| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Short Communication |
1 Laboratoire de Microscopie Electronique Structurale, Institut de Biologie Structurale, UMR 5075 CNRS-CEA-UJF, F-38027 Grenoble cedex, France
2 Laboratoire d'Enzymologie Moléculaire, Institut de Biologie Structurale, UMR 5075 CNRS-CEA-UJF, F-38027 Grenoble cedex, France
3 Institut de Virologie Moléculaire et Structurale, FRE 2854 CNRS-UJF, BP181, F-38042 Grenoble cedex 9, France
Correspondence
J. F. Conway
jxc100{at}pitt.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT MAIN TEXTREFERENCES |
|---|
Present address: Department of Immunology, University of Pittsburgh School of Medicine, W1052 Biomedical Science Tower, 200 Lothrop Street, Pittsburgh, PA 15261, USA. 
Present address: Department of Structural Biology, University of Pittsburgh School of Medicine, Room 2047, Biomedical Science Tower 3, 3501 Fifth Avenue, Pittsburgh, PA 15260, USA.
|
MAIN TEXT |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
).
Dodecahedra of adenovirus serotype 3 (Ad3) are observed in infected cells and assemble spontaneously upon expression of the 60 kDa penton base protein in insect cells as 12 pentameric penton bases (Fender et al., 1997; Norrby, 1966; Pettersson & Höglund, 1969; Schoehn et al., 1996) that are either partially or fully N-terminally proteolysed (Fuschiotti et al., 2006). Co-expressed fiber protein binds to the penton bases as in the virus, and combining fibers and penton bases from different adenovirus serotypes allows targeting of specific cell types (Mizuguchi & Hayakawa, 2004). The penton base protein is multifunctional: in addition to its structural role and binding the fiber, it is involved in viral cell entry through binding to cellular 
v integrins (Wickham et al., 1993) and in viral release from endosomes on the pathway towards the cell nucleus (Greber et al., 1993). Cell entry has been demonstrated by immunological localization of penton bases within target cells (Fender et al., 2003).
As structural analysis of the dodecahedron reveals that the internal cavity of 80 Å (8 nm) diameter is too small to accommodate a gene (Fuschiotti et al., 2006; Schoehn et al., 1996), we have pursued an alternative use for targeted drug delivery. Our first goal was to control assembly status of the dodecahedral particle, as production of intact particles was not reproducible. Our recent structural characterization (Fuschiotti et al., 2006) determined the importance of proteolysis of the N-terminal 37 residues, 60 copies of which cannot fit into the interior volume of the dodecahedron and are absent in assembled particles. We explored various conditions for promoting assembly, as listed in Table 1. Assembly status was screened by negative-stain electron microscopy (EM) (Fig. 1) and by native agarose-gel electrophoresis.
|
|
) and purified by anionic-exchange chromatography (Econo-Pac High Q Cartridge; Bio-Rad) as described previously (Fuschiotti et al., 2006). Disassembly was readily achieved by dialysing dodecahedra preparations at 4 °C in 50 mM Tris/HCl (pH 8.9) containing 5 mM EDTA for 3–4 days and applying several changes of buffer. The particle-assembly status was checked visually by negative-stain EM and biochemically by using a 15–40 % (w/w) sucrose-density gradient at pH 8.9 (50 mM Tris/HCl, 5 mM EDTA, 10 % glycerol). Centrifugation was performed in a Beckman ultracentrifuge using a TLS 55 rotor at 55 000 r.p.m. for 4 h. Disassembled dodecahedra were identified in low-molecular-mass fractions of the gradient by Western blot using a specific anti-dodecahedron antibody (Fender et al., 2003). The disassembled preparation was then redialysed for 3–4 days in 50 mM phosphate buffer at pH 6.6, including 750 mM ammonium sulphate (Braun et al., 1999; Salunke et al., 1986) with several buffer changes, to promote assembly. The assembly status was again checked by negative-stain EM and by sucrose density-gradient separation (high-molecular-mass fractions of 3–3.5 MDa) and Western blotting.
We established that pH and ionic strength are the major factors affecting assembly status. Titration in phosphate buffer at different pH values identified the limits between which the dodecahedra remained stable. Under conditions of high pH (8.9) and low ionic strength, penton bases were unassembled and most of the protein was present in the lighter fractions of the sucrose gradient (Fig. 1k
) and observed by EM as free bases or irregular clusters (Fig. 1l). Assembly into dodecahedra was observed after removal of chelating agents (EDTA) at high ionic strength and low pH. After dialysis against buffer containing 750 mM ammonium sulphate at pH 6.6, the preparation was recovered in the low part of the sucrose-density gradient (Fig. 1m), corresponding to heavier, assembled particles, as confirmed by EM (Fig. 1n). Control of particle assembly through manipulating pH promises an effective method for encapsidating therapeutic molecules, as well as establishing the feasibility of using this system at physiological conditions.
The effects of pH on particle status may be explained by examining the structural elements that maintain particle integrity. The subunit interactions within each penton base are relatively strong, as is evident from visualization of free penton bases in all conditions under which dodecahedra were not formed. For the Ad2 penton base, approximately 26 % of the subunit surface area is buried at this interface (Zubieta et al., 2005). However, only three small contact areas mediate the assembly of penton bases into the Ad3 dodecahedron (Fuschiotti et al., 2006). The amino acids at these interfaces may be inferred by analogy with the Ad2 penton base sequence and the Ad2 dodecahedron crystallographic structure (Zubieta et al., 2005). Most of the interface residues are the same or substituted conservatively, suggesting that the specific interactions will be similar. These interactions were narrowed down in the Ad2 crystal structure to four residues in each of the three regions, corresponding in the Ad3 sequence to aa 99–102 (NDFT) contacting aa 425–428 (RSTR), and aa 58–61 (SDVS) contacting a copy of itself. Of these in the Ad3 sequence, five are substituted conservatively compared with the Ad2 protein and six are identical. Two that are identical, D100 and R425, appear to make a salt bridge between pentons in the Ad2 structure (PDB id, 1X9P), the strength of which may be modulated by pH. Mutations at these sites will be necessary to establish the residues critical to dodecahedron stability, as well as to explore modifications that enhance particle integrity.
Our second goal was to demonstrate passive encapsidation of ‘cargo’ within the dodecahedron by shifting buffer conditions from disassembly to assembly after incubation with a marker molecule and subsequent imaging by cryo-EM to allow visualization of internal density, performed as described previuosly (Fuschiotti et al., 2006). The marker chosen, Nanogold (Nanoprobes Inc.), has a small core of gold atoms, 14 Å (1.4 nm) in diameter, surrounded by an organic shell of triphenylphosphines and is sufficiently electron-dense to be visible at the magnification used (x38 000). Unlike larger colloidial gold, Nanogold does not adhere spontaneously to protein, but is often used to specifically label thiols, such as the cysteine side chain, through maleimido derivitization of one of the phosphines and an appropriate incubation process. In this experiment, it is a passive marker chosen because it is small enough to fit inside the dodecahedron cavity. Dodecahedra were disassembled at basic pH (8.9) and mixed with Nanogold particles. Subsequently, the sample was dialysed at 4 °C into ‘assembly’ buffer. Several approaches were used to remove free gold particles, including filtration by 100K Ultrafree Millipore 0.5 centrifugal filters, dialysis (100 000 MWCO membrane; Perbio) and sucrose density-gradient separation.
For cryo-EM, samples were applied to holey carbon on copper Quantifoil grids, blotted and plunge-frozen into liquid ethane according to standard methods (Fuschiotti et al., 2006). Frozen grids were transferred onto a Gatan 626 cryoholder for imaging in an FEI CM200 microscope operating at 200 kV and a nominal magnification of x38 000. Low-dose techniques were used to focus and expose suitable areas onto Kodak SO163 film with an estimated dose of 6–12 e Å–2. Film was developed in full-strength Kodak D19 developer for 12 min.
Micrographs reveal gold particles that are not encapsidated by dodecahedra readily visible on the support film (Fig. 2a). Fortuitously, these free gold clusters are scavenged by the carbon film and none were observed free in the holes of frozen buffer, thus avoiding coincidental co-projection of gold particles and empty dodecahedra. The carbon-attached clusters are variable in size, suggesting that they have a tendency to aggregate, and we conclude that the smallest of the black dots probably represents one or two Nanogold clusters. We note that this behaviour has been observed before for a similar particle (Cheng et al., 1999).
|
) and the approximately 27 Å (2.7 nm) diameter of the entire Nanogold moiety including its organic shell, we do not expect to see encapsidation of large and highly visible aggregates, but rather of single or double Nanogold particles. Co-projection with surrounding dodecahedral density reduces their visibility further. We also expect only a limited number of successful encapsidations, unless there is an unlooked-for affinity between Nanogold and the penton base subunit. Nonetheless, evidently promising particles were selected for analysis – several are shown in Fig. 2(b) – and may be compared with control particles prepared in the absence of Nanogold (Fig. 2f). To enhance signal from any encapsidated moieties, the overlaid density due to the dodecahedron was removed by projecting equivalently oriented images from the reconstruction of the empty dodecahedra (Fuschiotti et al., 2006) and subtracting these from the EM images (Fig. 2c–e). Small cores of dark density that are significantly stronger than the noisy background remain after subtraction (Fig. 2e), indicating that gold clusters were encapsidated successfully in these particles. Control images subjected to the same procedure resulted only in background (Fig. 2f–i).
In conclusion, our demonstration of controlled assembly is an important basis for the development of Ad3 dodecahedra towards therapeutic application. Furthermore, successful encapsidation of a marker molecule indicates the feasibility of our approach. The following considerations remain for development of the Ad3 dodecahedron as a drug-delivery vector. Packaging of small molecules must avoid their escape by diffusion through the perforations in the dodecahedral shell, either by polymerization to increase the diameter of the moiety or by modifying the interior surface of the cavity to retain the cargo, such as at the truncated amino terminus (Fuschiotti et al., 2006). Additionally, a sufficient quantity of cargo molecules will need to be delivered into each target cell, which will depend both on packing density within the cavity and on the efficiency of dodecahedral entry (Fender et al., 1997; Garcel et al., 2006). These steps remain to be evaluated fully, but will build on the results presented here.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTMAIN TEXTREFERENCES |
|---|
Braun, H., Boller, K., Löwer, J., Bertling, W. M. & Zimmer, A. (1999). Oligonucleotide and plasmid DNA packaging into polyoma VP1 virus-like particles expressed in Escherichia coli. Biotechnol Appl Biochem 29, 31–43.
Cheng, N., Conway, J. F., Watts, N. R., Hainfeld, J. F., Joshi, V., Powell, R. D., Stahl, S. J., Wingfield, P. E. & Steven, A. C. (1999). Tetrairidium, a four-atom cluster, is readily visible as a density label in three-dimensional cryo-EM maps of proteins at 10–25 Å resolution. J Struct Biol 127, 169–176.[CrossRef][Medline]
Fender, P., Ruigrok, R. W. H., Gout, E., Buffet, S. & Chroboczek, J. (1997). Adenovirus dodecahedron, a new vector for human gene transfer. Nat Biotechnol 15, 52–56.[CrossRef][Medline]
Fender, P., Schoehn, G., Foucaud-Gamen, J., Gout, E., Garcel, A., Drouet, E. & Chroboczek, J. (2003). Adenovirus dodecahedron allows large multimeric protein transduction in human cells. J Virol 77, 4960–4964.
Fuschiotti, P., Schoehn, G., Fender, P., Fabry, C. M. S., Hewat, E. A., Chroboczek, J., Ruigrok, R. W. H. & Conway, J. F. (2006). Structure of the dodecahedral penton particle from human adenovirus type 3. J Mol Biol 356, 510–520.[CrossRef][Medline]
Garcel, A., Gout, E., Timmins, J., Chroboczek, J. & Fender, P. (2006). Protein transduction into human cells by adenovirus dodecahedron using WW domains as universal adaptors. J Gene Med 8, 524–531.[CrossRef][Medline]
Greber, U. F., Willetts, M., Webster, P. & Helenius, A. (1993). Stepwise dismantling of adenovirus 2 during entry into cells. Cell 75, 477–486.[CrossRef][Medline]
Mizuguchi, H. & Hayakawa, T. (2004). Targeted adenovirus vectors. Hum Gene Ther 15, 1034–1044.[CrossRef][Medline]
Norrby, E. (1966). The relationship between the soluble antigens and the virion of adenovirus type 3. II. Identification and characterization of an incomplete hemagglutinin. Virology 30, 608–617.[CrossRef][Medline]
Pettersson, U. & Höglund, S. (1969). Structural proteins of adenoviruses. III. Purification and characterization of the adenovirus type 2 penton antigen. Virology 39, 90–106.[CrossRef][Medline]
Salunke, D. M., Caspar, D. L. D. & Garcea, R. L. (1986). Self-assembly of purified polyomavirus capsid protein VP1. Cell 46, 895–904.[CrossRef][Medline]
Schoehn, G., Fender, P., Chroboczek, J. & Hewat, E. A. (1996). Adenovirus 3 penton dodecahedron exhibits structural changes of the base on fibre binding. EMBO J 15, 6841–6846.[Medline]
Wickham, T. J., Mathias, P., Cheresh, D. A. & Nemerow, G. R. (1993). Integrins v
3 and v5 promote adenovirus internalization but not virus attachment. Cell 73, 309–319.[CrossRef][Medline]
Zubieta, C., Schoehn, G., Chroboczek, J. & Cusack, S. (2005). The structure of the human adenovirus 2 penton. Mol Cell 17, 121–135.[CrossRef][Medline]
Received 13 March 2006;
accepted 30 May 2006.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
