Turnip Yellow Mosaic Virus as a Chemoaddressable Bionanoparticle

Apr 12, 2007 - Viruses and virus-like particles (VLPs) have been demonstrated to be robust scaffolds for the construction of nanomaterials. In order t...
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Bioconjugate Chem. 2007, 18, 852−859

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Turnip Yellow Mosaic Virus as a Chemoaddressable Bionanoparticle Hannah N. Barnhill,† Rachel Reuther,† P. Lee Ferguson,† Theo Dreher,‡ and Qian Wang*,† Department of Chemistry and Biochemistry and Nanocenter, University of South Carolina, 631 Sumter Street, Columbia, South Carolina 29208, and Department of Microbiology, Oregon State University, Corvallis, Oregon 97331. Received December 18, 2006; Revised Manuscript Received February 2, 2007

Viruses and virus-like particles (VLPs) have been demonstrated to be robust scaffolds for the construction of nanomaterials. In order to develop new nanoprobes for time-resolved fluoroimmuno assays as well as to investigate the two-dimensional self-assembly of viruses and VLPs, the icosahedral turnip yellow mosaic virus (TYMV) was investigated as a potential building block in our study. TYMV is an icosahedral plant virus with an average diameter of 28 nm that can be isolated inexpensively in gram quantities from turnips or Chinese cabbage. There are 180 coat protein subunits per TYMV capsid. The conventional N-hydroxysuccinimide-mediated amidation reaction was employed for the chemical modification of the viral capsid. Tryptic digestion with sequential MALDITOF MS analysis identified that the amino groups of K32 of the flexible N-terminus made the major contribution for the reactivity of TYMV toward N-hydroxysuccinimide ester (NHS) reagents. The reactivity was also monitored with UV-vis absorbance and fluorescence, which revealed that approximately 60 lysines per particle could be addressed. We hypothesized that the flexible A chain contains the reactive lysine because the crystal structure of TYMV has shown that chain A is much more flexible compared to B and C, especially at the N-terminal region where the Lys-32 located. In addition, about 90 to 120 carboxyl groups, located in the most exposed sequence, could be modified with amines catalyzed with 1-(3-dimethylaminopropyl-3-ethylcarbodiimide) hydrochloride (EDC) and sulfo-NHS. TYMV was stable to a wide range of reaction conditions and maintained its integrity after the chemical conjugations. Therefore, it can potentially be employed as a reactive scaffold for the display of a variety of materials for applications in many areas of nanoscience.

INTRODUCTION Viruses and virus-like particles (VLPs), also known as bionanoparticles, have recently emerged as promising building blocks for nanomaterials (1-7). For example, it has already been shown that semiconductive or conductive nanowires can be prepared with rod-like tobacco mosaic virus (TMV) as the template (2, 7-14). Spherical nanoparticles were produced using the hollow protein shell of icosahedral cowpea chlorotic mottle virus (CCMV) as a nanoreactor (4, 15). With genetically modified filamentous bacteriophage M13 as a template, inorganic, organic, and biological nanosized materials have been aligned into nanowires (16, 17), nanorings (18), nanofibers (19), films (20, 21), and other nanostructures (22-24). Because of increasing interest in using bionanoparticles for drug delivery and other biomedical applications (1, 25-27), it is of particular demand to develop methods to anchor functional groups on viral particles. Cowpea mosaic virus (CPMV) was the first virus to be demonstrated as a robust platform for the conjugation with a variety of molecules, ranging from small molecules such as fluorescent dyes (5, 28-31) to larger particles such as quantum dots (32). In addition, polymers (33, 34), stilbene derivatives (30), biotin (28, 29), DNA (35), peptides and proteins (36-38), and carbohydrates (34, 39) have been successfully conjugated to CPMV in an ordered and predictable manner. Following CPMV, the chemical reactivities of many other viruses have been studied, including CCMV (40), Nudaurelia capensis omega virus (41), TMV (42, 43), and bacteriophage MS2 (44). Moreover, it is possible to engineer multiple orthogonal reactive sites on the viral capsid using conventional * Corresponding author. Phone: ++001-803-777-8436. Fax: ++001803-777-9521. E-mail: [email protected]. † University of South Carolina. ‡ Oregon State University.

bioconjugation techniques or newly developed chemistries (5, 42, 44). In order to develop new nanoprobes for time-resolved fluoroimmuno assays as well as to investigate the twodimensional self-assembly of bionanoparticles (6, 45, 46), the icosahedral turnip yellow mosaic virus (TYMV) was explored as a potential building block in our study. As a member of the tymovirus group, TYMV is a plant virus with an average diameter of 28 nm and made of a single-stranded RNA of 1.9 × 106 Da and 180 chemically identical protein subunits of 20 133 Da. The subunits arrange into 60 trimeric asymmetric units loosely assembled in a T ) 3 icosahedral symmetry as shown in Figure 1 (47-49). The structure of TYMV has been exhaustively studied by X-ray crystallographic analysis (47, 48) and electron microscopy using negative staining (50). It can be isolated from the infected leaves of either the turnip or Chinese cabbage in gram quantities. Comparing with other plant viruses, TYMV has some unique advantages in chemistry, biomedical applications, and materials development. First, empty capsids can be isolated naturally from the host plant or generated artificially by treating under pressure (51), basic environment (52), or repeated freeze-thaw process (53). Second, some of these methods result in a hole in the otherwise intact capsid, which may provide a route for the introduction of useful materials, such as quantum dots or drugs, and opens the possibility of interior modification of the capsid. Third, TYMV is stable from 4 °C to RT for months and 60 °C for several hours. It can withstand, for a wide pH range (4-10), up to 50% organic solvent and a variety of reaction conditions. Finally, the capsid of TYMV represents a very rigid spherical assembly; therefore, we can predict the conformation of the coat proteins and attached molecules based on the X-ray structural data (48). In this paper, we will report the chemoselective modification of native TYMV at the lysine residues and carboxyl groups.

10.1021/bc060391s CCC: $37.00 © 2007 American Chemical Society Published on Web 04/12/2007

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Figure 1. (Left) Structure of TYMV protein capsid with an asymmetric unit cut out. Protein subunits are colored as blue (chain A), red (chain B), and green (chain C) (49). (Right) Enlarged ribbon diagram of TYMV asymmetric unit, which includes three subunits. The 5- and 6-fold axes are labeled where they form on the intact capsid (48). Scheme 1

EXPERIMENTAL PROCEDURES General. Most bioreagents were purchased from Bio-Rad or Fisher and used without further purification. Anti-TYMV antibody was raised in rabbits. Reactive dyes were purchased from Molecular Probes and used without further purification. Unless otherwise indicated, “buffer” refers to 10 mM potassium phosphate at pH 7.8. Size exclusion columns for purification of virus-containing reaction mixtures were prepared by preswelling 23 g of Bio-Gel P-100 (Bio-Rad) in 400 mL of buffer and loading the gel into Bio-Spin disposable chromatography columns (Bio-Rad). The columns were allowed to drain upon standing and were then further dried by centrifugation (3 min at 800 g). For 80 µL of virus solution (1 mg/mL), approximately 1 mL of prepared gel is required. Ultracentrifugation was performed at the indicated rpm values using a Beckman Optima L-90K Ultracentrifuge equipped with either SW41 or 50.2 Ti rotors. TEM analyses were carried out by depositing 20 µL aliquots of each sample at a concentration of 0.1 to 0.3 mg/mL onto 100-mesh carbon-coated copper grids for 2 min. The grids were then stained with 20 µL of 2% uranyl acetate and viewed

with a Hitachi H-8000 TEM electron microscope. FPLC analyses were performed on an AKTA Explorer (GE Biotech) using a Superose-6 size-exclusion column. Potassium phosphate buffer (0.05 M, pH 7.0) with 0.15 M NaCl was used as eluent, and the intact virions show retention volume of approximately 10 mL at an elution rate of 0.4 mL/min, whereas broken particles and individual subunit proteins elute after more than 20 mL. Western Blot analysis was carried out using a Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell with a 15% polyacrylamide gel. Purification of TYMV. Chinese cabbage was grown for 3 weeks after which it was inoculated with TYMV. After one week leaves began showing symptoms. After an additional 2 weeks, the leaves were picked and subjected to purification. Infected leaves were blended with 3× volume of buffer and 0.1% β-mercaptoethanol. The mixture was filtered and the filtrate subjected to centrifugation to remove bulk plant material. The supernatant was collected and clarified by adding an equal volume of CHCl3/1-butanol (v/v ) 1:1). The aqueous layer was collected, and the virus was precipitated with 8%

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Figure 2. (A) UV-vis spectra show virus absorbance of WT-TYMV (green line) and TYMV after modification with 1 (blue line) and 2 (red line) after purification and a 20-fold dilution. UV-vis absorbances of the dyes on TYMV directly after purification with no dilution are shown in the inset. (B) Loading of 1 (blue) and 2 (red) on TYMV related to concentration of reagents in the reaction mixture. (C) Size exclusion FPLC analysis of 2-modified TYMV: intact particles elute at 10 mL, denatured protein at 23.5 mL, and aggregated TYMV at 8 mL. Blue line is absorbance at 260 nm and red line is absorbance at 555 nm, indicating that 2 is attached to the intact particles. (D) TEM image of 1-modified TYMV, the scale bar ) 100 nm.

PEG 8K and 0.2 M NaCl. The pellet was centrifuged, resuspended in buffer, and purified by centrifuging over a sucrose gradient which showed two bands, one for the filled particle and one for the empty particle. In general, the virus was stored in buffer at a concentration of 10 mg/mL at 4 °C and stable for months. SDS-PAGE Analysis. Polyacrylamide gel electrophoresis was carried out in Bio-Rad MiniPROTEAN 3 gel electrophoresis cell. For analysis by coomassie blue staining, 1 µg of TYMV was denatured by heating at 95 °C for 5 min with Tris-HCl buffer containing β-mercaptoethanol, bromophenol blue, and glycerol. The proteins were then resolved on a 15% polyacrylamide gel at 200V for 1 h and stained with Bio-Rad Biosafe Coomassie Blue stain for 1 h and destained with distilled water. Fluorescently labeled TYMV was visualized with a UVP Epi Chemi II imager before staining. MALDI-TOF MS of TYMV Subunit. A solution of TYMV (1 mg/mL, 26 µL) was treated with guanidinium-HCl (6.0 M, 4 µL) for 5 min at RT. The denatured protein was spotted onto a MALDI plate using Millipore ZipTip µ-C18 tips to remove the salts. The samples were analyzed using a Bruker Ultra-Flex I TOF/TOF mass spectrometer with MS grade sinapinic acid in 70% acetonitrile and 0.1% TFA as the matrix. Trypsin in-Gel Digestion and MALDI Study. Pure viral protein (1 mg) was resuspended in Tris-HCl buffer (pH 7.4, 100 mM, 26 µL). Urea (6.0 M, 100 µL) and DTT (200 mM, 5 µL) were added, and the solution was incubated for 1 h at RT. Then the solution was diluted ten times and incubated with sequencing-grade modified trypsin (protein:enzyme ) 50:1) for

18 h at 37 °C. The pH was then adjusted to slightly below 6 to quench the reaction. Tryptic digests were then analyzed by MALDI-TOF MS and MALDI-TOF-TOF MS-MS using a Bruker Ultra-Flex I TOF/TOF mass spectrometer and mass spectroscopy grade trypsin in 70% acetonitrile and 0.1% TFA as the matrix. Reactivity Screening of TYMV. The reactivity of the lysine residues of TYMV was screened by incubating with varying concentrations of fluorescein N-hydroxysuccinimidyl (NHS) ester 1 or N,N,N′,N′-tetramethylrhodamine NHS ester 2 at a virus concentration of 1.0 mg/mL in a mixed solution of buffer and DMSO (v/v ) 80:20). Reactions were purified by size-exclusion chromatography using Bio-Gel P-100 (Bio-Rad) in Bio-Spin disposable chromatography columns as described above. The carboxylic acid residues of TYMV were screened for reactivity by incubating with varying concentrations of fluoresceinamine 3, EDC (10 mM), and sulf-NHS (5.0 mM) in buffer and purified by size exclusion chromatography. The loading of dyes per virion was determined by UV-visible spectroscopy. Virus concentrations were determined by measuring the absorbance at 260 nm; virus at 1.0 mg/mL gives a standard absorbance of 8.3. The average molecular weight of the TYMV capsid is 3.6 × 106. Dye concentrations were obtained by measurement of absorbance at λmax (495 nm for 1 and 555 nm for 2), with molar absorbtivity calibrated for each use by mixing known quantities of dye with TYMV (1.0 mg/mL). The integrity of the modified TYMV was determined by TEM, FPLC, and SDS-PAGE. Reactive residues were identified using tryptic digestion of

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Figure 3. (A) MALDI-TOF MS of whole subunit of WT-TYMV (blue line) and TYMV after modification with 2 (red line). WT peak ) 20 183 m/z, modified peak ) 20 713 m/z (the mass of modification with 2 ) 527 Da). Peak at 20 389 m/z is a matrix adduct. (B) SDS-PAGE of fluorescein-modified TYMV visualized under UV irradiation (left) or upon Coomassie Blue staining (right). (C) Tryptic digestion map of the coat protein of TYMV with the tryptic residues (K and R) highlighted in blue and the peptide sequence including reactive Lys-32 highlighted in red. (D) MALDI-TOF MS spectrum of tryptic digest, highlighting the peak corresponding to 2-modified peptide containing residues T13-R45. Theoretical m/z ) 3950.1, measured m/z ) 3949.9 indicating Lys-32 as the reactive residue on the basis that the dye adduct suppresses tryptic cleavage at K32.

Figure 4. Fluorescence vs dye loading for 1 modified (blue) and 2 modified (red) TYMV.

modified particles and MALDI-TOF mass spectrometry as described above.

RESULTS AND DISCUSSION TYMV was able to be purified in high yield from Chinese cabbage, i.e., about 0.5 mg of pure virus per gram of wet leaves. Purification yields from turnips were slightly lower, i.e., 0.2

mg of virus per gram of wet leaves. The integrity of virus through the purification was confirmed by FPLC, TEM, SDSPAGE, and Western-blot analysis. Fluorescein (FL) N-hydroxysuccinimidyl (NHS) ester 1 and N,N,N′,N′-tetramethylrhodamine (TMR) NHS ester 2, which are selective for reactions with amino groups of proteins, were utilized to screen the reactivity of TYMV at pH 7.8 to give TYMV-FL and TYMVTMR (Scheme 1). The reactions were purified over size exclusion chromatography and analyzed by UV-vis absorbance. As shown in Figure 2A, the absorbance of the virus, taken after a 20-fold dilution, gives the absorbance of viral genome and capsid proteins at 260 and 280 nm together with the absorbance of the dyes on TYMV. The UV-vis measurements were used to calculate the dye/virion ratios based on the molar absorbtivity of the dye and of the virus. When different concentrations of reagent 1 were used, up to 60 fluorescein molecules were attached on TYMV (see Figure 2B) although a loading of 40 dye molecules per virion was more typical. A consistent loading of 60 TMR were anchored on TYMV using dye 2 (Figure 2B). The different reactivities are likely due to the accessibility of the dyes to the reactive sites. Dye 2 has an additional five-carbon spacer between the bulky dye moiety and the NHS group, which makes it easier to access the reactive sites. The TYMV particles were stable to the reactions and remained intact as shown by FPLC and TEM (Figure 2C and 2D).

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Figure 5. (Top) Reactivity of the carboxylic acid residues of TYMV with 3 activated with EDC and sulfo-NHS. (Bottom) TEM analysis of 3-modified TYMV (scale bar ) 100 nm).

MALDI-TOF MS analysis of the subunit of TYMV (before and after the modification) confirmed a partial single modification (Figure 3A). The addition of dye motifs will alter the ionization efficiency of the protein in MALDI due to capping of basic residues; however, the presence of the peak at m/z )

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20713.1 corresponds to the addition of one TMR unit on the TYMV coat protein and is only present in the mass spectrum of the modified TYMV [The peak at m/z ) 20389.7 corresponds to a matrix adduct commonly seen in MALDI-TOF mass spectra of proteins and is present in both the WT-TYMV and the modified TYMV (54)]. Therefore, the absolute signal intensity could not precisely reflect the relative ratio of modified vs unmodified protein. SDS-PAGE of the fluoresceinmodified particles also showed the covalent attachment of dyes to the TYMV subunit (Figure 3B). Figure 3C shows tryptic map of the TYMV coat protein, with the reactive peptide highlighted in red and K32 underlined. Upon in-solution digestion of the protein bands with trypsin, the digested fragments were analyzed by MALDI-TOF MS and confirmed that K32 is the reactive residue as shown by appearance of the peak m/z ) 3949.9, indicating K32 as the reactive residue. This mass was calculated based on the attachment of the dye and on the fact that the dye adduct suppresses tryptic cleavage at K32 (Figure 3D). Using trypsin alone, we were able to achieve 88.8% to 93.7% sequence coverage for the WT protein; however, using V8 protease digestion in addition to trypsin gave us between 95.2% and 100% sequence coverage. Within each asymmetric unit of TYMV capsid, there are three different subunits chains termed A, B, and C as shown in Figure 1. Although they are chemically identical proteins, each subunit is located in a different microenvironment and adapts a slightly different configuration. While chains B and C make up hexameric structures on the virus, chain A makes up the pentameric units. Studies on the crystal structure of TYMV have shown that chain A is much more flexible compared to B and C, especially at the N-terminal region where the K32 located (55, 56). It is possible that the flexibility of A chain allows the access of NHS reagents; therefore, only about one-third of the K32 residues are chemically addressable. Other than trace amount of modification at K142, forced reaction conditions (longer reaction time or more concentrated starting reagents) could not accomplish quantitative modification of K32, as demonstrated by the existence of the unmodified peptide peak containing K32 at m/z ) 1961.4 in the tryptic digestion of modified TYMV.

Figure 6. TYMV coat protein highlighting reactive peptide sequence (D46 to R67). Aspartate and glutamate residues (D46, E48, and D57) are highlighted as wire models. The figure is generated with PyMol software.

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Scheme 2

Since the fluorescent dyes were covalently linked to the viral capsid at the defined position, they were isolated by the protein scaffold which prohibited the possible concentration quenching. As shown in Figure 4, for both 1 and 2, the fluorescence signals increased linearly over a wide range of dye-per-virus ratios, suggesting negligible quenching in the virus conjugates even at dye loadings up to ca. 40 dye/virion (a local concentration of dye equal to 4.6 mM considering TYMV occupies a space of 1.4 × 10-23 m3). This result is very similar to the recent report from Ratna, Soto, and co-workers (31), that fluorescent dyes can be anchored on CPMV with controlled distance which prevented the formation of nonfluorescent dimers and subsequent quenching and afforded highly fluorescent viral nanoparticles (31). Such kind of engineered viral particles can potentially be used as probes in microarray-based genotyping assays and sandwich immunoassays with improved sensitivities (31, 38). Many studies have shown that the carboxyl residues of various viral capsids are amenable to reaction with primary amines under activation (40, 42, 57). Besides the C-terminus of the protein, there are 13 carboxylic acid residues per TYMV subunit which belong to Asp and Glu side chains. As shown in Scheme 2, upon treating with fluorescein amine 3 under activation with 1-(3-dimethylaminopropyl-3-ethylcarbodiimide) hydrochloride (EDC) and sulf-NHS, about 90 carboxyl groups were derivatized after 24 h incubation. Under forcing conditions, i.e., longer reaction time, up to 120 units of 3 could be attached to TYMV without affecting the integrity of the capsid (Figure 5). The reactivity drops sharply at higher concentrations of 3 as the dye precipitates out of solution. In general, the reactivity of carboxyl groups greatly depends on their accessibility to solvent, and the modification often takes place at the most exposed residues or the most flexible loops. Proteomic studies have shown that the most exposed loop containing residues D46 through R67 is the reactive peptide. Shown in Figure 6 is the loop highlighting all the aspartate and glutamate residues. Work is ongoing to determine the exact position of the carboxylic acid modification, although fluorescent resonance energy transfer studies suggested the energy transfer takes place in a single environment, indicating a single reactive site (58).

CONCLUSION The bionanoparticle TYMV was purified in high yield from infected plant tissue and was shown to be stable to a variety of reaction conditions. On the basis of orthogonal chemical reactions, specific sites on the virion surface can be labeled selectively. Amine-targeted chemistry afforded functionalization at the Lys-32 lysine residue, as confirmed by the proteomic analysis. The use of dyes showed no self-quenching of fluorescence even at high labeling values. Targeting the chemical reaction toward carboxylate residues pointed to three possible reactive sites, Asp-46, Glu-48, and Asp-57. This could lead to dual-modified particles with multiple functionalities for the application of TYMV as a fluorescent biological probe (58).

Studies are underway toward the genetic manipulation of the TYMV scaffold, as well as the application of dual-modified TYMV nanoparticles in the development of new functional materials.

ACKNOWLEDGMENT This work was supported by USA NSF-NER program, DODDURIP, and the W. M. Keck Foundation. We are grateful to Alex McPherson for the TYMV inocula, Lisa Alexander and Venkata S. Kotakadi for the assistance of TYMV production, Mike Walla for the assistance of the proteomics study, and Loic Charbonnie`re for helpful discussions about the manuscript.

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