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Journal of Virology, February 2007, p. 1632-1640, Vol. 81, No. 4
0022-538X/07/$08.00+0     doi:10.1128/JVI.00960-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Interaction between a 54-Kilodalton Mammalian Cell Surface Protein and Cowpea Mosaic Virus{triangledown}

Kristopher J. Koudelka, Chris S. Rae, Maria J. Gonzalez, and Marianne Manchester*

Department of Cell Biology and Center for Integrative Molecular Biosciences, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, California 92037

Received 10 May 2006/ Accepted 14 November 2006


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ABSTRACT
 
Cowpea mosaic virus (CPMV), a plant virus that is a member of the picornavirus superfamily, is increasingly being used for nanotechnology applications, including material science, vascular imaging, vaccine development, and targeted drug delivery. For these applications, it is critical to understand the in vivo interactions of CPMV within the mammalian system. Although the bioavailability of CPMV in the mouse has been demonstrated, the specific interactions between CPMV and mammalian cells need to be characterized further. Here we demonstrate that although the host range for replication of CPMV is confined to plants, mammalian cells nevertheless bind and internalize CPMV in significant amounts. This binding is mediated by a conserved 54-kDa protein found on the plasma membranes of both human and murine cell lines. Studies using a deficient cell line, deglycosidases, and glycosylation inhibitors showed that the CPMV binding protein (CPMV-BP) is not glycosylated. A possible 47-kDa isoform of the CPMV-BP was also detected in the organelle and nuclear subcellular fraction prepared from murine fibroblasts. Further characterization of CPMV-BP is important to understand how CPMV is trafficked through the mammalian system and may shed light on how picornaviruses may have evolved between plant and animal hosts.


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INTRODUCTION
 
In the past several years, aside from understanding the natural life cycles of viruses as obligate intracellular pathogens, the power of viruses as tools for material applications has begun to be harnessed. There are several reasons why viruses are an excellent choice in this regard. First, rigid viral capsids provide natural molecular scaffolds that allow precise attachments for building nanostructures, with control over orientation and spacing that is not attainable using other materials, such as dendrimers or liposomes (14, 26, 29, 31, 46). Second, virus capsids use highly repeated structural motifs allowing for the polyvalent display of peptides (11), polysaccharides (22, 48), nucleic acids (54), or other synthetic structures (43). Self-assembly of virus capsids also ensures a lack of morphological polydispersity in the capsid size and shape, which is difficult to accomplish using synthetic materials (35, 36). Third, viral genomes are generally easy to manipulate, allowing the generation of mutants that can allow specific tailoring of the particle surface (12, 13, 60, 61). Fourth, procedures for inexpensive, efficient amplification of many such structures, e.g., plant viruses, virus-like particles, and bacteriophages, are already well defined (24, 25, 41, 49, 67).

Plant viruses are especially attractive for development in material science, nanotechnology, and vaccine applications because of their ease of production and purification. In particular, cowpea mosaic virus (CPMV) has been studied increasingly as a material for these purposes. CPMV is the type member of the genus Comovirus, which is part of the Picornaviridae superfamily spanning the plant and animal kingdoms and including Poliovirus and Rhinovirus. CPMV has a single-stranded, positive-sense bipartite RNA genome, and its icosahedral pseudo-T=3 structure is solved to 2.8 angstroms. CPMV capsids are comprised of two proteins, the large (L) (42 kDa) and small (S) (24 kDa) proteins, and 60 copies of each protein make up a mature capsid (34-36). CPMV grows to high yields in the black-eyed pea plant (Vigna unguiculata), and purification of virus at a yield of 1 mg/gram of leaves is straightforward and inexpensive (62).

The natural properties of CPMV make it an attractive nanoscale building block for biomedical and material science applications. Wang et al. first showed that the presence of natural reactive lysine residues on the particle surface and the inherent stability of the particles to temperature, pH, and organic solvents facilitated chemical conjugation (60). This method has been expanded through the use of azide-alkyne chemistry, often called click chemistry (22, 59). The ability to create infectious chimeras by insertion of residues and peptides on the capsid surface further allows the multivalent display and controlled chemical modification of the particles (13, 61). Genetically engineered CPMVs displaying immunogenic epitopes have also been used for vaccine development and induce immune responses that protect against viral or bacterial infection (4-6, 15, 17, 56).

Although the development of CPMV for vaccines and therapeutics has been well documented, few studies have been performed to investigate how CPMV interacts with cells in vivo. We recently showed that CPMV is bioavailable in the mouse, with a several-day retention time following either oral or intravenous inoculation, suggesting that the virus particles were taken up into cells in vivo (47). In separate intravital imaging studies, we showed that fluorescently labeled CPMV particles are readily internalized in vascular endothelial cells in live mouse or chick embryos, and this uptake can be blocked by polyethylene glycol coating of the virus (33). This study also demonstrated internalization of CPMV into endolysosomes, as evidenced by colocalization with the lysosomal and trans-Golgi markers LAMP-2 and ß-COP (33).

Since CPMV appears to interact specifically with mammalian cells, it is important to determine how the virus binds and is taken up into cells. To date, little is known about how CPMV interacts with mammalian cell membrane components. In this study, we quantified the interaction between fluorescently labeled CPMV and a mouse fibroblast cell line through fluorescence-activated cell sorting (FACS) analysis. We then studied whether CPMV could bind to specific membrane proteins by using a virus overlay protein blot assay (VOPBA). The VOPBA technique has been used to identify several virus receptors, including those for adenoviruses, arenaviruses, and coronaviruses (3, 7, 19, 40, 55, 65). Characterization of the requirement of proteoglycans or glycosylation for protein binding by CPMV was also performed using a heparan sulfate-deficient cell line, endoglycosidases, and glycosylation inhibitors. These studies will be helpful for further enabling the specific targeting of CPMV nanoparticles in vivo, since current biological applications using CPMV depend on better understanding these cell-surface interactions.


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MATERIALS AND METHODS
 
Cell culture. Murine BALB/Cl7 cells were grown and maintained in minimal essential medium supplemented with 7% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin-streptomycin. Murine MC 57 cells were grown and maintained in RPMI medium supplemented with 7% FBS, 1% L-glutamine, and 1% penicillin-streptomycin. Human KB epidermal carcinoma cells were grown and maintained in minimal essential medium supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin. CHO and heparan sulfate-deficient CHO cells obtained from the ATCC were grown and maintained in F-12 (Ham) nutrient medium supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin. Murine bone marrow-derived dendritic cells were isolated and cultured as described previously (23).

Protoplast preparation. California Blackeye 5 (Vigna unguiculata) protoplasts were isolated from 10-day-old plants by mechanically removing the lower cuticles of primary leaves, followed by incubating whole leaves in an enzyme solution consisting of 0.45% (wt/vol) cellulase, 0.05% (wt/vol) pectolyase, and 0.6 M mannitol at pH 5.5 for 3.5 h at 25°C in an Innova 44 incubator shaker at 100 rpm. The entire solution was filtered through a monolayer of sterile cheesecloth and centrifuged at 700 x g for 2 min. The pellet containing the protoplasts was collected and combined with a second pellet resulting from an additional 2-minute centrifugation of the supernatant. Protoplast pellets were combined, resuspended, and washed by centrifugation three times at 600 x g in 0.6 M mannitol.

Cell membrane isolation. BALB/Cl7, MC 57, KB, CHO, heparan sulfate-deficient CHO, and primary mouse dendritic cells were prepared by washing cells three times, with 0.9% sodium chloride irrigation (Baxter). Cell monolayers were then scraped from tissue culture flasks into a homogenization buffer containing 250 mM sucrose, 20 mM HEPES, 2 mM EDTA, 2 µg/ml aprotinin, and 2 µg/ml leupeptin at pH 7.0. All cells, including protoplasts, were then separately pelleted at 500 x g for 10 min and resuspended in additional homogenization buffer. Cells were homogenized and disrupted by a 15-ml Bellco Dounce homogenizer with a pestle clearance of 0.001 to 0.003 in., and then organelles and nuclei were pelleted at 720 x g for 10 min. The organelle and nucleus pellets were solubilized in 10 mM Tris-HCl, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.5% Triton X-100 at pH 8.0 and stored at –80°C (org/nuc fraction). The plasma membrane fraction of the remaining supernatant was isolated by ultracentrifugation in a Beckman SW41 rotor for 65 min at 32,500 rpm at 4°C (PM fraction). Pelleted membranes were resuspended and stored in the same fashion as the organelle and nucleus fractions. The protein concentration for each sample was determined with a Bio-Rad protein assay.

Viruses. CPMV was grown in California Blackeye 5 seeds obtained from The Burpee Company. Plants were grown and mechanically inoculated with wild-type CPMV as previously described (18). CPMV was harvested 7 days later as described previously (51). Cowpea chlorotic mottle virus (CCMV) and rabbit anti-CCMV polyclonal antibody were provided by Mark Young (20, 27).

Chemical coupling of Alexa Fluor 488 dye to CPMV (CPMV-A488). To conjugate dye molecules to lysines on the wild-type CPMV capsid, 1 mg Alexa Fluor 488 carboxylic acid, 2,3,5,6-tetrafluorophenyl ester (Molecular Probes) was suspended in 0.1 M potassium phosphate buffer and mixed with 5 mg of CPMV in a total volume of 1 ml, using a molar ratio of 10 dye molecules per asymmetric unit. The virus-dye suspension was incubated at room temperature in a rolling shaker for 72 h. After incubation, the samples were initially purified by ultracentrifugation at 42,000 rpm (3 h, 4°C) and resuspended in 1 ml of the same buffer. To eliminate free dye, the sample was further purified by sucrose gradient (10% to 30% [wt/vol] in phosphate-buffered saline [PBS]) ultracentrifugation at 28,000 rpm (2 h, 4°C). After collection of the labeled CPMV fraction, the virus was concentrated by ultracentrifugation at 42,000 rpm (3 h, 4°C). The final pellet was resuspended in PBS (Gibco-BRL) and filtered through a 0.2-µm membrane (Costar) to eliminate aggregate particles and sterilize the sample. The number of dye molecules per particle was calculated as follows: number of dye molecules/particle = {(A495 x dilution)/{varepsilon}Alexa Fluor 488}/(concentration of CPMV/molecular weight of CPMV), where {varepsilon}Alexa Fluor 488 is 71,000 and the molecular weight of CPMV is 5.6 x 106 grams/mole. Free Alexa Fluor 488 dye (A488) was created by allowing 100 µg of Alexa Fluor 488 carboxylic acid, 2,3,5,6-tetrafluorophenyl ester (Molecular Probes) to hydrolyze at room temperature in 1 ml of 0.1 M potassium phosphate buffer overnight on a rolling shaker.

Isolation of polyclonal anti-CPMV rabbit antibody. Purified CPMV was mixed 1:2 with complete Freund's adjuvant for the primary immunization of a rabbit. The rabbit was boosted with CPMV and incomplete Freund's adjuvant at weeks 6, 8, 10, 12, and 15. Ear bleeds were performed on weeks 8, 10, 12, and 14. The total blood volume of the animal was collected on week 19. Total immunoglobulin G containing CPMV-specific antibody was isolated from the harvested rabbit serum by using a MabTrap G II system from Pharmacia Biotech, following the manufacturer's instructions.

Flow cytometry. Confluent BALB/Cl7 and KB cells were collected by trypsinization, counted, and resuspended in their respective growth media to a concentration of 5 x 105 cells per ml. Two hundred microliters of cell suspension was placed in 400-µl V-bottomed wells positioned in 96-well plates. To each well, various concentrations of CPMV-A488, A488, or CPMV-A488 that was allowed to preincubate overnight with isolated polyclonal anti-CPMV rabbit antibody were added. Tubes were then allowed to incubated at 4°C or 37°C for 0.5, 1.5, 3.0, 8.0, or 24.0 h. To remove unbound virus, cells were washed three times in FACS buffer (PBS, 1 mM EDTA, 25 mM HEPES, 1% FBS at pH 7.0), followed by centrifugation at 500 x g for 6 min. After these washes, cells were fixed in 1% paraformaldehyde in PBS, washed two more times in PBS, and stored at 4°C in a PBS solution until FACS analysis. Twenty thousand events were then acquired for each sample, using a FACSCalibur machine (BD Biosciences), and data were analyzed with FlowJo software (Tree Star Inc.).

VOPBA. Cell membrane isolates were incubated with 50 mM dithiothreitol (DTT) and loading buffer at 95°C for 10 min prior to being loading into a 4 to 12% bis-Tris NuPage gel (Invitrogen). Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon P; Millipore). PVDF membranes were incubated for two rounds of 10 minutes each in denaturing buffer consisting of 6 M guanidine-HCl, 2 mM EDTA, 50 mM DTT, and 50 mM Tris-HCl at pH 8.3. PVDF membranes were then incubated for 10 minutes each at 4°C in renaturing buffer consisting of 10 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 2 mM DTT, and 0.1% Triton X-100 at pH 7.3, with sequentially reduced amounts of guanidine-HCl, at 4 M, 3 M, 2 M, 1 M, and 0 M. Following the guanidine-HCl treatment, the membranes were blocked using 5% nonfat dry milk (Carnation; Nestle) in renaturing buffer overnight at room temperature; this temperature was maintained for the rest of the procedure. Membranes were then incubated with a virus suspension consisting of 1% nonfat dry milk, 5% glycerol, and 10 µg/ml of purified virus (CPMV or CCMV) for 90 min, followed by four 5-min washes in wash buffer consisting of PBS with 0.2% Triton X-100. The membranes were incubated with appropriate purified rabbit antivirus primary antibody at a 1:1,000 dilution in 5% nonfat dry milk in PBS wash buffer for 1 h and then washed four times for 5 min each in wash buffer. The membrane was then incubated with a 1:10,000 dilution of goat anti-rabbit-horseradish peroxidase in 5% milk in wash buffer for 1 h and then washed four times for 5 min each in wash buffer. All of the incubations and washes in the VOPBA procedure were carried out using gentle shaking on a rotating shaker platform. Bands were visualized using chemiluminescence detection (SuperSignal; Pierce) and exposed to X-ray film (CL-Xposure; Pierce). Molecular weights were determined by computer-assisted comparison to the SeeBlue Plus 2 molecular weight standards (Invitrogen), using the AlphaEaseFC program (Alpha Innotech).

Trypsin treatment of cells. Confluent BALB/Cl7 cells in a 175-mm2 flask were washed three times, with 0.9% sodium chloride irrigation (Baxter), and then allowed to incubate for 10 min with 5 ml of 0.05% trypsin-EDTA (Gibco). Cells that were no longer adherent to the flask were collected, spun at 500 x g for 10 min, and resuspended in homogenization buffer, and PM fractions were isolated as described above. Twenty-microgram PM fractions from trypsinized cells and cells collected by cell scraping were run in a 4 to 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to a PVDF membrane for VOPBA as described above.

PNGase F treatment of membrane samples. Digestion of membrane proteins using peptide-N-glycosidase F (PNGase F) (New England Biolabs) was carried out using 20 µg of cell membrane isolate as described by the manufacturer. All 20 µg of the sample was run in 4 to 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane for VOPBA as described above. A sample of purified PNGase F (500 units) and its buffers was also loaded into the gel.

Inhibition of glycosylation in tissue culture cells. To one semiconfluent T175 flask of BALB/Cl7 cells with 20 ml of culture medium (described above), 1 µg/ml of tunicamycin (Sigma) or 3 mM benzyl N-acetyl-alpha-D-galactosaminide (Sigma) was added and incubated for 46 h to inhibit N-linked or O-linked glycosylation, respectively. Cell membranes from these cells were harvested as previously described. Ten micrograms of the membrane fraction was run in 4 to 12% SDS-PAGE and transferred to a PVDF membrane for VOPBA as described above.


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RESULTS
 
CPMV binds to mammalian fibroblast cells. Before biochemical analysis of a possible mammalian cellular receptor could be performed, CPMV binding had to first be demonstrated. Quantifying cellular binding and internalization of CPMV was accomplished through the use of a chemically modified CPMV and flow cytometry. A 2,3,5,6-tetrafluorophenyl ester of Alexa Fluor 488 was conjugated to the reactive lysines on the surface of CPMV, and the resulting virus, CPMV-A488, was determined to be labeled at a density of 70 fluorophores per particle by absorbance spectroscopy, as discussed in Materials and Methods. Following purification, CPMV-A488 particles were incubated at 37°C with the mouse fibroblast cell line BALB/Cl7 at ratios of 104, 105, and 106 viruses per cell (V/C). Representative data demonstrating a clear fluorescence shift (3.97% versus 97.7% positive) were observed for cells that bound or internalized CPMV-A488 after 1.5 h at 106 V/C compared with cells with no virus addition (Fig. 1A). These data are summarized in Fig. 1B, showing a dose-dependent shift in fluorescence intensity with increasing amounts of CPMV. Quantifying the cells that were positive for CPMV showed that at a dose of either 105 or 106 CPMV-A488, the V/C ratio could be detected by as little as 30 min of incubation and continued to increase for 24 h of incubation. While a relatively large ratio of V/C was required to detect binding, it is important that individual fluorescent virus particles are detected in this assay, rather than amplification of viral proteins following replication. Thus, mammalian cells were able to take up CPMV in a dose- and time-dependent manner.


Figure 1
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  		FIG. 1. CPMV is bound and internalized by BALB/Cl7 fibroblasts. (A) CPMV-A488 was incubated with BALB/Cl7 cells at 37°C, with either no virus (white histogram) or 106 V/C (gray histogram). (B) Percentages of CPMV-A488-positive cells at 0 (gray bars), 104 (striped bars), 105 (white bars), and 106 (black bars) V/C, with incubation times indicated on the x axis. (C) BALB/Cl7 cells were incubated with 106 V/C at 37°C or 4°C for 1.5 or 3 h (white and black bars, respectively). (D) CPMV-A488 that was precoated with polyclonal anti-CPMV rabbit antibody (white bars) or control CPMV-A488 (black bars) was added to BALB/Cl7 cells and incubated at 37°C for 3 h at either 104, 105, or 106 V/C.

To compare surface binding and internalization, labeled CPMV-A488 particles at a concentration of 106 V/C were incubated with BALB/Cl7 cells, as discussed above, for 1.5 or 3 h. One set of cells was incubated at 4°C, which is known to limit internalization of viruses (1, 39), and the other set of cells was incubated at 37°C. Cells incubated at 37°C showed a much more rapid accumulation of fluorescence (Fig. 1C).

CPMV capsid mediates binding and uptake in mammalian cells. Next, it had to be determined if the binding and internalization of CPMV were specific. The specificity of this interaction was first probed through flow cytometry with antibody-coated CPMV-A488 particles. When CPMV-A488 particles were allowed to preincubate with polyclonal rabbit anti-CPMV antibodies prior to introduction to BALB/Cl7 cells, the ability of the cells to bind and internalize CPMV-A488 was impaired compared to the ability to bind and internalize CPMV-A488 that was not coated with antibodies (Fig. 1D). This experiment shows that CPMV-A488 uptake is not caused by random cellular sampling of the extracellular environment but, rather, via specific interaction with the CPMV particle.

Binding and uptake studies mirroring those done with BALB/Cl7 cells and shown in Fig. 1B were carried out with human KB epidermal carcinoma cells. Similar trends of both time- and dose-dependent uptake were observed (Fig. 2 [data shown are for the highest concentration used, i.e., 106 V/C]). Interestingly, KB cells internalized less CPMV than did BALB/Cl7 cells, indicating that uptake may also be specific to the cell type.


Figure 2
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  		FIG. 2. CPMV uptake is dependent upon the cell line. KB cells (white bars) or BALB/Cl7 cells (black bars) were incubated at 37°C with CPMV-A488 for various incubation times, as noted on the x axis. Binding of an equimolar concentration of the A488 fluorophore alone to BALB/Cl7 cells is also shown (gray bars).

To confirm that the Alexa Fluor 488 modification of CPMV did not mediate cellular interactions, an equimolar concentration of Alexa Fluor 488 alone was incubated with BALB/Cl7 cells (Fig. 2). Alexa Fluor 488 by itself did not significantly label these cells, indicating that the fluorophore itself was not responsible for the binding and uptake of the CPMV-A488 particle. In additional tests, a 1,000-fold molar excess of Alexa Fluor 488 was unable to compete for CPMV-A488 binding (data not shown). Furthermore, competition experiments performed at 4°C showed that preincubation with unlabeled CPMV could reduce CPMV-A488 binding by an average of 64% when used at a 100-fold molar excess (P = 0.00003 by Student's t test [data not shown]). Together, these results indicate that the interaction of CPMV with mammalian cells is specific and is mediated by a direct interaction between the CPMV capsid and the cell surface.

CPMV binds to cell membrane proteins in mammalian cells. The results for CPMV binding to mammalian cells, in combination with our previous studies showing that CPMV is bioavailable in the mouse (33, 47), suggested that CPMV might interact specifically with mammalian cell surface proteins. Since we already determined that CPMV was taken up by a variety of cell types in vivo (47), we asked whether this was a specific or nonspecific interaction and whether it occurred via membrane proteins or other membrane components. The specificity of CPMV binding to membrane proteins was explored through the use of the VOPBA technique. VOPBA has been used extensively to identify cell surface receptors for other viruses (3, 7, 19, 40, 55, 65). In these experiments, cells were homogenized and separated into a fraction enriched for plasma membranes (PM fraction) as well as a separate fraction containing enriched organelles and nuclei (org/nuc fraction). Membranes were isolated from several cell lines, including murine fibroblast cell lines BALB/Cl7 and MC 57, human KB epidermal tumor cells, and murine dendritic cells. Enriched PM proteins were separated by SDS-PAGE, transferred to PVDF membranes, and prepared for detection by VOPBA, using purified, unlabeled wild-type CPMV and an anti-CPMV polyclonal antibody.

To further validate the flow cytometry results using the VOPBA procedure, BALB/Cl7 cell PM fractions were incubated with either wild-type CPMV or CPMV that had been coated with anti-CPMV antibody, as summarized in Fig. 1C. Wild-type CPMV reacted positively in the VOPBA, revealing a single, sharp band. CPMV that had been coated with antibody did not interact with the PM fraction from BALB/Cl7 cells (Fig. 3A). When PM fractions were treated with proteinase K prior to VOPBA treatment, no bands were seen, indicating that the binding entity is of a proteinaceous nature (data not shown).


Figure 3
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  		FIG. 3. CPMV binding proteins are detected in multiple mammalian cell lines. Cell fractions were separated by SDS-PAGE, transferred to PVDF membranes, and prepared for detection by VOPBA, using wild-type CPMV or CPMV that was precoated with polyclonal anti-CPMV rabbit antibody (Ab-CPMV). (A) The PM-enriched fraction from murine fibroblast BALB/Cl7 cells was analyzed by VOPBA, using wild-type CPMV (lane 1) or CPMV precoated with anti-CPMV antibody (lane 2). (B) PM fractions from murine fibroblast cell lines MC 57 and BALB/Cl7, human epidermal tumor cell line KB, and murine primary dendritic cells all contain a sole conserved 54-kDa protein, which strongly bound CPMV in VOPBA. (C) The organelle and nucleus fraction derived from BALB/Cl7 cells also contains the 54-kDa binding protein, as well as an additional CPMV binding protein of 47 kDa. (D) The PM fraction of Vigna unguiculata protoplasts does not contain a specific binding protein for CPMV, and PM fractions from the BALB/Cl7 and MC 57 cell lines contain the 54-kDa protein.

This CPMV binding protein (CPMV-BP) was detected in PM fractions from all of the mammalian cell lines tested and was determined, by comparison to molecular size markers, to be a 54-kDa protein (Fig. 3B). Membranes from human KB tumor cells and primary mouse dendritic cells contained the 54-kDa CPMV-BP, although these cells displayed a reduced signal compared to those from the murine BALB/Cl7 and MC57 fibroblast cell lines when equal amounts of protein were loaded in SDS-PAGE gels. This result was consistent with the reduced CPMV binding observed in KB cells in Fig. 2. In addition, subcellular fractionation experiments comparing the org/nuc fractions of BALB/Cl7 and MC 57 cells to the PM fractions showed that in addition to CPMV-BP, another CPMV binding protein also appeared that migrated at 47 kDa (Fig. 3C). No other proteins displaying significant CPMV reactivity were detected. When membrane fractions were subjected to VOPBA but the CPMV was excluded, no signal was detected (data not shown).

Screening for CPMV binding proteins by VOPBA, using membranes from cowpea protoplasts. To ask whether CPMV binding proteins could be detected in membrane samples from plant cells, VOPBA was performed using a PM fraction purified from protoplasts that had been prepared from 10-day-old, uninfected Vigna unguiculata seedlings. In contrast to the case for mammalian cells, no specific CPMV binding bands were observed (Fig. 3D). A faint band at 29 kDa was detected, but this band also appeared on a Western blot using anti-CPMV antiserum, suggesting that this protein may have cross-reacted with the polyclonal antiserum raised against CPMV that had been purified from plant tissue, rather than specifically interacting with CPMV particles. The 29-kDa band only appeared when >150 µg of total protoplast PM fraction was loaded and exposed to the X-ray film for two to four times longer than the exposure times for VOPBA on mammalian cell fractions. No other significant bands were observed.

CPMV-BP is specific for CPMV. To show that the interaction with CPMV-BP was specific to CPMV, VOPBA analysis was performed using purified particles from a similarly sized plant virus, CCMV. CCMV is a bromovirus with a 26-nm-diameter T=3 capsid that also infects the V. unguiculata host (53). CCMV did not interact with plasma membrane proteins from BALB/Cl7 cells in VOPBA (Fig. 4A, lane 1).


Figure 4
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  		FIG. 4. CPMV-BP is specific for CPMV capsid and is expressed on the surfaces of mammalian cells. (A) CCMV (lane 1) is not reactive with PM fractions of BALB/Cl7 cells in the VOPBA technique, unlike CPMV (lane 2). (B) Intact BALB/Cl7 cells were treated with or without trypsin prior to PM fraction isolation. CPMV-BP interacts with CPMV in the VOPBA when not treated with trypsin (lane 1) and does not appear when cells are preincubated with trypsin (lane 2).

Trypsin eliminates CPMV-BP reactivity in VOPBA. To confirm whether CPMV-BP was exposed on the surface of the cell, BALB/Cl7 cells were collected by trypsinization rather than scraping prior to homogenization and PM isolation. Trypsin effectively removed CPMV-BP reactivity from the PM fraction of intact cells (Fig. 4B), suggesting that CPMV-BP is cleaved on the cell surface by trypsin or that an alternative protein that bridges CPMV-BP binding to the cell membrane is removed under these conditions. This result is also consistent with the binding studies outlined in Fig. 1B, showing that 1.5 h of incubation at 37°C was required following trypsinization for CPMV to be taken up efficiently into BALB/Cl7 cells.

Effects of heparan sulfate and N- and O-glycosylation on CPMV-BP. To determine whether heparan sulfate proteoglycans participate in CPMV binding to CPMV-BP, PM fractions were isolated from the normal CHO cell line, which expresses heparan sulfate, and a CHO cell line that does not, and then both were subjected to VOPBA. There was no significant difference in band intensity for CPMV-BP in the VOPBA comparing normal CHO cells (Fig. 5A, lane 2) and heparan sulfate-deficient CHO cells (Fig. 5A, lane 3); therefore, heparan sulfate does not play a role in CPMV binding to the 54-kDa CPMV-BP.


Figure 5
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  		FIG. 5. CPMV-BP lacks heparan sulfate modification and N- or O-glycosylation. (A) VOPBA using PM fractions isolated from BALB/Cl7 cells (lane 1), CHO cells (lane 2), and heparan sulfate-deficient CHO cells (lane 3), with all having similar recognition of CPMV-BP. No other bands were observed. (B) VOPBA using PM fraction isolated from BALB/Cl7 cells. The membrane fraction was left untreated (lane 1) or treated with PNGase F (lane 2). The PNGase F-only lane contains 500 units of enzyme (lane 3). No other bands were observed. (C) VOPBA using PM fractions isolated from BALB/Cl7 cells grown in the presence of benzyl N-acetyl-alpha-D-galactosaminide to inhibit O-linked glycosylation (lane 1) or tunicamycin to inhibit N-linked glycosylation (lane 2) or from a no-drug control (lane 3). No other bands were observed.

To determine whether the 54-kDa CPMV binding protein is a glycoprotein, the PNGase F endoglycosylase was first used to remove N-linked glycans from cell membrane components. In this experiment, 20 µg of the PM fraction from BALB/Cl7 cells was treated with PNGase F, and the samples were then subjected to VOPBA. Figure 5B shows CPMV binding to CPMV-BP whether or not PNGase F was added, indicating that CPMV-BP is not N-glycosylated. In addition, CPMV bound to a band of 29 kDa which was also found in the PNGase F lane, where 500 units of PNGase F alone was loaded. Thus, either CPMV interacts directly with PNGase F (29 kDa), or this represents a nonspecific interaction between CPMV and the large amount of enzyme loaded into the gel.

In a second series of experiments, metabolic glycosylation inhibitors were used in cell culture prior to preparation of membranes to determine whether N- or O-glycosylation was required for CPMV binding. BALB/Cl7 cells were treated with the glycosylation inhibitor tunicamycin (inhibiting N-linked glycosylation) or benzyl N-acetyl-alpha-D-galactosaminide (inhibiting O-linked glycosylation) prior to membrane isolation as performed previously. When equal protein concentrations of these membrane fractions were subjected to VOPBA, CPMV again interacted with CPMV-BP as previously shown, with no other bands appearing (Fig. 5C). The drug-inhibited samples did show significant reductions in signal, although equal amounts of protein were loaded in each lane. When quantified using densitometry, the benzyl N-acetyl-alpha-D-galactosaminide-treated cells (Fig. 5C, lane 1) showed a 20% reduction in signal, and the tunicamycin-treated cells (Fig. 5C, lane 2) showed a 75% reduction in signal, compared to that of the control sample with no drug added (Fig. 5C, lane 3). In the org/nuc fractions prepared from inhibitor-treated BALB/Cl7 cells, the 47-kDa band was also present at a lower level (data not shown). Since the migration of the CPMV protein appeared not to be affected by either deglycosylation or production in the presence of glycosylation inhibitors, these results indicate that the CPMV-BP is neither N- nor O-glycosylated. Since O-linked endoglycosylases were not tested, an alternative possibility is that in the presence of benzyl N-acetyl-alpha-D-galactosaminide, a lower-molecular-weight form of the protein is generated, lacking O-glycans, and that this deglycosylated form does not interact with CPMV.


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DISCUSSION
 
Here we show that the plant virus CPMV interacts with mammalian cell membranes, and we identify two CPMV binding proteins that were detected in several mammalian cell lines. These results were obtained through flow cytometry and, biochemically, through the use of VOPBA. For isolated membrane fractions examined by VOPBA, it was shown that CPMV interacts with a 54-kDa protein (CPMV-BP) that is present in the PM-enriched fractions from several mammalian cell types, as well as a 47-kDa protein found within the org/nuc fraction. Studies using a heparan sulfate-deficient cell line, glycosidases, and metabolic glycosylation inhibitors indicated that the 54-kDa protein was not modified by heparan sulfate or N- or O-linked glycosylation.

All mammalian cell lines tested expressed the 54-kDa CPMV-BP, including human KB cells. Interestingly, murine BALB/C17 fibroblasts showed a stronger signal in the VOBPA than did KB cells, and this difference was also observed by direct binding in flow cytometry assays. This difference could reflect a diversity of expression levels of CPMV-BP between the cell lines or changes in affinity for CPMV when the protein is produced from different cell lines. The 47-kDa band localized to the org/nuc fraction of membranes could be detected in both BALB/Cl7- and MC 57-derived samples. This 47-kDa protein may be a cleavage product or isoform of the 54-kDa protein or an entirely different protein. Strong 54-kDa CPMV-BP signals in VOPBA using both cell membrane fractions and the nuclear and organelle fractions for the fibroblast cell lines showed relatively equal distributions of this protein. Further biochemical fractionation studies are required to definitively localize the 47-kDa protein intracellularly. At present, affinity purification and tandem mass spectrometry experiments are under way to identify and characterize both the 54-kDa and 47-kDa proteins.

The 54-kDa CPMV-BP appears to be abundant and also present on the cell surface, as shown by the fact that trypsin treatment of intact cells prior to membrane isolation eliminates binding to the 54-kDa band. The fact that the 54-kDa protein can interact with CPMV in VOPBA indicates that the affinity is probably quite high. Indeed, for other cellular receptor proteins that are capable of interacting with virus capsids or capsid glycoproteins in VOPBA experiments, the affinity between virus and receptor reflects an equilibrium dissociation constant (KD) in the low micromolar to nanomolar range (30, 50). Quantification of the binding of CPMV by flow cytometry showed significant binding and uptake of CPMV-A488 in both a dose- and time-dependent manner. At the 24-hour time point, using 106 virions per cell, almost every cell bound or internalized a large enough amount of CPMV-A488 to achieve an almost 2-log increase in fluorescence. The interaction appears to be specific to the CPMV capsid and not the A488 fluorophore; indeed, when CPMV labeled with fluorescein was used in place of wild-type CPMV in the VOPBA, many nonspecific interactions with the cell membrane were observed, as previously noted (M. G. Finn, personal communication), but when CPMV-A488 was used in the VOPBA, the single CPMV-BP band was seen, and few, very faint nonspecific interactions were observed (data not shown). A488 also did not compete for CPMV binding by flow cytometry (Fig. 2).

The 54-kDa CPMV-BP showed no signs of glycosylation by several methods. First, the sharpness of the 54-kDa band on VOPBA was consistent with an unglycosylated receptor or a single glycosylated form. Second, the CPMV-BP intensity visualized through VOPBA was not affect between heparan sulfate-positive and heparan sulfate-negative CHO cells. Third, the PNGase F glycosidase treatment yielded no reactive cleavage products or reduction in the original signal. Fourth, metabolic inhibition of mammalian cells revealed no CPMV-reactive deglycosylated products, and the reduction in signal observed in Fig. 5C was most likely associated with cytotoxicity observed when the cells were grown in the presence of either glycosylation inhibitor. Fifth, preliminary studies showed that when the PM fraction of BALB/Cl7 cells was passed over a concanavalin A lectin-Sepharose column, CPMV-BP was not retained and, instead, flowed through the column (K. J. Koudelka, preliminary results). The role of PNGase F binding to CPMV (Fig. 5B, lane 3) is unknown but likely reflects nonspecific binding because of the large amount of PNGase F loaded into the gel.

Intravital imaging studies from our laboratory recently demonstrated the uptake of fluorescently labeled CPMV in the vascular endothelia of both mouse and chick embryos (33). Cellular uptake of CPMV was also indicated when a broad biodistribution of CPMV in mouse organs and tissues was observed following oral or intravenous dosing (47). Interestingly, a similar CPMV-BP was not identified in plant protoplasts derived from mature leaf tissue. If there is a plant homolog of CPMV-BP, it might be expressed only in specific regions in the plant phloem, or it is possible that other putative receptor proteins may mediate specific virus uptake in other regions of the infected plant. Alternatively, the procedure for isolation of protoplast membranes may release plant proteases that inhibit binding to a homolog of CPMV-BP.

A specific CPMV receptor in plants has not been identified. Trafficking of CPMV within leaves of its native host plant, Vigna unguiculata, occurs through the use of a virus-carried movement (M) protein, which forms tubules between cells through adjacent cell walls, forming a tunnel through which whole virions can pass and regulating movement between cells in infected leaves (2, 8, 21, 32, 44, 45, 52, 58). To achieve systemic replication, plant viruses must typically travel via the phloem, the vascular system of the plant; generally, this pathway is not mediated by movement protein and could possibly require a specific cellular receptor (16). Thus, it is possible that CPMV-BP represents a homolog of a plant receptor that is expressed in only a small number of cells within vascular structures in plants and is not expressed in leaf protoplasts.

The fact that a plant virus is specifically taken up by animal cells is intriguing. There are several examples of viruses having the ability to cross phylogenetic barriers, such as flock house virus (insects, plants, and mice) (10) and bluetongue virus (insects and ruminants) (38, 63). The similarities in genetic organization and structure between the plant and animal picornaviruses are significant, and it is unlikely that picornavirus-like viruses spontaneously arose separately in both plants and animals. For example, using the Enterovirus genus within the Picornaviridae family, the genome arrangements of animal Poliovirus (28) and Coxsackievirus (9) are very similar to that of CPMV (37, 57) (Fig. 6A to C). All of these infectious RNAs have the same placement of VPg, capsid proteins (VP1-3, L, and S), nonstructural proteins (2A-C, 3A, CPRO, and NBSP), proteases (3C and PRO), and polymerases. These viruses all share a similar capsid structure, where VP1 is analogous to the S subunit of CPMV. The L subunit of CPMV can be divided into two separate ß-jellyroll domains that directly correlate to VP2 and VP3 (34, 64, 66). Our previous studies demonstrating the systemic bioavailability of orally delivered CPMV (47) and the discovery of specific binding proteins for CPMV in animal cells suggest that plant viruses may gain access to cells of mammalian hosts at a higher frequency than previously thought. A full characterization of these new mammalian binding proteins for CPMV promises to yield a wealth of knowledge on the mammalian uptake of CPMV, and perhaps even the evolution of the picornavirus superfamily.


Figure 6
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  		FIG. 6. Genome arrangements and capsid structures of several picornaviruses from the genus Enterovirus. (A) Arrangement of poliovirus genomic RNA, which is 7,440 nucleotides long (28). (B) Genomic arrangement of coxsackievirus, which is 6,621 nucleotides long (9). (C) Genomic arrangement of CPMV. RNA1 and RNA2 are 5,889 and 3,481 nucleotides long, respectively (37, 57). Corresponding proteins or domains for the viruses are indicated by dotted lines. (D) Capsid structures of three viruses in the Picornaviridae superfamily. For poliovirus (PDB accession no. 1ASJ), the diameter is 325 Å, and for coxsackievirus (PDB accession no. 1Z7S), the diameter is 323 Å; the capsid VP1, VP2, and VP3 subunits are indicated in black, gray, and white, respectively. For CPMV (PDB accession no. 1NY7), the diameter is 317 Å, the L subunit is shown in white, and the S subunit is shown in black. All structures were rendered using Chimera from UCSF (42).

Understanding the interaction of CPMV with mammalian vasculature is important for further development of CPMV and similar viruses as tools for nanobiotechnology. In order to specifically redirect the viruses to tissues or locations of disease in vivo, it is critical to understand the mammalian uptake pathway. The 54-kDa and 47-kDa mammalian binding proteins are highly specific, have high affinities, and likely mediate endocytosis of CPMV. Full characterization of these proteins will provide opportunities to precisely tailor the targeting of virus-based nanoparticles in vivo.


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ACKNOWLEDGMENTS
 
This work was supported by grant CA112075 from the National Institutes of Health to M.M.

We thank members of the Manchester and Schneemann laboratories for helpful discussions and Mark Young for his generous gifts of purified CCMV and rabbit anti-CCMV polyclonal antibody.


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FOOTNOTES
 
* Corresponding author. Mailing address: The Scripps Research Institute, CB262, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Phone: (858) 784-8086. Fax: (858) 784-2139. E-mail: marim{at}scripps.edu. Back

{triangledown} Published ahead of print on 22 November 2006. Back


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Journal of Virology, February 2007, p. 1632-1640, Vol. 81, No. 4
0022-538X/07/$08.00+0     doi:10.1128/JVI.00960-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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