5:1 ratio of VP7 to VP3 as determined by single particle cryo-EM, giving a mass extinction coefficient of 1.075 (mg/mL)−1cm−1. Concentrations were derived from the Beer−Lambert law: A = εcS , where ε is the molar extinction coefficient; c is the concentration of those species; and S is the path length. Dynamic Light Scattering. Dynamic light scattering (DLS) was performed using a Dynapro plate reader (Wyatt Technology, www. wyatt.com) and 384-well polystyrene plates. Samples at 250 μg/mL were measured in triplicate with 10 acquisitions per well at 25 °C, and the Dynamics software (Wyatt Technology) was used for both scheduled data acquisition and cumulants analysis of the intensity autocorrelation function. Hydrodynamic radius (RH) and percent polydispersity are reported as averaged triplicate measurements and given with standard deviation for the radius. Electron Microscopy. Samples were initially diluted to 50 μg/mL; 5 μL was pipetted onto 200-mesh carbon-coated copper grids and allowed to settle for 90 s. Following two washes in water, grids were negatively stained with 2% uranyl acetate for 60 s. TEM was carried out on a JEOL 1010 at 80 kV. For subsequent cryo-EM of the CLPs and GFP-CLPs, samples were diluted to 200 μg/mL and acclimated to ambient temperature. Immediately prior to vitirifcation, 4 μL of sample was allowed to adsorb on either 300-mesh lacey holey carbon (CLPs) or Quantifoil 300 R2/2 (GFP-CLPs) grids for 10−30 s at ambient temperature and plunge frozen in liquid ethane at near liquid nitrogen temperature using a Vitrobot (FEI). Low-dose imaging of empty CLPs was performed on a Tecnai F30 TEM (FEI) operating at a high-tension of 300k V and nominal magnification of 59000×. Unbinned digital micrographs were recorded using a 4K × 4K LC1100 lens coupled CCD camera resulting in a pixel size corresponding to 2.43 Å at the specimen level. Low-dose imaging of the GFP fused CLPs (GFP-CLPs) was performed on a Tecnai T12 TEM (FEI) operating at a high-tension of 120 kV and nominal magnification of 67000×. Unbinned images were recorded on a 4K x 4K LC-1100 lens coupled CCD camera (Direct Electron) resulting in a pixel size of 2.17 Å at the specimen level. Image Processing. Single particles were selected from cryo-EM micrographs using the Swarm mode55 (Woolford et al.) within e2boxer of the EMAN2 package.56 The contrast transfer function (CTF) of each micrograph was estimated using CTFFIND3.57 2D and 3D classification and subsequent 3D refinement was carried out in Relion 1.4.58 Initial reconstructions were produced without any explicitly imposed symmetry. Subsequently, icosahedral symmetry was imposed during refinement. The CLP structure was calculated from a total of 18808 particles selected from an initial data set of 41098 particles. GFP-CLP was refined from a data set of 4422 particles with 1570 particles used in the final refinement. A second CLP reconstruction was generated at a resolution comparable to the GFP-CLP structure using a subset of the CLP data set comprising 2081 particles. Resolution was estimated by Fourier Shell Correlation (FSC) following gold standard refinement,59 and local resolution of the CLP 3D reconstruction was estimated using ResMap.60 Post reconstruction analysis was handled using Chimera61 for molecular

visualization and ImageJ62 for the radial density analysis of the CLP and GFP-CLP maps. Solid-State Binding Assay. Black 96-well plates were coated overnight at 4 °C with Integrin αvβ3 diltuted to 5 μg/mL in 20 mM Tris-HCL (pH 7.4) containing 150 mM NaCl, 2 mM CaCl2, 1 mM MnCl2, and 1 mM MgCl2. Wells were blocked with 1% (w/v) bovine serum albumin for 90 min at 37 °C before CLPs were applied at 5 μg/ mL with or without 60 min preincubation with various concentrations of c-RGD (10−3−10−7 M) and incubated for 30 min at 37 °C. Following three washes with phosphate-buffered saline (PBS), fluorescence was measured on a TECAN Pro plate reader. Excitation/emission wavelengths of 488/530 nm and 646/675 nm were used for GFP and Cy5 containing particles, respectively. Confocal Microscopy. MCF-7 cells (ATCC) were seeded onto coverslips in 24-well plates and grown at 37 °C with 5% CO2 to 80% confluency (1−2 days). CLP samples were diluted to (0.5 μg/mL) in DMEM media before incubation with MCF-7 cells overnight at 37 °C. The cells were fixed using 4% formaldehyde in PBS and permeabilized with 0.1% Triton X-100 in PBS followed by blocking with 2% BSA in PBS before staining actin with Alexa Fluor 488 phalloidin and cell nuclei with Hoechst stain. Coverslips were then mounted onto glass slides using Fluoroshield mounting medium. Fluorescence was generated and recorded using an LSM Zeiss 710 confocal microscope (Zeiss; www.zeiss.com) equipped with a Plan-Apochromat 63x/1.4 Oil M27 objective (Zeiss). Lasers of 405, 488, and 633 nm were used for the excitation of cell nuclei, actin, and Cy5, respectively. Acquired images were processed and arranged using Adobe Phototshop.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b07747. 3D reconstruction of the BTV CLP; densitometric analysis of the ratio of VP7 to VP3 in recombinant BTV CLPs; 3D reconstruction of the GFP-CLP and comparable CLP; location of solvent-exposed lysine residues on BTV CLPs (Figures S1−S4) (PDF)

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Frank Sainsbury: 0000-0001-8152-3820 Author Contributions #

P.V.J. and N.D. contributed equally.

Notes

The authors declare the following competing financial interest(s): F.S. and G.P.L. declare that they are named inventors on granted patent US8674084 which describes the plant transient expression system used for the work described in this manuscript.

ACKNOWLEDGMENTS F.S. acknowledges funding from the Australian Research Council in the form of a Discovery Early Career Researcher Award (DE140101553). Work at the John Innes Centre was supported by the UK Biotechnological and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Grant “Understanding and Exploiting Plant and Microbial Secondary Metabolism” (BB/J004596/1), BBSRC Grant BB/ L014130/1 and the John Innes Foundation. We acknowledge the facilities and the scientific and technical assistance of the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of 3482

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