1 Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59717, USA
2 Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
3 Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717, USA
4 Department of Microbiology, Montana State University, Bozeman, MT 59717, USA
Correspondence
Mark Young
myoung{at}montana.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
The spherical plant virus Cowpea chlorotic mottle bromovirus (CCMV) has been a model system for virus assembly studies for over 30 years (for review, see Ahlquist, 1999) and is therefore an advantageous system for use as a constrained reaction vessel. The CCMV capsid is comprised of 180 copies of a 20 kDa coat protein subunit that can self-assemble in vitro into an empty T=3 icosahedral protein cage (Zhao et al., 1995
). The
280 Å external diameter capsid defines a protein cage with an
180 Å diameter internal cavity (Johnson & Speir, 1997
; Speir et al., 1995
). In addition, the CCMV protein cage is known to undergo a reversible, pH- and metal-dependent structural transition that results in the creation of 60
20 Å pores allowing access between the exterior and interior of the cage (Schneemann & Young, 2003
; Speir et al., 1995
).
The utility of viral protein cages is greatly enhanced by the ability to modify the cage genetically to impart new architectures. It is critical to separate the assembly of these cages from other viral processes, because many modifications will disrupt essential viral activities. For example, some modifications to the CCMV coat protein severely affect viral nucleic acid packaging, limiting particle accumulation and thus purification from their plant hosts. Viral structural proteins have been successfully expressed in a variety of heterologous expression systems (Schneemann & Young, 2003). A few of these expression systems have resulted in the assembly of virus-like particles (VLPs). The two most widely used are E. coli and baculovirus-based heterologous expression. However, both of these systems have limitations. E. coli-based expression often results in proteins that do not self-assemble or are insoluble, and baculovirus-based expression is cumbersome and expensive.
The P. pastoris system offers an attractive alternative for expression of VLPs. This eukaryotic yeast-based system is easy to manipulate and expresses high levels of protein. The P. pastoris system has been shown to be able to assemble wild-type VLPs in at least one case (Sugrue et al., 1997). This system has the advantage of providing a method for expressing a range of viral protein mutants using large-scale fermentation technology. We have investigated the ability of a wide variety of CCMV coat protein mutants to be expressed and assemble into VLPs using this system. We were interested in determining whether the P. pastoris system could be used to assemble protein cages with new functionality.
To explore the ability of the P. pastoris expression system to assemble a broad range of genetically modified protein cages, the CCMV coat protein gene was amplified from an infectious cDNA (Allison et al., 1988) and cloned into the pPICZA plasmid vector (Invitrogen EasySelect Pichia Expression Kit). This plasmid was used as a template for introducing mutations into the coat protein gene by oligonucleotide-directed PCR mutagenesis (Stratagene). Three general classes of modification were generated to test the range of expression and assembly. These modifications were designed to alter the interior, exterior or the interface between adjacent subunits in the assembled protein cage. To accomplish this, mutagenic primers were used to: (i) alter the interior surface charge of the viral nucleic acid binding region by replacing eight basic residues (K, R) in the N terminus with acidic glutamic acids (SubE mutant) (Douglas et al., 2002b
); (ii) delete interior residues 437 from the N terminus (N
34 mutant); (iii) insert a cDNA encoding an 11 amino acid polypeptide cell-targeting sequence (Graf et al., 1987
) into a surface-exposed loop (CPPep11 mutant); and (iv) modify interactions between viral subunits by altering the metal-binding sites (81/148 mutant). All mutations were confirmed by DNA sequencing.
P. pastoris was prepared for electroporation according to protocols for intact yeast cells (Saccharomyces cerevisiae) (Meilhoc et al., 1990). The resulting yeast colonies were screened by PCR and analysed for protein expression according to the manufacturer's recommendations (Invitrogen). Coat protein expression was monitored by immunoblots (Sambrook et al., 1989
) using specific CCMV polyclonal antibodies (Zhao et al., 1995
) and by transmission electron microscopy (TEM).
Clones expressing high levels of coat protein during small-scale production were chosen for fermentation scale-up in a 4 l BioFlow 3000 fermentation vessel (New Brunswick Scientific) according to established protocols (Stratton et al., 1998). The gas distribution system was modified by the addition of a stone diffuser to increase dissolved oxygen (DO). Eight ml trace elements (PMT 1) was added to basal salts media and the pH brought up to 5·0 with ammonium hydroxide. Approximately 300 ml mid-exponential phase yeast culture was added to the vessel. Conditions were set to 30 °C with agitation at 1000 r.p.m. and maintained at pH 5·0. DO was maintained at 35 % and cells were batch-fed glycerol until they began to starve, as indicated by the decrease in oxygen input. At this point, oxygen input was maintained at 100 % and glycerol was added for 4 h to increase biomass. Cells were then starved for 30 min prior to induction by a slow dripping of methanol. At least 5 h was required for the cells to become adapted to the use of methanol as the sole carbon source. The rate of methanol addition was then slowly increased during the next 48 h until a steady rate was achieved that maintained DO at
20 %. Fermentation was allowed to continue for an additional 48 h, during which approximately 4 l methanol was consumed. Cells were harvested by centrifugation and stored at -80 °C.
Cells were disrupted by blending 100 g cells, 235 g glass beads and 150 ml homogenization buffer (0·2 M sodium acetate, pH 4·8, 10 mM EDTA, 10 mM ascorbic acid) in a BeadBeater (BioSpec Products) for 5 min. Cell debris was removed by centrifugation (10 000 g) and the supernatant was centrifuged at 73 360 g (Beckman Type 30 rotor) for 2 h to pellet VLPs. The pellet was resuspended in virus buffer (0·1 M sodium acetate, pH 4·8, 1 mM EDTA) and VLPs were further purified by isopycnic banding in caesium chloride by loading 2 ml of a clarified VLP suspension on to 10 ml 39 % CsCl solution (w/v) in virus buffer and spinning in a SW41 rotor (Beckman) at 234 745 g for 1820 h. After centrifugation, the banded VLPs were removed, extensively dialysed against virus buffer and stored at -20 °C.
Nucleic acid content and size of VLPs purified from yeast was determined. Purified VLPs were subjected to UV/visible spectroscopy to estimate the concentration (A280 extinction coefficient of 24 075 M-1 cm-1) and to monitor the nucleic acid component (A260 : A280). Extracted nucleic acids were also analysed by Northern blotting using DIG-dUTP-labelled PCR probes (Roche Applied Science) specific for the P. pastoris 18S rDNA gene or the CCMV coat protein gene. The nature of the VLPs was determined by (i) dynamic light scattering (DLS) using a Brookhaven 90Plus particle size analyser; (ii) size exclusion chromatography (SEC) on a Superose 6 analytical column (Amersham Biosciences); and (iii) TEM analysis including a cryo-EM and image reconstruction of the SubE mutant. Purified VLPs stained with uranyl acetate were visualized by TEM (Leo 912 AB) and particle sizes measured from recorded images. The SubE mutant VLPs were vitrified and examined with a Phillips CM 100 TEM equipped with a Gatan 626 cryo-holder using methods previously described (Yeager et al., 1999). Images were recorded on film at a magnification of x45 000 under low-dose conditions (
510 e- Å-2). The software program Spider (Frank et al., 1981
, 1996
) was used to obtain the three-dimensional reconstruction at 25 Å resolution using
356 particles.
In contrast to expression of the CCMV coat protein in E. coli, which resulted in insoluble, non-assembled coat protein, the P. pastoris fermentation-based expression of wild-type and mutant forms of the coat protein gene of CCMV under the control of a methanol-inducible promoter resulted in the expression of soluble coat protein and the assembly of VLPs. While all constructs resulted in nearly equivalent levels of protein accumulation as determined by immunoblot assays, the amount of assembled VLPs was construct dependent. Typical isolated yields of VLPs ranged from 0·05 to 0·5 mg (g wet cell mass)-1. Wild-type virus and the SubE mutant typically resulted in the highest yields, while the CPPep11 mutant resulted in low yields. TEM analysis of CCMV VLPs demonstrated intact protein cages for the wild-type and mutant VLPs (Fig. 1). Particles indistinguishable from plant-derived wild-type virus particles were observed for all constructs except the N
34 VLPs, which showed a variety of sizes.
|
Purification to near homogeneity of the VLPs was accomplished by cell disruption and banding on CsCl gradients. SEC and DLS confirmed the assembly of homogeneous VLPs for all the mutations except the N34 mutant (discussed below). SEC showed a retention time equivalent to plant-derived wild-type CCMV (Fig. 2
). DLS analysis of these particles confirmed the size (29·9 nm±0·5) and indicated a homogeneous population of particles (Fig. 2
, inset).
|
We utilized cryo-TEM and image reconstruction to compare SubE mutant VLPs with plant-derived wild-type particles. Image reconstructions at 25 Å resolution of the SubE particles produced in the P. pastoris system demonstrated that they assembled into T=3 particles that were indistinguishable from particles produced in plants (Fig. 3). This showed that even fairly drastic coat protein mutations can still result in assembled cages within P. pastoris.
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Allison, R. F., Janda, M. & Ahlquist, P. (1988). Infectious in vitro transcripts from cowpea chlorotic mottle virus cDNA clones and exchange of individual RNA components with brome mosaic virus. J Virol 62, 35813588.[Medline]
Douglas, T. & Young, M. (1998). Hostguest encapsulation of materials by assembled virus protein cages. Nature 393, 152155.[CrossRef]
Douglas, T. & Young, M. (1999). Virus particles as templates for materials synthesis. Adv Mater 11, 679681.[CrossRef]
Douglas, T., Allen, M. & Young, M. (2002a). Self-assembling protein cage systems and applications in nanotechnology. In Biopolymers, pp. 405426. Edited by A. Steinbüchel. Weinheim: Wiley-VCH.
Douglas, T., Strable, E., Willits, D., Aitouchen, A., Libera, M. & Young, M. (2002b). Protein engineering of a viral cage for constrained nanomaterials synthesis. Adv Mater 14, 415418.[CrossRef]
Flenniken, M., Allen, M., Young, M. & Douglas, T. (2003). Viruses as host assemblies. In Encyclopedia of Supramolecular Chemistry. Edited by J. Atwood & J. Steed. New York: Marcel Dekker.
Frank, J., Shimkin, B. & Dowse, H. (1981). SPIDER a modular software system for electron image processing. Ultramicroscopy 6, 343358.
Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M. & Leith, A. (1996). SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol 116, 190199.[CrossRef][Medline]
Graf, J., Iwamoto, Y., Martin, G. R., Kleinman, H. K., Robey, F. A. & Yamada, Y. (1987). Identification of an amino acid sequence in laminin mediating cell attachment, chemotaxis, and receptor binding. Cell 48, 989996.[Medline]
Johnson, J. E. & Speir, J. A. (1997). Quasi-equivalent viruses: a paradigm for protein assemblies. J Mol Biol 269, 665675.[CrossRef][Medline]
Krol, M. A., Olson, N. H., Tate, J., Johnson, J. E., Baker, T. S. & Ahlquist, P. (1999). RNA-controlled polymorphism in the in vivo assembly of 180-subunit and 120-subunit virions from a single capsid protein. Proc Natl Acad Sci U S A 96, 1365013655.
Meilhoc, E., Masson, J. M. & Teissie, J. (1990). High efficiency transformation of intact yeast cells by electric field pulses. Biotechnology 8, 223227.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). In Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schneemann, A. & Young, M. (2003). Viral assembly using heterologous expression systems and cell extracts. Adv Prot Chem 64, 136.
Speir, J. A., Munshi, S., Wang, G., Baker, T. S. & Johnson, J. E. (1995). Structures of the native and swollen forms of cowpea chlorotic mottle virus determined by X-ray crystallography and cryo-electron microscopy. Structure 3, 6378.[Medline]
Stratton, J., Chiruvolu, V. & Meagher, M. (1998). High cell-density fermentation. Methods Mol Biol 103, 107120.[Medline]
Sugrue, R. J., Fu, J., Howe, J. & Chan, Y. C. (1997). Expression of the dengue virus structural proteins in Pichia pastoris leads to the generation of virus-like particles. J Gen Virol 78, 18611866.[Abstract]
Yeager, M., Unger, V. M. & Mitra, A. K. (1999). Three-dimensional structure of membrane proteins determined by two-dimensional crystallization electron cryomicroscopy, and image analysis. Methods Enzymol 294, 135180.[Medline]
Zhao, X., Fox, J. M., Olson, N. H., Baker, T. S. & Young, M. J. (1995). In vitro assembly of cowpea chlorotic mottle virus from coat protein expressed in Escherichia coli and in vitro-transcribed viral cDNA. Virology 207, 486494.[CrossRef][Medline]
Zlotnick, A., Aldrich, R., Johnson, J. M., Ceres, P. & Young, M. J. (2000). Mechanism of capsid assembly for an icosahedral plant virus. Virology 277, 450456.[CrossRef][Medline]
Received 2 October 2003;
accepted 9 December 2003.