Blue Fluorescent Antibodies as Reporters of Steric Accessibility in

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Bioconjugate Chem. 2003, 14, 38−43

Blue Fluorescent Antibodies as Reporters of Steric Accessibility in Virus Conjugates Qian Wang,† Krishnaswami S. Raja,† Kim D. Janda,† Tianwei Lin,*,‡ and M. G. Finn*,† Departments of Chemistry and Molecular Biology, The Skaggs Institute for Chemical Biology, and the Center for Integrative Molecular Biosciences, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037. Received July 29, 2002; Revised Manuscript Received November 13, 2002

Nonenveloped viruses provide the chemist with large, preassembled polyvalent protein scaffolds for modification. These structures are typically porous to small molecules but not to large ones. The solution-phase structures and reactivities of such assemblies may be substantially different than indicated by X-ray crystal structures. Here, the attachment of organic compounds to either the inside or outside surface of the cowpea mosaic virus (CPMV) coat protein was verified with an indicating antibody-antigen interaction. Antibody binding was subsequently blocked by the installation of poly(ethylene glycol) chains. These results typify the type of site-specific control that is available with CPMV and related virus building blocks.

INTRODUCTION

Cowpea mosaic virus (CPMV) is a prototypical icosahedral plant virus having qualities that render it useful as a supramolecular chemical compound as well as a biological entity. It can be made and purified in large quantities (1, 2), is structurally characterized to nearatomic resolution (3), is stable to a variety of conditions compatible with both hydrophobic and hydrophilic molecules (4), and can be manipulated at the genetic level to introduce mutations at desired positions (5-9). Sixty identical asymmetric protein units, each composed of “large” and “small” polypeptide chains, comprise the CPMV coat protein (capsid). The virion is approximately 300 Å in diameter, with an average capsid thickness of 30 Å and RNA packaged in the interior. The structure therefore possesses inside and outside protein surfaces, which are chemically different. The most obvious distinction is the disposition of multiple positively charged residues on the interior surface, which presumably serve as points of association with the encapsidated polynucleotide. Thus, X-ray crystallography shows more than 10 lysines per asymmetric unit to be arrayed on the capsid interior, whereas only five lysine residues per asymmetric unit are found on the exterior surface. We have taken advantage of some of these properties to demonstrate the attachment of lysine- and cysteine-reactive small molecules to specific positions on the CPMV capsid (4, 10, 11). While the positions of attachment to the coat protein sequence may be engineered and/or elucidated, such experiments do not provide independent information about the location of the reactive site relative to other structural elements of the virion in solution. In other words, the crystal structure of a virus provides a useful initial guide to its chemistry, but such a structure offers little information about capsid dynamics, which may bring apparently blocked residues to the surface. In fact, * To whom correspondence should be addressed. Fax: (858) 784-8850. e-mail: [email protected], [email protected]. † Department of Chemistry. ‡ Department of Molecular Biology.

dynamic structural transitions occur in many virions (12). For example, cleavage of an N-terminal sequence of flock house virus by added trypsin occurs readily, although the crystal structure shows this sequence to be attached to the interior surface of the capsid (13). Another example of dynamic behavior is afforded by cowpea chlorotic mottle virus, which undergoes reversible pH-dependent swelling, changing the virus diameter by approximately 10% (14). To evaluate the solution-phase chemical behavior of the lysine and cysteine residues, we require solution-phase methods to determine if the chemical linkages have been made on the inside vs the outside capsid surface. Here we describe the use of a LernerJanda blue fluorescent antibody to report on the position of its stilbene hapten (15, 16) and the modulation of this antibody-antigen interaction by covalent modification of the virus surface. The result of an initial version of this experiment was included in a previous report (4). EXPERIMENTAL PROCEDURES

trans-4-Aminostilbene, 5-aminofluorescein, and bromoacetyl bromide were purchased from Aldrich. Poly(ethylene oxide) reagents “mPEG-succinimidyl propionate5000” (MW 5000) and “mPEG-succinimidyl propionate2000” (MW 2000) were purchased from Shearwater Polymer Inc. Wild-type and mutant (VEFCysR) viruses were prepared as previously described (10, 11). Unless otherwise noted, “buffer” refers to 0.1 M potassium phosphate, pH 7.0. The virus was stored in buffer at a concentration of about 10 mg/mL. Size exclusion columns for purification of virus from reaction mixtures were prepared by preswelling Bio-Gel P-100 Gel (BioRad, 23 g) in buffer (400 mL, degassed overnight for use with mutants displaying exterior cysteine residues) and loading the gel into Bio-Spin disposable chromatography columns (BioRad). For 80 µL of virus solution (1 mg/mL), approximately 1 mL of the prepared gel is required. Sucrose gradient ultracentrifugation separation of virus samples was performed on 30 mL gradients [made of 20% (w/w) sucrose solution in buffer, frozen at -20 °C, and defrosted just before use] with centrifugation at 38000 rpm for 3 h (Beckman SW41 Ti rotor), giving rise to wellseparated bands.

10.1021/bc025587g CCC: $25.00 © 2003 American Chemical Society Published on Web 12/20/2002

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Figure 1. Plots showing the amount of the covalently attached fluorescein to (a) wild-type CPMV, and (b) the mutant virus VEFCysR, as a function of the ratio of reagent 6 to virus. Note that there are 60 asymmetric units per virus particle. Each data point shown is the average of three independent experiments with maximum error of ( 9% of the reported attachment value.

TEM analyses were performed by depositing 20 µL aliquots of each sample 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 Philips CM100 electron microscope. FPLC analyses were performed with AKTA Explorer (Amersham Pharmacia Biotech) equipment, using Superose-6 size-exclusion or Hitrap-Q ion-exchange columns. In the former case, 0.05 M potassium phosphate buffer (pH 7.0) was used as eluent; intact virions show retention times of approximately 25 min at an elution rate of 0.4 mL/min, whereas broken particles and individual subunit proteins elute only after 50-60 min. Ion-exchange FPLC was performed with 0.05 M potassium phosphate as the low-salt buffer and 1.0 M NaCl in 0.05 M potassium phosphate as highsalt eluent. 4-(4-E-Styryl-phenylcarbamoyl)butyric Acid (1). trans-4-Aminostilbene (426 mg, 2.18 mmol) and glutaric anhydride (250 mg, 2.19 mmol) were stirred in CH2Cl2 (10 mL) at room temperature, the solution depositing a white precipitate within a few minutes. After 5 h, the solvent was removed to give carboxylic acid 1 as a pale yellow solid (671 mg, 99% yield). 1H NMR (DMSO-d6) δ 12.02 (br s, 1H), 9.97 (s, 1H), 7.6 (m, 6H), 7.35 (t, J ) 7.9 Hz, 2H), 7.23 (d, J ) 7.9 Hz, 1H), 7.15 (s, 2H), 2.32 (m, 4H), 1.80 (m, 2H). 13C NMR (DMSO-d6) δ 174.8, 171.3, 139.5, 137.9, 132.5, 129.3, 128.7, 127.9, 127.6, 126.9, 119.8, 36.2, 33.7, 21.2. 4-(4-E-Styryl-phenylcarbamoyl)butyric Acid 2,5Dioxo-pyrrolidin-1-yl Ester (2). A solution of 1 (200 mg, 0.65 mmol) in 1:1:0.1 CH2Cl2:MeCN:pyridine was treated with disuccinimidyl carbonate (182 mg, 0.71 mmol), and the reaction was stirred at room temperature under a drying tube. The small amount of solid that was undissolved at the beginning of the reaction was solubilized after several hours. After 12 h, the solvent was removed to give a wet, off-white solid; CH2Cl2 was added and then removed to give a dry white solid. This material was purified by column chromatography on silica gel, using 2:1 CH2Cl2:MeCN as eluent. The desired product, 2, eluted near the solvent front and was isolated as a white solid (186 mg, 70% yield). 1H NMR (CDCl3) δ 7.96 (br s, 1H), 7.5 (m, 6H), 7.35 (m, 2H), 7.28 (m, 1H), 7.05 (s, 2H), 2.89 (s, 4H), 2.74 (t, J ) 7.0 Hz, 2H), 2.49 (t, J ) 6.8 Hz, 2H), 2.22 (m, 2H).

(E)-4-Bromoacetamidostilbene (3). A suspension of trans-4-aminostilbene (0.98 g, 5 mmol) and NaHCO 3 (1.3 g, 15 mmol) in dry tetrahydrofuran (15 mL) was stirred at 0 °C for 15 min, followed by dropwise addition of a solution of bromoacetyl bromide (2.0 g, 10 mmol) in dry tetrahydrofuran (5 mL). After the mixture was stirred for 30 min, diethyl ether (50 mL) and saturated aqueous NaHCO3 (50 mL) were added. The organic layer was separated and concentrated to afford 1.4 g (91%) of the desired product as a brown powder. Further purification was performed by recrystalization from acetone. 1H NMR (CDCl3): δ 8.16 (br,1 H), 7.53-7.26 (m, 9 H), 7.08 (s, 2 H), 4.04 (s, 2 H). 6-r-Bromoacetamidofluorescein (6) (17). This compound was prepared analogously to 3, using 6-aminofluorescein (1.74 g, 5 mmol). The crude product was purified by column chromatography over silica gel, eluting with ethyl acetate, giving 6 as a yellow powder (1.85 g, 79%). 1H NMR (CDCl3, 500 Hz): δ 9.95 (s, 1 H), 7.987.70 (m, 4 H), 6.75-6.61 (m, 6 H), 4.00 (s, 2 H). General Procedure for Modification of CPMV with Chemical Reagents. Organic reagents in DMSO solution were introduced into a solution of virus, such that the final solvent mixture was composed 80% buffer and 20% DMSO. Following incubation at 4 °C for 24-48 h, the mixture was purified by passage through a P-100 size exclusion column (centrifugation at 800 g for 3-5 min). This filtration was repeated (typically two or three times) with fresh columns until all the excess reagents were removed. Purification of larger quantities of derivatized virus (>1 mg) was performed by ultracentrifugation at 42000 rpm (Beckman 50.2 Ti rotor) through a 2 cm sucrose cushion, followed by solvation of the resulting material in buffer. Mass recoveries of derivatized viruses were typically 60-90%; all such samples were composed of >95% intact particles as determined by analytical sizeexclusion FPLC. Virus concentrations were measured by absorbance at 260 nm; virus at 0.1 mg/mL gives a standard absorbance of 0.8. The average molecular weight of the CPMV virion is 5.6 × 106. Stoichiometry of Virus Derivatization (Figure 1). The reactions were performed at constant virus concentration (1 mg/mL) under the above conditions. After purification, the concentration of viruses and dyes were determined by absorbance spectroscopy. Fluorescein

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Scheme 1. Preparation of Virus-Stilbene Conjugates

concentrations were obtained by measurement of absorbance at 495 nm, using a calibrated value of molar absorbtivity (77 000 cm-1 M-1) determined by mixing known quantities of dye with CPMV (1 mg/mL). Each data point is the average of values obtained from three independent parallel reactions. The average deviation was 5%, and the maximum deviation was 9%. Most samples were also analyzed by anion-exchange FPLC to verify the integrity of the particles and the quantitation of their dye attachments. Wild-Type CPMV Lysine-Stilbene Conjugate (4) and Cysteine-Stilbene Conjugates (5a, 5b). Wildtype CPMV (10 mg) and 2 (20 µmol, ca. 200-fold excess relative to viral protein) in a solution of DMSO (1 mL) and buffer (4 mL) were shaken gently at 4 °C for 24 h. Initial separation of virus from reagent was accomplished by adding another 4 mL of buffer to induce precipitation of the organic reagent and spinning at 16000g for 2 min. The supernatant was further purified by ultracentrifugation at 42000 rpm over a 2 cm 40% (w/w) sucrose cushion, and the resulting transparent pellets were dissolved in 1 mL buffer. Measurement of OD260 provided a concentration of virus 4 of 9.1 mg/mL (91% yield). The syntheses of 5a and 5b were performed analogously, using wild-type or mutant CPMV (5 mg) and 3 (100 µmol, ca. 2000-fold excess relative to viral protein), with the only difference being the reaction time (48 h for 5a and 12 h for 5b), reflecting the difference in reactivity between the two viruses. PEGylation of 5b. Conjugate 5b (2 mg) and 7a or 8a (20-fold excess with respect to viral protein) were incubated in 0.1 M NaHCO3 buffer (pH 8.4) at 4 °C for 48 h. The resulting viruses were purified by sucrose gradient sedimentation. The bands corresponding to virus were collected, concentrated by ultracentrifugation through a 2 cm sucrose cushion, and dissolved in PBS buffer. Formation of Antibody 19G2 Complexes. All samples were dialyzed into PBS buffer at 4 °C before mixing with antibody 19G2. The concentration of 19G2 was 1.5 mg/mL and virus concentrations were made constant at 1.0 mg/mL. For denaturation, the viruses were heated for 1 h at 60 °C in 50 mM ammonium carbonate buffer containing 20% acetonitrile. RESULTS AND DISCUSSION

Preparation of CPMV-Stilbene Conjugates. Stilbene derivatives 2 and 3, prepared from trans-4-amino-

stilbene, were used to modify wild-type and mutant CPMV, as shown in Scheme 1. Reactions with 2 were performed under the conditions previously shown with dye-NHS esters to provide attachment of the small molecule predominantly at a single lysine residue of the coat protein (K38 in the small subunit), giving 60 stilbenes per virus particle (virus 4) (10). Stilbene bromoacetamide 3, a readily available reagent for specific modification of sulfhydryl groups, was chosen for the derivatization of the cysteine mutant viruses and the modification of the cysteines of wild-type CPMV (Scheme 1). The use of 3 required a preliminary assessment of the reactivity of a model bromoacetamide with the virus. Thus, fluorescein derivative 6 was employed to establish the proper conditions for stilbene attachment to thiol residues of CPMV. Wild-type CPMV proved to be poorly reactive toward 6, showing no dye attachment in a 6-h reaction and modest levels of covalent modification of the virus after 2 days. The dose-response for attachment under the latter conditions is shown in Figure 1; under the most forcing conditions (2000:1 molar ratio of 6 to viral protein), a maximum of 10 dye molecules per capsid were attached. The CPMV mutant designated VEFCysR was chosen for comparison to the wild-type particle for reasons of stability and spacing of the inserted cysteine moieties. Figure 2 shows the site of the five-residue (GGCGG) insert made in the βE-βF loop of the large subunit, between residues G98 and K99, and the roughly equidistant disposition of these sites about the external surface of the particle. The preparation and chemistry of this virus has been described elsewhere (11). As observed previously with maleimides, this mutant virus displayed much greater reactivity, in this case loading fluorescein bromoacetamide to the maximum value of 60 per particle within 12 h (Figure 1). On the basis of these results, stilbene bromoacetamide 3 was used to derivatize both wild-type and mutant CPMV at a reagent:protein ratio of 2000:1, to give viruses 5a and 5b (Scheme 1) in excellent chemical yield. Recognition of trans-Stilbene on CPMV with Blue-Fluorescent Antibody. The addition of the monoclonal antibody 19G2 to a solution of virus 5b, which bears stilbene groups at the designed cysteine residues on the exterior βE-βF loop, gave an intense blue fluorescence under ultraviolet irradiation (Figure 3). No fluo-

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Figure 2. (Left) Ribbon diagram of the asymmetric unit of the CPMV coat protein, showing the two component proteins (small subunit ) domain A; large subunit ) domains B + C), and the βE-βF loop in red. (Right) View down the 2-fold axis of the wild-type CPMV structure (R carbons only), depicting the pattern created on the surface of the particle by mutational insertion between R97G98 and K99-Y100 of the βE-βF loop, shown in red.

Figure 4. Black-and-white photographs taken under ultraviolet illumination of antibody 19G2 complexed with stilbenedecorated wild-type CPMV (A1) 4 + 19G2; (B1) 5a + 19G2; (A2) denatured 4 + 19G2; (B2) denatured 5a + 19G2. Sample volumes and conditions were as described in Figure 3.

Figure 3. (Top) Black-and-white photographs taken under ultraviolet illumination of the following 50 µL samples in PBS buffer, pH 7.4: (A) virus 5b + 19G2; (B) 5b alone; (C) 19G2 alone; (D) VEFCysR + 19G2 (concentrations: 19G2, 1.5 mg/mL; viruses, 1.0 mg/mL). (Bottom) representative TEM pictures (negative staining with uranyl acetate) showing the binding of 5b with 19G2. (A) 5b. (B) 5b + 19G2; approximately 90% of all virus particles visualized in an exhaustive examination of these samples were found in the type of small clusters shown here. (C) 5b + 19G2, subsequently treated with 1 (10 mg/mL, approximately 3500 molar equivalents relative to viral protein, at 4 °C for 12 h). All samples were diluted to a virus concentration of 0.2 mg/mL before TEM analysis.

rescence was observed for the underivatized virus in the presence of 19G2. The relatively low binding affinity of 19G2 for the stilbene hapten (in the µM range) (15) precludes the chromatographic purification of such adducts. However, the interaction could be independently visualized by transmission electron microscopy (TEM) as shown in Figure 3. The polyvalent display of hapten on the virus and the presence of two binding sites per

immunoglobulin gave rise to clusters of virus particles formed only in the presence of both components; the addition of soluble, monomeric stilbene 1 breaks up these clusters. The blue fluorescent response was similarly observed for four other cysteine-containing mutants (11) of CPMV derivatized with reagent 2 (data not shown). The basic function of the outer protein shell is to protect the fragile nucleic acid genome from physical, chemical, or enzymatic damage (18). While small molecules can pass through the capsid of CPMV easily, it should be difficult for proteins or other big molecules to reach the virus interior. The ability of blue-fluorescent antibodies to recognize trans-stilbene on the exterior CPMV surface thereby offers a convenient way to distinguish between attachments made to the inside vs the outside surface of CPMV, when the site of attachment is less certain than it is for 5b. Incubation of virus 4 with antibody 19G2 resulted in strong blue fluorescence under UV irradiation (Figure 4), confirming that NHS ester 1 reacts to a significant extent at exterior residues, consistent with the expected site of attachment (K38). Furthermore, the antibody should not give a strong positive response when presented with a hapten structure that is accessible only in a transient sense by virtue of an energetically unfavorable dynamic

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Scheme 2. Preparation of Virus-Stilbene-PEG Conjugates

conformational change, unless the antibody-hapten binding energy (relatively weak in these systems) (15) is sufficient to overcome the conformational cost. In other words, residues recognized by this method as being on the virus exterior are likely to reside there in the most stable solution-phase structures of the capsid. In contrast, assays that involve a rapid and irreversible chemical reaction can more easily intercept an energetically disfavored subpopulation of conformers (12). As shown in Figure 4, the S-alkylated product from wild-type CPMV, 5a, was not recognized by the antibody, and thus the reactive residues are assigned to a position inside the particle. This is consistent with the X-ray crystal structure of CPMV, in which no cysteines are visibly exposed on the exterior surface. In the case of both 4 and 5a, denaturing the derivatized virus allows for antibody binding and strong fluorescence with 19G2 (Figure 4). The capsid is thereby found to be sufficiently permeable to allow small molecules such as 2 and 3 to diffuse through, but the antibody is too large to do likewise. Position-Dependent Blocking of Antibody-Antigen Interaction. Many therapeutic proteins have been coupled to poly(ethylene oxide) [commonly referred to as poly(ethylene glycol), or PEG] in order to prolong their circulating lifetimes and increase their potencies in vivo (19, 20). PEG attachment is also commonly used to passivate the immune response toward proteins (21, 22) and viruses (23). Here we show that PEGylation can be used to block the binding of a blue fluorescent antibody to its antigen. The decoration of CPMV with PEG produces substantial changes in the physical and chemical properties of the virus, which will be discussed elsewhere (24). The cysteine-added mutants we have described thus far all retain the natural chemical reactivity of exposed lysines, and especially K38 (11). Derivatization of 5b at lysine with commercially available N-hydroxysuccinimide PEG reagents 7a and 7b afforded virus conjugates 8a and 8b, as shown in Scheme 2. In this case, the virus products were purified by filtration through size-exclusion resin to remove the excess PEG reagent, followed by sucrose gradient sedimentation, unltracentrifugation pelleting, and resuspension in 0.1 M potassium phosphate buffer (pH 7.0). From previous studies under identical conditions with wild-type CPMV, we have established that approximately 60 copies of PEG-2000 are attached per virion with 7a, and approximately 29 copies of PEG-5000 are attached per virion in the presence of 7b (24). The products here were shown to be composed of intact virus particles by their sedimentation properties in sucrose gradients as well as by electron microscopy (data not shown). Upon incubation with 19G2, neither PEG-decorated virus showed fluorescence under

Figure 5. Steady-state fluorescence emission spectra with excitation at 327 nm: (a) 5b + 19G2; (b) 8a + 19G2; (c) 8b + 19G2. For all samples, concentrations were 1.5 mg/mL in antibody and 1.0 mg/mL in virus.

Figure 6. Stereoimage of the 19G2 Fab fragment (green and orange) containing hapten 1 (red) docked with the CPMV coat protein asymmetric unit (small subunit in dark blue; large subunit in light blue), as described in the text. The orientation is chosen to minimize the distance between the carboxylate group of 1 and the site of VEFCysR mutation (residue 99 of the large subunit, shown in white). Lysine 38 of the small subunit is shown in purple.

UV irradiation (Figure 5), presumably because the PEG chains block the binding of antibody with the attached trans-stilbene on the virus. An appreciation for the qualitative aspects of the interactions between antibody and a virus-hapten conjugate can be obtained by considering the crystal structure of the 19G2-1 complex (involving only the antibody Fab fragment (Protein Data Bank structure 1FL3, www.rcsb.org/pdb/). The hapten is bound in the expected planar, trans-configuration with the long axis directed toward the center of the mAb and the carboxy-terminated

Virus-Based Antigen−Antibody Interactions

linker at its periphery. Most of the side chains adjacent to the stilbene moiety are nonpolar, forming an uncharged, hydrophobic pocket (15). While not intended to be a detailed model, docking of this structure with that of the wild-type CPMV asymmetric unit, as seen in Figure 6, shows that the stilbene unit of 5b should be easily accessible to the binding pocket of 19G2. The structures are positioned such that the carboxylic acid group of the hapten 1 (in red) is as close as possible to the location of the GGCGG insert (residue 99 of the large subunit, shown in white). Note the close approach of these two fragments, suggesting that the stilbenedecorated virus should suffer few steric barriers to forming a complex with the antibody. Furthermore, the attachment of a PEG chain to lysine 38 (purple) is likely to result in an unfavorable interaction with a loop of the antibody structure (marked with an arrow) that overhangs this position, preventing antigen-antibody binding, as observed. CONCLUSIONS

We have shown here that decoration of CPMV by bromoacetamide reagents is specific and selective for displayed cysteine residues, with the degree of loading controlled by reaction conditions in a predictable manner. It is noteworthy that these electrophilic reagents are quite hydrophobic, demonstrating that the virus survives the attachment of such species and the use of aqueousorganic cosolvent mixtures. Cysteine residues on the wild-type capsid are at least 6-fold less reactive than those displayed on the inserted loop of the VEFCysR mutant. Most importantly, the blue fluorescent antibody can be employed as a convenient probe of the location of its stilbene hapten on a nanoparticle scaffold, and that this interaction can be inhibited by the site-directed attachment of a PEG chain. These tools and insights will be employed in the design of virus-displayed antigens and other species. ACKNOWLEDGMENT

We thank The Skaggs Institute of Chemical Biology and the David and Lucille Packard Foundation (Interdisciplinary Science Program) for support of this work. Q.W. and K.S.R. are Skaggs Postdoctoral Fellows. We are grateful to Dr. L. Tang for assistance with representations of the CPMV-antibody interactions shown in Figure 6. LITERATURE CITED (1) Goldbach, R., and van Kammen, A. (1985). Structure replication and expression of the bipartite genome of cowpea mosaic virus. In Molecular Plant Virology (J. Davies, Ed.) pp 83-120, CRC Press, Boca Raton. (2) Spall, V. E., Porta, C., Taylor, K. M., Lin, T., Johnson, J. E., and Lomonossoff, G. P. (1998). Antigen Expression on the Surface of a Plant Virus for Vaccine Production. In Engineering Crops for Industrial End Uses (P. R. Shewry, J. A. Napier, and P. Davis, Eds.) pp 35-46, Portland Press, London. (3) Lin, T., Chen, Z., Usha, R., Stauffacher, C. V., Dai, J.-B., Schmidt, T., and Johnson, J. E. (1999). The Refined Crystal Structure of Cowpea Mosaic Virus at 2.8 Å Resolution. Virology 265, 20-34. (4) Wang, Q., Lin, T., Tang, L., Johnson, J. E., and Finn, M. G. (2002). Icosahedral Virus Particles as Addressable Nanoscale Building Blocks. Angew. Chem., Int. Ed. 41, 459-462. (5) Lin, T., Porta, C., Lomonossoff, G., and Johnson, J. E. (1996). Structure-Based Design of Peptide Presentation on a Viral Surface: the Crystal Structure of a Plant/Animal Virus Chimera at 2.8 Å Resolution Fold. Des. 1, 179-187.

Bioconjugate Chem., Vol. 14, No. 1, 2003 43 (6) Porta, C., Spall, V. E., Lin, T., Johnson, J. E., and Lomonossoff, G. P. (1996). The Development of Cowpea Mosaic Virus as a Potential Source of Novel Vaccines. Intervirology 39, 7984. (7) Johnson, J., Lin, T., and Lomonossoff, G. (1997). Presentation of Heterolgous Peptides on Plant Viruses. Annu. Rev. Phytopathol. 35, 67-86. (8) Taylor, K. M., Lin, T., Porta, C., Mosser, A., Giesing, H., Lomonossoff, G. P., and Johnson, J. E. (2000). Influence of 3-Dimensional Structure on the Immunogenicity of a Peptide Expressed on the Surface of a Plant Virus. J. Mol. Recog. 13, 71-82. (9) Lomonossoff, G. P., and Hamilton, W. D. O. (1999). Cowpea Mosaic Virus-Based Vaccines. Curr. Top. Microbiol. Immun. 240, 177-189. (10) Wang, Q., Kaltgrad, E., Lin, T., Johnson, J. E., and Finn, M. G. (2002). Natural Supramolecular Building Blocks: WildType Cowpea Mosaic Virus. Chem. Biol. 9, 805-811. (11) Wang, Q., Lin, T., Johnson, J. E., and Finn, M. G. (2002). Natural Supramolecular Building Blocks: Cysteine-Added Mutants of Cowpea Mosaic Virus. Chem. Biol. 9, 813-819. (12) Johnson, J. E., and Speir, J. A. (1997) Quasi-equivalent viruses: A paradigm for protein assemblies. J. Mol. Biol. 269, 665-675. (13) Bothner, B., Dong, X. F., Bibbs, L., Johnson, J. E., and Siuzdak, G. (1998). Evidence of Viral Capsid Dynamics Using Limited Proteolysis and Mass Spectrometry. J. Biol. Chem. 273, 673-676. (14) Speir, J. A., Munshi, S., Wang, G., Baker, T. S. and Johnson, J. E. (1995) Stuctures of the native and swollen forms of cowpea chlorotic mottle virus determined by X-ray crystallography and cryo-electron microscropy. Structure 3, 63-68. (15) Simeonov, A., Matsushita, M., Juban, E. A., Thompson, E. H. Z., Hoffman, T. Z., Beuscher, A. E. I., Taylor, M. J., Wirsching, P., Rettig, W., McCusker, J. K., Stevens, R. C., Millar, D. P., Schultz, P. G., Lerner, R. A., and Janda, K. D. (2000). Blue-Fluorescent Antibodies. Science 290, 307-313. (16) Chen, D. W., Beuscher, A. E., Stevens, R. C., Wirsching, P., Lerner, R. A., and Janda, K. D. (2001). Preparation of stilbene-tethered nonnatural nucleosides for use with blue -fluorescent antibodies. J. Org. Chem. 66, 1725-1732. (17) The designation of this compound as the 6-substituted isomer corresponds to common usage by vendors and in other publications; the structure is more properly described as the 4-substituted isomer. The use of this reagent in bioconjugation is described in Heiman, D. F., Yang, H. H. Y., and Flentge, C. A. (1986) Eur. Pat. Appl., EP199042. (18) Cann, A. J. (1997) Principles of molecular virology, pp 2249, 2nd ed., Academic Press, New York (19) Zalipsky, S. (1995). Chemistry of poly(ethylene glycol) conjugates with biologically active molecules. Adv. Drug Delivery Rev. 16, 157-182. (20) Abuchowski, A., Van Es, T., Palcank, N. C., and Davis, F. F. (1977). Alteration of immunological properties of bovine serum albumin by covalent attachment of poly(ethylene glycol). J. Biol. Chem. 252, 3578-3581. (21) Zalipsky, S., and Lee, C.-H. (1992). Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical Applications (J. M. Harris, Ed.) pp 347-367, Plenum Press, New York. (22) Delgado, C., Francis, G. E., and Fisher, D. (1992). The uses and properties of PEG-linked proteins. Crit. Rev. Ther. Drug Carrier Syst. 9, 249-304. (23) O’Riordan, C. R., Lachapelle, A., Delgado, C., Parkes, V., Wadsworth, S. C., Smith, A. E., and Francis, G. E. (1999). PEGylation of Adenovirus with Retention of Infectivity and Protection from Neutralizing Antibody in Vitro and in Vivo. Hum. Gene Ther. 10, 1349-1358. (24) Raja, K. S., Wang, Q., Gonzalez, M., Manchester, M., Johnson, J. E., Finn, M. G., unpublished results.

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