Bioconjugate Chem. 2004, 15, 807−813
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Chemical Conjugation of Heterologous Proteins on the Surface of Cowpea Mosaic Virus Anju Chatterji,† Wendy Ochoa,† Lara Shamieh,‡ Shant P. Salakian,† Sek Man Wong,§ Gail Clinton,‡ Partho Ghosh,| Tianwei Lin,*,† and John E. Johnson*,† Department of Molecular Biology and Center for Integrative Molecular Biosciences and The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, Center for Bio/Molecular Sciences and Engineering, 4555 Overlook Avenue SW, Oregon Health Sciences University, Portland, Oregon, Department of Biological Sciences, National University of Singapore, Science Drive 4, Singapore, 117543, and Department of Chemistry and Biochemistry, University of California, San Diego, 9500, Gilman Drive, La Jolla, California 92093. Received March 13, 2004; Revised Manuscript Received May 18, 2004
Genetic economy leads to symmetric distributions of chemically identical subunits in icosaherdal and helical viruses. Modification of the subunit genes of a variety of viruses has permitted the display of polypeptides on both the infectious virions and virus particles made in expression systems. Icosahedral chimeric particles of this type often display novel properties resulting in high local concentrations of the insert. Here we report an extension of this concept in which entire proteins were chemically crosslinked to lysine and cysteine residues genetically engineered on the coat protein of icosahedral Cowpea mosaic virus particles. Three exogenous proteins, the LRR domain of internalin B, the T4 lysozyme, and the Intron 8 gene product of the of the HER2 tyrosine kinase receptor were derivatized with appropriate bifunctional cross-linkers and conjugated to the virus capsid. Characterization of these particles demonstrated that (1) virtually 100% occupancy of the 60 sites was achieved; (2) biological activity (either enzyme or binding specificity) of the attached protein was preserved; (3) in one case (LRR-internalin B) the attached protein conformed with the icosahedral symmetry to the extent that a reconstruction of the derivatized particles displayed added density with a shape consistent with the X-ray structure of the attached protein. Strategies demonstrated here allow virus particle targeting to specific cell types and the use of an icosahedral virus as a platform for structure determination of small proteins at moderate resolution.
INTRODUCTION
Several rod shaped and icosahedral viruses have been used for expression and display of foreign peptides (110). Cowpea mosaic virus (CPMV) offers novel advantages for presentation of foreign epitopes on its surface (1-6). The virus capsid is highly permissive to genetic alterations, accumulates to high levels in plants, and the virus is stable over a wide range of pH and temperature conditions, making it amenable for chemical modification. The structure of the virus has been resolved to 2.8 Å resolution, allowing rational designing of peptide presentation on the virus surface (11-15). The inherent nature of the viral symmetry coupled with the polyvalent display of exogenous protein opens up additional venues for structure determination of the foreign epitope being displayed (16-18). Surface presentation of foreign polypeptides that are introduced genetically into the capsid, however, is restricted to approximately 30 amino acids (4, 18-19). A useful extension of this technology will be to display full* To whom correspondence should be addressed. Ph: (858)784-9705. Fax: (858)-784-8660. E-mail:
[email protected] (J.E.J.); Ph: (858)-784-9730. Fax: (858)-784-7775. E-mail: twlin@ scripps.edu (T.L.). † Department of Molecular Biology and Center for Integrative Molecular Biosciences and The Scripps Research Institute. ‡ Oregon Health Sciences University. § National University of Singapore. | University of California.
length proteins and protein domains on the virus capsid. We explored the option of chemically coupling exogenous proteins to the virus capsid using a variety of readily available homo-and heterobifunctional cross-linkers and investigated the suitability of CPMV to serve as a scaffold for structure determination of the conjugated proteins by directly imaging the altered virus particle with methods that make use of the icosahedral redundancy. The CPMV genome consists of two separately encapsidated RNA molecules of 6.0 kb (RNA 1) and 3.5 kb (RNA2) (20, 21). The viral capsids contain 60 copies each of the large and small coat protein subunits arranged with icosahedral symmetry (22). Any attachment on the viral capsid therefore translates into the heterologous protein being presented on the virus surface 60 times, assuming 100% occupancy. To illustrate the versatility of CPMV as a polyvalent display platform, we used proteins with different biological functions as well as size range for cross-linking (8-22 kDa) to the virus surface. Some of these proteins were chosen to provide the necessary proof of principle that the structural information derived from the virus-protein-conjugated complexes is authentic while others were selected to determine the effect of enhanced local concentration from the multivalent presentation of the protein on the virus capsid. The attachment sites for chemical derivatization of the viral capsid were engineered by site-specific mutagenesis of four of the five surface lysine residues to arginine thereby retaining only one reactive lysine on the virus (23). Further, wild-type CPMV does not have any surface
10.1021/bc0402888 CCC: $27.50 © 2004 American Chemical Society Published on Web 07/01/2004
808 Bioconjugate Chem., Vol. 15, No. 4, 2004
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Figure 1. Left. Schematic diagram of CPMV capsid. The reference asymmetric unit is framed and the symmetry elements are labeled. Small (S) subunits labeled A are in blue, and the large (L) subunits formed by two domains are in red (B domains) and in green (C domains). The oval represents a 2-fold axis; the triangle is a 3-fold axis and the pentagon a 5-fold axis. Right. Ribbon diagram of the asymmetric unit comprised by three jellyroll β sandwiches with surface Lys residues represented as spheres in cyan. Residue numbers are preceded by a 1, if they are in the small subunit and a 2, if they are in the large subunit. K138 and K182 are in the A domain, K299 and K234 are in the C domain, while K2199 is in the B domain. Two mutants were used for the conjugation. Mutant vK299 contains the unique K299 (encircled) and mutant vEFB contains a unique cysteine engineered in the βE-βF loop of the C domain (magenta). The figure is drawn with MOLSCRIPT.
exposed cysteines on the virus capsid; hence, it was straightforward to engineer a cysteine residues at a unique location on the virus surface (24, 25). These CPMV mutants were used for directed modification of the virus with foreign proteins (Figure 1). EXPERIMENTAL PROCEDURES
Propagation of the Virus in Plants. The primary leaves of cowpea seedlings were mechanically inoculated with 10 µg each of cDNA plasmids encoding RNA1 (pCP1) and RNA2 (pCP2) (26). The initial virus inoculum was extracted from infected cowpea leaves with 0.1 M potassium phosphate, pH 7.0 (phosphate buffer) 7 days post infection. Typically 50 plants were infected with the plant extract, and the symptomatic leaves were harvested after three weeks. For the lysine and cysteine mutants of CPMV, the respective mutant cDNA clones of RNA 2 in conjunction with pCP1 were used to initiate the infection. Purification of LRR-internalin B, T4 Lysozyme, and Intron 8. The LRR domain of internalin B (LRRInlB, 27) and the Intron 8 proteins (Int 8, 28) were cloned as plasmids in E coli expression vectors (pet 28 and pet 30, respectively, Novagen) and overexpressed in bacterial cells BL21 under the control of T7 promoter. Both proteins have the 6xHIS tag and therefore can be purified readily on Ni-NTA beads using the well-developed metal affinity chromatography (29). The cys mutant of T4 lysozyme was purified by ion exchange chromatography on a Mono S column (Amersham Pharmacia Biotech). The proteins were further purified on Superdex 200 (Amersham Pharmacia Biotech) columns by size exclusion chromatography and confirmed by immunoblots using anti HIS antibody (Clontech). Chemical Conjugation of Heterologous Proteins on the Virus Capsid. Both LRR-InlB and the T4 lysozyme have cys residues that were used as the reactive groups to attach a homo- or a heterobifunctional crosslinker with its reactive maleimide or NHS ester group cross-linked to the unique cysteine or lysine residues on the virus, respectively. There are no unique cysteines present on the Int 8; therefore, thiolation reagent SATA
was used to modify the N-terminal amine of the protein to introduce a protected thiol group (30). The protected thiol group is treated with hydroxylamine hydrochloride just before addition of the cross-linked virus to deprotect the introduced thiol and used for conjugation. Briefly, the conjugation reaction was completed in two steps. In the first step, the heterobifunctional crosslinker, 50 µL of 5 mg/mL solution of sulfo SMCC (Pierce) dissolved in DMSO (Pierce), was added to the virus sample (4-5 mg) and stirred gently at room temperature for 1 h. The samples were later purified on a Sephadex G-25 column to get rid of the excess cross-linker and added to the protein samples in 50× excess molar ratios of protein to the virus. The samples were incubated at room temperature for 2 h or overnight at 4 °C. The conjugated samples were finally purified on superose6 column and the virus specific peak was collected and analyzed for the conjugated reaction products by western blots. Biochemical Analysis of the Conjugated Samples. Typically, the retention time of wt CPMV on Superose6 column (Pharmacia) is 12 min (at flow rate 0.4 mL/min), but the conjugated samples eluted at approximately 8.7 min, indicating that the virus size was larger than the wild type. The virus conjugate peak was collected and analyzed on 4-12% Bis Tris gels (NuPAGE, Invitrogen) and probed with both CPMV and anti HIS antibodies using standard procedures for immunoblotting (31). Bioassays for T4 Lysozyme and Int 8. T4 Lysozyme. The rate of lysis of Micrococcus lysodeikticus is determined as suggested by Shugar et al. (32). The cells were prepared by suspending 9 mg of dried M. lysodeikticus (Sigma) cells in 25 mL of 0.1 M potassium phosphate buffer, pH 7.0. A 100 µL aliquot of the virus-T4conjugated sample (4 mg/mL) was mixed with 1 mL of M. lysodeikticus cell suspension. The samples were then analyzed on a UV-vis spectrophotometer to record the decrease in the absorbance of the samples at 450 nm as a function of time. Intron 8. Monolayer cultures of ≈5 × 106 NIH-3T3 cells stably transfected with HER-2 (17-3-1) or parental
Bioconjugation of Proteins on CPMV Capsid
NIH-3T3 cells were plated in 10-cm tissue culture plates and incubated with the virus-Int 8 conjugate or wild-type CMPV in serum-free DMEM for 30 min at 37 °C. The cells were washed two times with phosphate buffered saline (PBS) and extracted in MTG (50 mM Tris‚HCl (pH 7.0), 1.0% NP-40, 0.1% deoxycholic acid, 30 mM Na3VO4, 1 mM PMSF). Lysates were cleared by centrifugation and quantified using the Bradford Assay (BioRad). Equal amounts (20 µg total protein per lane) were resolved by SDS-PAGE in a 4-12% gradient polyacrylamide gel and transferred onto nitrocellulose. Ponceau stain was used to assess protein transfer and protein amounts in loaded samples. Membranes were blocked in 5% milk and then probed with R-CPMV or R Int 8 antibody, to assess virusInt 8 binding, or with R-phosphotyrosine (Sigma) to assess receptor activation. Electron Microscopy and Image Reconstruction of the Conjugates. Bioconjugated samples at a concentration of 1 mg/mL in 100 mM potassium phosphate, pH 7.0 (phosphate buffer), were placed on glow-discharged carbon film and stained for 1 min with 2% uranyl acetate. The grids were observed under a Philips CM120 microscope operated at 100 kV at a magnification of 45 000×. The micrographs were recorded under minimal dose conditions (
