Structure-Based Multifunctionalization of Flexuous Elongated Viral

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Article Cite This: ACS Omega 2019, 4, 5019−5028

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Structure-Based Multifunctionalization of Flexuous Elongated Viral Nanoparticles Carmen Yuste-Calvo,† Ivonne Gonzaĺ ez-Gamboa,†,‡ Luis F. Pacios, Flora Sań chez, and Fernando Ponz* Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid - Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (CBGP, UPM-INIA), Campus Montegancedo, Autopista M-40, km 38. Pozuelo de Alarcón, 28223 Madrid, Spain

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ABSTRACT: Nanoparticle multifunctionalization has become an important goal in many nanoscience-related areas because it allows different simultaneous applications with the same nanoparticle. To develop viral nanoparticles (VNPs) derivatized with different compounds, structural models for Turnip mosaic virus coat protein and viral particles (VNPs) were generated. The models were used as guides toward conjugation-prone amino acid targets. A single cysteine, internal in the coat protein folding, was subjected to different conjugations with several small compounds. Lysines, very abundant on the virion surface, turned out to be suitable for multiderivatization with larger compounds, including a human peptide. The set of locations in the VNP for multifunctionalization was completed by combining chemical and genetic derivatizations of the assembled viral coat protein. This VNP multiple derivatization opens the path toward complex goals in nanoscience, which require the need for different, possibly unrelated but complementary, added functions.

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INTRODUCTION Over the last few years, the development of different nanomaterials for applications in multiple fields has reached substantial growth, and consequently, a major present trend is the simultaneous multifunctionalization of these nanomaterials. Most multifunctionalization efforts have taken place in inorganic nanoparticles, and they often require the use of linkers with different functional groups (one per derivatization) or the use of only one linker to conjugate different compounds with the same chemical reaction, an approach in which the control of the different derivatizations becomes a handicap.1,2 It is also important to take into consideration that the use of inorganic particles involves certain limitations related to toxicity and biodegradability, which can be solved with the use of bioparticles, where biocompatibility appears as an important advantage.3,4 Focusing on bionanoparticles, proteinbased nanoparticles (PBNs) are specially interesting because they are biodegradable and the production of fusion proteins by genetic modification is widely used.5 In addition, there are multiple possibilities for surface modification through covalent attachment to different amino acidic residues, which provide a high range of functional groups.6,7 Within PBNs, the use of viral nanoparticles (VNPs) for biotechnological applications has emerged for a myriad of applications in many areas. VNPs offer the potential for different derivatization routes, related to © 2019 American Chemical Society

not only chemical conjugation but also genetic fusion. A highly exploited characteristic of VNPs is the possibility to obtain noninfectious particles known as viruslike particles (VLPs), with similar structural characteristics but not containing nucleic acids.8−11 In this context, the use of plant viruses as VNPs because of the extra advantages of biosafety; low production costs; and diversity in shape, size, and nature is gaining wide acceptance and developement.12−14 In biotechnological applications, viruses with a helicoidal symmetry like tobamoviruses (such as Tobacco mosaic virus, TMV) and potexviruses (mostly Potato virus X, PVX) provide a high number of coat protein (CP) subunits susceptible to be functionalized. Those with an elongated flexuous architecture, such as PVX or Papaya mosaic virus (PapMV), are particularly interesting for a high aspect ratio or a better performance in tumor homing and tissue penetration, in addition to the flexuosity trait.15−17 Somewhat surprisingly, members of the Potyvirus genus of plant viruses have not received comparable attention and publications about them in this context are scarcer.18−21 Potyvirus particles share with potexviruses the elongated flexuous architecture, being even longer (with more CP Received: October 11, 2018 Accepted: December 10, 2018 Published: March 8, 2019 5019

DOI: 10.1021/acsomega.8b02760 ACS Omega 2019, 4, 5019−5028

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Figure 1. TuMV structural aspects. (A) TEM images of purified virion particles (upper panel) or VLPs (bottom panel) with no genetic or chemical modifications (wild type). (B) Structural TuMV model based on the WMV structure: two assembled particle representations (3 turns, monomer in gray) and one of the CP monomer (bottom). The monomer representation allows us to distinguish the core region and both amino and carboxy domains. These representations are repeated marking the cysteine residue (blue) and the lysines (red), three of them at the represented region of the amino domain (Lys69, Lys74, and Lys82; other 12 are not visible) exposed on the VNP surface.

3D model for the viral particle of TuMV based upon the recent CryoEM structure of WMV also added valuable information regarding the spatial location of chemical conjugation products. In this work, we show the capacity to conjugate different molecules to these residues, obtaining multiderivatized VNPs. Our aim is to show the potential of TuMV particles for chemical functionalization using their natural residues, which in addition to genetic fusion should enable VNP multiderivatization. This approach can enhance the VNP properties and tailor them to several different specific applications.

subunits), which can be advantageous for some applications. The structure of a member of this family, Watermelon mosaic virus (WMV), has recently been determined by CryoEM, thus opening the possibility of modeling three-dimensional (3D) structures of viral particles for related potyviruses.22 This study, a similar one related to the potexvirus Pepino mosaic virus (PepMV),23 and other previous studies24−28 have revealed a common architecture between these families of plant viruses. This structure consists of a CP with both amino and carboxy domains projected from a central core region, assembled with less than 9 subunits per turn. Turnip mosaic virus (TuMV) is a flexuous filamentous plant virus that belongs to the Potyvirus genus. The virions about 750 nm long and 12 nm diameter are made up of approximately 2000 identical copies of 33 kDa CP subunits that cover a molecule of viral RNA of approximately 10 kb in size.29 Until now, potyviruses have only been modified genetically to expose epitopes on their surface, with biomedical applications such as immunization or hypersensitive diagnosis.20,21,30 To our knowledge, no chemical conjugation has been reported. Viruses in this genus exhibit a good homology in amino acid sequence in their coat proteins; the greatest differences are found in the N-terminal region, but always with a high number of lysine residues in the region. Several conserved sites are found, and one of them, the WCIEN amino acid motif, is located in the CP core region.31 Each CP contains this cysteine residue, likely playing a role in structural stability and which can be a used for chemical conjugation. In the overwhelming majority of potyvirus CPs, the WCIEN cysteine is the only one in the protein. Lysines are also potential targets for chemical functionalization. In the particular case of TuMV, the CP has 21 lysines, so this residue is a good candidate for multiderivatization with more than 2000 copies of the conjugated compound. Therefore, the use of cysteine and lysine residues present in the capsid for chemical derivatization was attempted. The construction of a

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RESULTS AND DISCUSSION TuMV Structure-Based VNP Functionalization Strategies. An alignment of 49 sequences of different species of the potyvirus genus was carried out to determine the conserved sites and the presence of cysteines. The WCIEN motif, located in the coat protein (CP) region of the potyvirus genome, is one of the most conserved sites, appearing in all potyviruses studied, being in most cases the only cysteine in the whole CP (Figure S1). Intrigued by the presence of only one cysteine, we decided to study its role in virion assembly and the reactivity of this cysteine for possible conjugations. To assess a possible role of the cysteine in the assembly process or in the preservation of the VNP structure, a point mutation in the CP was performed, replacing the cysteine with a serine. Upon plant agroinfiltration, we observed flexuous elongated particles, similar to those from wild-type VLPs (Figure S2), indicating that a free cysteine is not an absolute requirement for assembly and/ or particle stability, so it could be a convenient target for bioconjugation processes. Comparing with the sole cysteine, the high number of lysine residues (21 in TuMV) and its prevalence in different potyvirus members was considered as a great chance to functionalize VNPs with a higher number of molecules than 5020


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the approximately 2000 per particle obtained by genetic fusion.21,30 This residue has been used in other viruses to exploit its target potential for bioconjugation because of its ability to form very stable amide bonds spontaneously under physiological conditions and also because this residue is very abundant and usually solvent-exposed.32 These features make this residue a good candidate for surface coating. A better understanding of the potyviral architecture was required to localize these residues on the VNP structure, as well as their structural implications. Other structural studies in elongated or elongated and flexuous viruses have provided a notable level of structural knowledge.23−25,27,33 Fiber diffraction and immunological analysis suggested that the N- and Cterminus were exposed on the particle surface, but recent CryoEM studies on a similar potyvirus have only been able to sustain the exposed amino region.28,29,34,35 During our research, a potyvirus structure was published,22 that of Watermelon mosaic virus (WMV). Based on this structure, we could develop our own model for TuMV (Figure 1), which allowed us to place the cysteine and lysine residues on the CP and on the surface of the assembled particle. It is important to consider the lack of amino acids 1−65 in the model, corresponding to the part of the N-terminal domain (which includes 12 lysines, no cysteine), and 272−288, the last 16 amino acids from the C-terminal domain (none of them were lysines or cysteines). The reason is that there is no density attributable to these regions in the cryoEM map, presumably due to its flexibility. The model shows that the cysteine residue is located inside the core region of the CP, not exposed to the surface, whereas lysines are distributed all along the CP (mostly at the N-terminal region), except the Cterminal domain (located inside the VNP filament). A high number of lysines (15 residues) are modeled to be exposed on the VNP surface, thus more solvent-accessible than the cysteine residue. Based on these structural studies, both residues were considered for chemical derivatization strategies: cysteine for internal conjugation of small molecules and lysines for surface coating with larger compounds, including macromolecules like peptides and proteins. The only type of chemical functionalization attempted so far with TuMV was using glutaraldehyde to create nanonets displaying immobilized Candida antarctica lipase B.36 Individual CP conjugations of TuMV have not been tried before this work. To obtain multifunctional particles, bioconjugation to cysteine and lysines was attempted using two different compounds, representatives of reactive groups (iodoacetamide for cysteine conjugation and NHS ester in lysines) present in different chemical linkers. Iodoacetyl reacts with free sulfhydryls by nucleophilic substitution of iodine with a thiol group, resulting in a stable thioether linkage. NHS ester, at pH 7.0−8.5, hydrolyzes the nonprotonated amine groups, creating very stable amide bonds and releasing NHS. Additionally, genetic fusion of peptides provided extra functionalization; thus, on combining all of the strategies, we got three different ways to functionalize each particle (Figure 2). Addressing CP Internal Location for Chemical Derivatization. Because of its free sulfhydryl group, the cysteine residue in the CP seemed to be a good choice. It would readily and spontaneously form disulfide bonds with other sulfhydryl-containing ligands under oxidative conditions or form thioether linkages by alkylation using maleimide or haloacetyl molecules.37,38 According to the model, the sole

Figure 2. Functionalization strategies. On the top, chemical conjugation between the sulfhydryl group (present in the cysteine) and iodoacetamide group (present in different conjugating compounds). The different compounds were I-AEDANS (fluorescent dye), iodoacetyl-PEG2-biotin (for immunoassays), and sulfo-SIAB (heterobifunctional linker between CP and other proteins/peptides). In the middle section, chemical conjugation between the amine group (present in lysines and the N-terminus of the CP) and NHS ester group (present in different conjugating compounds). In this case, the linked compounds were Alexa Fluor 555 NHS ester (fluorescent dye) and NHS-phosphine (linker between amines present in lysines and other compounds with an azido group, Staudinger ligation). At the bottom, genetic fusion of peptides.

cysteine of the CP is in the central domain and appears difficult to reach for conjugation. Nonetheless, it was attempted 5021


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Figure 3. Cysteine chemical derivatization. (A) SDS-PAGE of I-AEDANS-treated denatured VNPs stained with Coomassie Blue or illuminated with UV-light; (1) virions and (2) VLPs. (B) SDS-PAGE of nonconjugated (1) or conjugated (1*) denatured VNPs. Detection by Coomassie Blue or Western blot. Antibodies used in the Western blot: anti-poty (potyvirus general) or anti-biotin. (C) Electron microscopy was performed for purified biotinylated TuMV particles without (TEM) or with (ISEM) anti-biotin antibody immunodecoration. (D) Comparative enzyme-linked immunosorbent assay (ELISA) for biotin detection in plates coated with either free biotin (red columns) or biotin-conjugated TuMV virions (blue columns). Positions at the x-axis represent serial dilutions of anti-biotin antibodies. The black line (threshold) represents the A405 value over which the response of a given solution is considered as positive (three times over background).

After chemical accessibility of the cysteine was shown, we used a biotinylated compound (EZ-Link iodoacetyl-PEG2biotin) detectable in immunoassays with anti-biotin antibody. The conjugated VNPs were characterized first by SDS-PAGE and Western blot with anti-poty and anti-biotin antibodies (Figure 3B). As expected, SDS-PAGE showed a 33 kDa band, consisting of the TuMV CP and biotinylated TuMV CP. The Western blot analysis exposed to anti-poty antibody showed a band in both the conjugated and nonconjugated TuMV CPs. The conjugation of the biotin linker did not interfere with the recognition of the virus by a potyvirus monoclonal antibody. Likewise, an anti-biotin antibody Western blot was performed, which showed a protein band in the lane of biotinylated TuMV CP with a molecular weight of ∼33 kDa. No band appeared in the absence of conjugation. Another important aspect of the conjugation strategy was to maintain the integrity of TuMV particles. To visualize the nanoparticle structure of the conjugate, transmission electron microscopy was used. TEM and ISEM images showed that the filamentous shape of TuMV particles remained intact after the conjugation (Figure 3C). The overall architecture of the biotinylated particles retained the characteristics of a potyvirus, similar to TuMV wild-type VNPs. The enzyme-linked immunosorbent assay (ELISA) was performed using plates coated with biotin-conjugated TuMV

because a free cysteine is rarely available at CP surfaces, and if reactive, it would constitute an excellent choice for chemoselective modification.39 Three compounds were tried to test the possibility of chemical derivatization of the cysteine residue with molecules different in size and nature: a fluorescent dye (I-AEDANS), a biotinylated compound (EZ-Link iodoacetyl-PEG2-biotin), and a bifunctional linker for secondary peptide conjugation (sulfo-SIAB). The fluorescent dye, I-AEDANS, was used to determine whether the TuMV CP had accessible sulfhydryl groups. Protein alkylation was visualized by UV illumination of a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel prior to staining with Coomassie brilliant blue (Figure 3A). After incubation, it was evident that the TuMV samples (virions and VLPs), as well as the positive control (BSA, 35 cysteine residues), had reacted with IAEDANS. By UV illumination, we could observe the 33 kDa band expected for the TuMV CP. A tobamovirus (Pepper mild mottle virus, PMMV) was used as control because tobamoviruses have one cysteine but nonreactive, so cysteine residues need to be introduced by genetic engineering for conjugation.40−42 This tobamovirus CP did not conjugate to IAEDANS and therefore not displaying a band at the expected 17 kDa position. 5022


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virions or with free biotin. Biotin was detected using different dilutions of anti-biotin antibody (Figure 3D). The results indicated that the method can successfully expose small molecules onto the VLPs. Not only did we prove conjugation by immunoassays, as shown earlier with genetic modifications,21 but also an enhancement in ELISA sensitivity was observed by the repetitive multimeric display of biotin in the CP. A 15-fold increase over the free iodoacetyl-PEG2-biotin was obtained. In this case, we proved the possibility of detecting small molecules. This approach can easily be extended to load other small molecules. Examples of this could be haptens, difficult to use in normal immunoassays.43 To further functionalize VNPs and provide them with a wider spectrum of applications, we decided to attempt the conjugation of a peptide. To functionalize TuMV VNPs by peptide conjugation to the sole cysteine, several linkers were considered to find an optimal one. An amine- and sulfhydryl-reactive heterobifunctional cross-linker was chosen: sulfo-SIAB (Figure 2).44 This crosslinker was chosen because of having a similar chemistry as the one with biotin. Both contain iodoacetyl, which worked well in the previous conjugation. The peptide linkage was through NHS esters present in the cross-linker. NHS esters react with primary amino groups (−NH2) on the side chain of lysine residues and the N-terminus of polypeptides. We selected the vasoactive intestinal peptide (VIP), a neuropeptide described as a potent anti-inflammatory factor and also related to autoimmune diseases.45 It was a good candidate due to its amino acid composition (HSDAVFTDNYTRLRKQMAVKKYLNSILN) because it has three lysine residues in only 28 amino acids, favoring the conjugation (besides the N-terminus), and no cysteines that could directly attach covalently to the VNP (making it a good control of linker efficiency). It also had a very attractive structure, a simple α-helix with an optimum size to get inside the assembled particle.46 Moreover, this is a peptide that is problematic to express on the surface of either virions or VLPs by a genetic approach (our unpublished observations), so we decided to try to overcome these problems by chemical conjugation. Conjugation between VIP and VNPs was confirmed by SDSPAGE and Western blot (Figure 4A). Results showed 33 kDa bands, consistent with the CP, in both cases: chemical conjugation (free peptide and VNPs with linker) and negative control (free peptide and VNPs without linker). Western blot using antibodies against the virus showed the same bands as obtained in SDS-PAGE, but using antibodies against VIP, just the one corresponding to conjugation (with linker and peptide) was VIP-positive. Several bands above the 33 kDa bands appeared in the VIP conjugation lane. This is consistent with the molecular weight increase after conjugation of one, two, or three VIP peptides per CP. This might occurs because the linker attaches itself to the VIP peptide through the lysine residues also, allowing another peptide to be linked through the haloacetyl part of the linker. Although the reactivity of haloacetyl-activated supports toward sulfhydryls is relatively selective, these also can react with methionine, histidine, or tyrosine under appropriate conditions.44 Electron micrographs (Figure 4B) showed that conjugation with sulfo-SIAB gave small aggregates, probably because this heterobifunctional linker reacted not only with the lysines present in VIP but also with the ones present in other VNPs, creating a nanonet with both VNPs and peptides.

Figure 4. Chemical internal TuMV functionalization with a peptide. (A) SDS-PAGE and Western blot of denatured virions (1) and VLPs (2) conjugated (1* and 2*) or nonconjugated (1 and 2) with the VIP peptide through cysteine. Antibodies used in the Western blot: antipoty (potyvirus general) or anti-VIP. (B) TEM showing the nanonets formed in the conjugation reaction.

Addressing the Nanoparticle Surface. Once we showed that an inner derivatization through cysteine−iodoacetyl conjugation was feasible, coating the VNP surface through lysine bioconjugation was attempted, lysines being located mostly on the VNP surface. In this case, the group chosen was NHS ester, which generates very stable amide bonds. Focusing on lysines, the free amino groups and the abundance on the virion surface seem to make this residue the best candidate to obtain a high degree of derivatization, even more than 2000 theoretical units of the conjugated compound with no size restrictions, limitations present in the genetic functionalization aproach.9,47−49 For addressability of these residues, a fluorescent dye was used: Alexa Fluor 555 NHS ester.50 The conjugation was also followed by UV illumination of a SDS-PAGE gel, as described previously (Figure 5). Conjugation was tested in both virions and VLPs, obtaining conjugation in both. Once the addressability of lysine residues was shown, we tried other lysine-mediated functionalizations. Several reactions were considered. The Staudinger ligation (Figure S3) was chosen to get a spontaneous conjugation under physiological conditions by phosphine−azide linkage. In this reaction, triphenylphosphine reacts with azides to form an intermediate iminophosphorane with the release of nitrogen gas. This intermediate quickly breaks down in aqueous environments to yield triphenylphosphine oxide and a primary amine.47 We used a phosphine-NHS-ester cross-linker. In this type of crosslinker, the phosphine reacts with azido groups (present in the compounds to conjugate) and the NHS ester reacts with the amino groups present in lysines. After conjugating the VNP with the phosphine-ester linker, the first Staudinger ligation was done with a fluorescent-azido compound (Alexa Fluor 488 azide) so as to be able to check the reaction by fluorescence of SDS-PAGE gels (Figure 5B) and to evaluate the stability of the VNP structure by TEM (Figure 5C). Results showed a successful conjugation with nonstructural modifications in the VNP after conjugation. 5023


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VNP (virion or VLP). Usually, above 70% of the conjugated CPs bore only one VIP peptide. Western Blot analysis with anti-poty antibody showed the 33 kDa band corresponding to the unconjugated CP (Figure 6A, lanes 1 and 2). In the case of the conjugated sample, additional bands appeared, corresponding to different amounts of conjugated peptides, 1−5 per CP (Figure 6A, lanes 1* and 2*). With anti-VIP antibody, the 33 kDa band did not appear, and only bands corresponding to CPs conjugated with VIP molecules (Figure 6A, lanes 1* and 2*). Electron micrographs showed that conjugation with Staudinger ligation did not alter VNP structural characteristics (Figure 6B). Depending on the application, different strategies can be deployed: nanonets by cysteine conjugation or free VNPs by lysine conjugation. VIP-functionalized nanoparticles could be a major boost in antibody detection and/or treatment of different pathologies. Multifunctionalization. To show the possibility of simultaneous chemical conjugation in both residues, a double-conjugation was developed. Also, a triple-derivatization was developed using a previous construct bearing a genetically fused peptide to the CP.21 Cysteine was functionalized with the biotinylated compound and lysines with the fluorescent dye, obtaining different double-derivatizations (double-chemical conjugation or genetic−chemical modification) and also a triple-derivatization (double-chemical conjugation and genetic fusion). Western blot analyses showed that double-chemical conjugation took place in both wild-type and genetically modified VNPs (Figure 6A). TEM showed that there were no structural modifications in the multifunctionalized VNPs (Figure 6B). In this work, we evidenced the potential of TuMV VNPs as scaffolds for multimeric display of two and even three different compounds, providing multifunctionalization. Also, the different nature of these compounds should allow developing different applications, using not only peptides but also fluorophores, drugs, ligands, etc. In addition, the possibility of producing VLPs that are noninfectious constitutes an advantage for several fields. These studies will allow the production of TuMV VNPs as scaffolds to display novel functions for enhanced applications. Some of the applications we are currently developing, related to the biomedical field, need to consider stability in physiological conditions of temperature30 (Figure S4) and pH (Figure S5). The absence of cytotoxicity is an important issue too, and results related to this aspect are to be published somewhere else. The referred applications include sensitive diagnostics based on VNP high antibody-sensing, directed drug delivery by means of VNP diconjugation (drug and cell-specific signaling molecule), or bioimaging of specific cell types, just to mention some of the many possibilities of multifunctionalization. This work lays the foundation for Turnip mosaic virus-derived nanoparticles to achieve these goals.

Figure 5. Lysine chemical derivatization. (A) SDS-PAGE Coomassiestained gel and UV illumination of the same gel, showing conjugation with fluorescent dye through lysine in virions and VLPs. Virions conjugated (1*) or nonconjugated (1) and VLPs conjugated (2*) or nonconjugated (2). (B) Characterization of Staudinger ligation with Alexa Fluor; SDS-PAGE Coomassie-stained gel and UV illumination of the same gel. Virions conjugated (1*) or nonconjugated (1) and VLPs conjugated (2*) or nonconjugated (2) with NHS-phosphine and Alexa Fluor 488 azide. (C) TEM of conjugated VNPs; M, molecular weight marker (kDa).

After assessing the effectiveness of the Staudinger ligation, we also tried to conjugate VIP. The linker was the same as that used in indirect conjugation of a fluorochrome: NHSphosphine, being the azide molecule a peptide modified with an azidopentanoic acid conjugated to the N-terminus. Based on the band intensity obtained in the SDS-PAGE analysis with the conjugated VNPs (Figure 6A, lanes 1* and 2*), 30−60% of the CPs were peptide-conjugated, depending on the type of

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CONCLUSIONS We derivatized successfully viral nanoparticles with three different compounds, allowing multiple functions simultaneously. The development of multiderivatized nanoparticles is the beginning of novel nanostructures, capable to combine several tools in the same space. This implies the emergence of new nanotechnologies capable of taking current nanoscience research a step further.

Figure 6. Surface chemical coating with a peptide. SDS-PAGE Coomassie-stained gel and Western blot using anti-poty and anti-VIP antibodies. (A) Virions (1) or VLPs (2) conjugated with NHSphosphine and VIP peptide (*) compared with nonconjugated VNPs; M, molecular weight marker (kDa), showing bands corresponding to 31 and 38 kDa. (B) TEM of conjugated VNPs. 5024


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EXPERIMENTAL PROCEDURES Production and Purification of TuMV VNPs. Turnip mosaic virus (isolate UK 1)51 was propagated in plants of Indian mustard (Brassica juncea), which were harvested 30 days post-inoculation. For VLP production, 5 week old Nicotiana benthamiana plants were agroinfiltrated for CP transient expression. Agrobacterium tumefaciens (LBA4404 strain) transformed with the CP construction was subcultured and grown overnight, pelleted at 2000g, resuspended to OD600 = 1.2 in MMA (10 mM MES buffer, pH 5.6; 10 mM magnesium chloride; 450 μM acetosyringone), and then infiltrated into the leaves using a blunt-ended 2 mL syringe. Tissue was harvested 10−12 days post-agroinfiltration. VNPs were purified either from Indian mustard (150 g) or N. benthamiana (100 g) plant material as described.52 Briefly, plant tissue was finely ground in 0.5 M potassium phosphate, pH 7.5, 1:2 (w/v), in an electrical tissue grinder, at 4 °C. The resulting suspension was extracted with one volume of chloroform at 4 °C. Phases were separated by centrifugation; the aqueous phase was filtered through Miracloth. After that, VNPs were precipitated with 6% PEG 6000 (w/v) and 4% NaCl (w/v). They were allowed to precipitate for 90 min at 4 °C. The particles were recovered by centrifugation for 10 min at 12 000g. The pellet was resuspended overnight in 0.5 M potassium phosphate, pH 7.5, and 10 mM EDTA. The solution was clarified by centrifugation (10 min at 9000g), and the VNPs were pelleted (2 h at 80 000g). The pellet was resuspended in 0.25 M potassium phosphate, pH 7.5, and 10 mM EDTA, and CsCl was added to a final density of 1.27 g/ cm3. The resulting solution was subjected to centrifugation at 150 000g for 18 h at 4 °C. A visible band in the gradient containing the particles was recovered by punching the tube with a syringe and needle. It was diluted in 0.25 M potassium phosphate, pH 7.5, and 10 mM EDTA and pelleted by centrifugation (2 h at 80 000g). Finally, the pellet was resuspended in 50% glycerol (v/v), 5 mM Tris, pH 7.5, and 5 mM EDTA at a final concentration of 1 mg/mL and stored at −20 °C until further use. VNP concentration was determined spectrophotometrically considering an absorption coefficient (A0.1%, 1 cm at 260 nm) of 2.65. TuMV Structure. The 3D structure of the TuMV coat protein was obtained by homology modeling using SwissModel53 in “user template mode”. The CryoEM geometry of the WMV coat protein (PDB code 5ODV),22 which has 62.80% sequence identity with TuMV, was used as a template in input. Quality scores for the TuMV coat protein model were GMQE = 0.65 and QMEAN = −2.99, values that guarantee a high reliability of the modeled structure. This model included the sequence range of residues 66−272 of TuMV protein (0.72 coverage).22 A 3D model of the 24-subunit viral particle of TuMV was obtained upon superposition of 24 copies of the homologymodeled structure of the TuMV coat protein with every chain in the CryoEM geometry of the 24-subunit assembly of the WMD coat protein. Chemical Conjugation to Cysteine. Different compounds were conjugated to the VNPs by the same functional group, iodoacetyl. Alkylation with I-AEDANS. The alkylation reaction was initiated by addition of a 30-fold molar excess of I-AEDANS to VNPs, followed by incubation at room temperature for 30 min

and 2 h in the dark. The alkylation reaction was terminated by the addition of a 10-fold molar excess of dithiothreitol (DTT) to the samples, followed by a 5 min incubation. Then, samples were stored at 4 °C for further uses. Biotinylation. For the chemical conjugation of the EZ-Link iodoacetyl-PEG2-biotin to TuMV virions, 4× molar excess of the Iodoacetyl Biotin reagent was added to the virus preparation. The reaction mixture was stirred and incubated in the dark for 90 min at room temperature. Then, samples were stored at 4 °C for further uses. Vasoactive Intestinal Peptide (VIP) Conjugation. The VIP peptide (HSDAVFTDNYTRLRKQMAVKKYLNSILN)54 was conjugated to VNPs via the heterobifunctional cross-linking reagent sulfo-SIAB (Thermofisher). First, a 30-fold molar excess of the cross-linking reagent was added to the VNP solution, followed by incubation in the dark for 2 h at room temperature. Afterward, a 10-fold molar excess of VIP was added to the reaction, which was left overnight at room temperature without light. For mock samples, the same protocol was applied without the addition of cross-linker. Then, samples were kept at 4 °C for further use. Chemical Conjugation to Lysine. Different compounds were conjugated to the VNPs through the same functional group, ester. Previously, a buffer exchange was required because of the amines present in Tris buffer. VNPs were centrifuged (50 min, 50 000g, 4 °C) and then resuspended in 10 mM HEPES, pH 7.5, to a final concentration of 2 mg/mL. Fluorochrome Conjugation. For the chemical conjugation of the Alexa Fluor 555 NHS ester (Thermofisher) to TuMV VNPs, a 20-fold molar excess of the Alexa Fluor reagent was added to the VNP preparation. The reaction mixture was stirred and incubated overnight in the dark at room temperature. Then, samples were stored at 4 °C for further use. Fluorochrome Indirect Conjugation by Staudinger Ligation. For further conjugations of different compounds, Staudinger ligation was tested by the addition of Alexa Fluor 488 azide (Sigma).47,55,56 The ligation started with the addition of a 10-fold molar excess of Thermo Scientific Pierce NHS-phosphine to the VNP preparation at 2 mg/mL, followed by overnight incubation in the dark at room temperature. The excess of NHS-phosphine was eliminated with the supernatant obtained by centrifugation (50 min, 50 000g, 4 °C). The pellet was then resuspended to a final concentration of 1 mg/mL. The resulted solution was incubated with the Alexa-azide reagent at a 20-fold molar excess. The reaction mixture was stirred and incubated overnight in the dark at room temperature. Then, samples were stored at 4 °C for further use. VIP Conjugation. VIP was synthetized with one molecule of azidopentanoic acid conjugated to the N-terminus (Caslo ApS, Denmark). The conjugation followed the same conditions as previously described, using NHS-phosphine to conjugate the peptide by Staudinger ligation. After the reaction between VNPs and the phosphine reagent, a 10-fold molar excess of the modified VIP was added, and the reaction mixture was stirred and incubated overnight in the dark at room temperature. Then, samples were stored at 4 °C for further use. Multifunctionalization. Wild-type VNPs and VNPs genetically modified with a peptide derived from the human vascular endothelial growth factor receptor 3 (VEGFR-3)21 were chemically conjugated with two different compounds in two different residues: iodoacetyl-PEG2-biotin conjugated to the cysteine and Alexa Fluor 555 NHS ester conjugated to lysines. VNPs in 10 mM Tris, pH 7.5, and 10 mM EDTA were 5025


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borate buffer, pH 8.1; incubated 1 h with anti-poty antibody (Agdia), anti-biotin antibody (Thermofisher), anti-VIP (GeneTex) antibody, or anti-VEGFR-3 (Abcam 27278) antibody, respectively; and diluted 1:50 for decoration. Then, grids were washed with distilled water and stained with 2% uranyl acetate. Samples were examined on a transmission electron microscope (JEM 1400, the Centro Nacional de Microscopı ́a Electrónica, Madrid).

incubated with a 4-fold molar excess of iodoacetyl-PEG2biotin, mixed, and incubated 90 min at room temperature. The excess of biotinilated compound was eliminated with the supernatant obtained by centrifugation (50 min at 50 000g, 4 °C). The pellet was resuspended in 10 mM HEPES, pH 7.5, to a final concentration of 2 mg/mL. The resulting VNPs were incubated with a 10-fold molar excess of the Alexa Fluor compound, mixed, and incubated overnight in the dark at room temperature. Then, samples were stored at 4 °C for further use. Characterization of Conjugated VNPs. Labeled TuMV particles were characterized using a combination of denaturing gel electrophoresis (SDS-PAGE), Western blot, ELISA, and transmission electron microscopy (TEM). Gel Electrophoresis (SDS-PAGE). Denatured VNP samples (1000−2000 ng) were loaded on 12% PAGE gels. Protein bands were visualized by UV illumination of the PAGE gel (for fluorescent-labeled VNPs) and white light after staining with Coomassie brilliant blue. Protein quantification based on band intensity was performed with Photoshop. Western Blot. SDS-PAGE-fractionated proteins were transferred to a poly(vinylidene difluoride) (PVDF) membrane (Trans-Blot Transfer Medium, Bio-Rad). The membrane was then blocked with 2% skimmed milk in PBS. CP was detected (wild type, biotin-conjugated, or VIP-conjugated) by incubation with the primary monoclonal antibody: anti-POTY (Agdia) diluted 1:500, anti-biotin (Thermofisher) diluted 1:500, anti-VIP (GeneTex) diluted 1:2000, or anti-VEGFR-3 (Abcam 27278) diluted 1:500 in PBS, pH 7.4, and 0.05% (v/ v) Tween 20, followed by binding the anti-mouse secondary antibody (Agdia) diluted 1:200 (wild type, biotin-conjugated), or anti-rabbit (Chemicon) diluted 1:1500 (VIP, VEGFR-3) conjugated to alkaline phosphatase. Incubation with the primary and the secondary antibodies was performed at room temperature for 1 h. Membranes were developed with NBT-BCIP. Enzyme-Linked Immunosorbent Assay (ELISA). An indirect enzyme-linked immunosorbent assay (ELISA) was performed to assess the maximum antibody detection sensitivity provided by the VNPs, in comparison with free biotinylated compound, at equal biotin amounts. Plates (Nunc maxisorp) were coated as follows:

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b02760. Figure S1, alignment of 49 potyvirus species in the region containing the WCIEN motif; Figure S2, micrographs of TuMV VLPs carrying a C140S mutation and the cysteine present in the WCIEN motif; Figure S3, Staudinger ligation to TuMV VNPs; Figure S4, structural stability of TuMV VNPs at 37 °C; Figure S5, structural stability of TuMV VNPs (virus particles) in several buffers at different pHs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Fernando Ponz: 0000-0003-0344-4363 Present Address

Centro de Biotecnologi á - FEMSA, Tecnológ ico de Monterrey, Ave. Eugenio Garza Sada 2501, Col. Tecnológico, CP 64849 Monterrey, Nuevo León, México (I.G.-G.). ‡

Author Contributions †

C.Y.-C. and I.G.-G. are co-authors.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was partially supported by funds RTA201500017 (INIA) and APCIN2016-00014-00-00 (Nanobioagri, ArimNet 2). I.G.G. would like to thank CONACYT and Nuevo Leon Institute for Innovation and Technology Transference for the PhD student grant (No. 216571/CVU 313028). We thank George Lomonossoff for providing the pEAQ vectors and Luciá Zurita for her technical assistance.

• 0.25 μg of TuMV virions resuspended in 50 mM sodium carbonate buffer, pH 9.6; • 1.25 μg of biotin-conjugated TuMV virions resuspended in 50 mM sodium carbonate buffer, pH 9.6; • 1 μg of EZ-Link iodoacetyl-PEG2-biotin (ThermoFisher) dissolved in the same buffer.

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ABBREVIATIONS VNP, viral nanoparticle; VLP, viruslike particle; CP, coat protein; VIP, vasoactive intestinal peptide; VEGFR-3, vascular endothelial growth factor 3; TuMV, Turnip mosaic virus; NHS, N-hydroxysuccinimide; PBN, protein-based nanoparticle

Plates were incubated overnight at 4 °C, washed intensively, and incubated for 2 h with anti-biotin antibody (Thermofisher) [different dilutions in PBS, pH 7.4, 0.05% Tween 20 (v/v), and 2% PVP-40 (w/v)]. Then, anti-mouse secondary antibody (AGDIA) diluted 1:200 in the same buffer was added and incubated for 1 h at room temperature. Alkaline phosphatase activity was detected after adding p-nitrophenylphosphate. The optical density of samples was determined at 405 nm (TECAN Genios Pro, Switzerland). Transmission Electron Microscopy (TEM). Grids (nickel formvar-coated, 400 mesh and copper/carbon-coated, 400 mesh) were placed on one drop of the conjugated VNPs (wild type, biotin-conjugated, and VIP-conjugated) and incubated at room temperature for 10 min. They were washed with 50 mM

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