Protein Engineering, Design & Selection vol. 24 no. 1 2 pp. 5363, 2011 Published online October 5, 2010 doi:10.

1093/protein/gzq069

REVIEW

Virus engineering: functionalization and stabilization

Mauricio G.Mateu 1
Centro de Biologa Molecular Severo Ochoa (CSIC-UAM), Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain
1

To whom correspondence should be addressed. E-mail: mgarcia@cbm.uam.es Received September 8, 2010; revised September 8, 2010; accepted September 9, 2010 Edited by Javier Sancho

Chemically and/or genetically engineered viruses, viral capsids and viral-like particles carry the promise of important and diverse applications in biomedicine, biotechnology and nanotechnology. Potential uses include new vaccines, vectors for gene therapy and targeted drug delivery, contrast agents for molecular imaging and building blocks for the construction of nanostructured materials and electronic nanodevices. For many of the contemplated applications, the improvement of the physical stability of viral particles may be critical to adequately meet the demanding physicochemical conditions they may encounter during production, storage and/or medical or industrial use. The rst part of this review attempts to provide an updated general overview of the fast-moving, interdisciplinary virus engineering eld; the second part focuses specically on the modication of the physical stability of viral particles by protein engineering, an emerging subject that has not been reviewed before. Keywords: nanotechnology/nanoparticle/protein engineering/ thermal and mechanical stability/virus capsid

Introduction Viruses can be regarded as nucleoprotein-based, supramolecular ensembles that evolved into biological nanomachines capable of self-replication within cells and propagation between cells and organisms. A typical infectious virus particle (virion) must be able to recognize specic cells, enter and navigate in them, and undergo the conformational rearrangements required for the productive release of its genome. After replication of its macromolecular components in the host cell, the typical virion must be able to selfassemble, mature into a stable, infectious entity, navigate again and exit the cell and withstand severe physical and chemical aggressions in the extracellular environment (Chiu et al., 1997). In response to so many selection pressures, the blind process of virus evolution has managed to pack a remarkable ensemble of features and many complex functions into relatively simple nucleoprotein structures. A detailed, albeit still incomplete, knowledge of the structure, dynamics, properties and functions of many different virions and their protein capsids has been obtained by

structural and molecular virologists during the last decades. This knowledge and the development of chemical and genetic technologies to modify proteins and nucleic acids have opened up many possibilities for the exploitation of virus particles in medicine and the industry. The recent advent of nanotechnology has led to a growing general awareness of those possibilities, and suggested exciting new ones (Douglas and Young, 2006; Singh et al., 2006a,b; Saini et al., 2006; Fischlechner and Donath, 2007; Steinmetz and Evans, 2007; Young et al., 2008; Flenniken et al., 2009; Manchester and Steinmetz, 2009; Steinmetz et al., 2009). Viruses are also providing inspiration for the design and fabrication of non-viral nanoparticles, and becoming a focus of truly multidisciplinary research. Natural viruses and their capsids do not have all of the several properties and functionalities required, or at least are not optimized, for any of their contemplated applications. Thus, viral particles have to be modied by direct chemical means (Gillitzer et al., 2002; Strable and Finn, 2009) and/or by protein engineering using genetic techniques. A noncomprehensive list of present or developing applications of virus engineering (Fig. 1) may include: (i) fundamental structure function studies of viruses; (ii) phage display techniques for the selection of peptides and proteins; (iii) new vaccines, including heterologous vaccines based on viral particles carrying foreign epitopes (virus chimeras); (iv) delivery of therapeutic genes into specic cells (gene therapy); (v) targeted delivery of drugs using viral particles as nanocarriers or nanocontainers, allowing for specic chemotherapeutic treatments; (vi) target-specic contrast agents for molecular imaging; (vii) building blocks for the construction of nanomaterials and nanostructures; and (viii) improvement of the physical stability of viral particles. This later aspect may be critical to adequately meet the severe physicochemical demands viral particles may encounter during production, chemical functionalization, synthesis of nanomaterials, storage and/or medical or industrial use. In the rst part of this review, I will attempt to provide an overview of current virus engineering approaches for the applications outlined above; in the second part, I will focus specically on the modication of the physical stability of viral particles by protein engineering, an emerging subject that has not been reviewed before.

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Engineering virus particles for nano/biotechnological applications Since the pioneering study by Winter, Fersht, Wilkinson, Zoller and Smith nearly 30 years ago (Winter et al., 1982), protein engineering using molecular genetics techniques has been extensively developed and used in many laboratories worldwide, including dedicated institutes such as the MRC Centre for Protein Engineering in Cambridge, UK (Leatherbarrow and Fersht, 1986; Brannigan and Wilkinson, 53

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Fig. 1. Some applications of virus engineering in biotechnology and nanotechnology. Fundamental structurefunction relationship studies of viruses and viral capsids using protein engineering are being used for the development of many applications. Physical stabilization of viral particles by protein engineering may be critical for many of those applications.

2002; Leisola and Turunen, 2007). Protein engineering has already led to an unprecedented and dramatic advance in the understanding of protein structure function relationships. In addition, some of the engineered proteins have found important commercial uses, especially in scientic research, the biopharmaceutical industry or as biocatalysts for industrial processes; many more are at different stages of development. For conceptual and technical reasons, comparatively few studies have been aimed at the engineering of large supramolecular (nucleo)protein complexes such as virus capsids or complete virions, especially for biotechnological applications. This situation is rapidly changing, partly because of the recently perceived need to learn how to adequately modify virus particles, if they are to really fulll so many expectations regarding their biomedical and technological potential. Many of the current and emerging application-oriented (bio)chemical modications of viruses have been previously reviewed separately. This section provides a schematic (and necessarily non-comprehensive), but updated overview of the fast-moving virus engineering eld; references to some representative examples and many recent, more specialized revisions have been included.

technique is based on the physical coupling of genotype and phenotype of a peptide or protein of interest in a modied bacteriophage virion. The DNA sequence coding for the ( poly)peptide is fused with a viral capsid protein gene using recombinant DNA techniques. As a result, the ( poly)peptide can be expressed and presented on the virion surface as a covalently linked, terminal extension of the capsid protein. By coupling a large combinatorial library of peptides or proteins, those ( poly)peptide variants with a desired property, such as a higher binding afnity for a ligand, chemical or conformational stability or even modied enzymatic activity, may be selected. The individual phages carrying the selected ( poly)peptide variants can be plaque-puried and amplied, and the mutations responsible for the variant phenotype identied by partial sequencing of the viral genome. The insertion sites as well as procedures have been standardized for different phages by considering the need to minimize deleterious effects on virion assembly and infectivity. Limitations of phage display include the size of the peptide or protein (which depends on the display system chosen), the size of the library and the availability of an adequate selection method. In spite of those limitations, in the past two decades, a very large variety of target peptides and proteins have been successfully subjected to selection by phage display (Forrer et al., 1999; Wittrup, 1999; Amstutz et al., 2001; Smothers et al., 2002; Kehoe and Kay, 2005; Paschke, 2006). In addition, the later application of the same principles has led to the development of other molecular display systems (Levin and Weiss, 2006). Recently, the use of molecular display techniques has expanded for the engineering of non-viral and viral vectors useful in targeted gene therapy or drug delivery (Schaffer et al., 2008). Display techniques are also being used in materials science to select for peptides able to bind to non-biological components (Kehoe and Kay, 2005; Kriplani and Kay, 2005; Fischlechner and Donath, 2007).

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Engineering chimeric virus particles for presentation of heterologous peptides and proteins
Another display-related, early approach to virus engineering is the construction of virus particle chimeras, originally developed for presentation to the immune system of peptide epitopes from other pathogens (Palucha et al., 2005; Inoue et al., 2008; Roy and Noad, 2008; Steinmetz et al., 2009). Chimeras have been derived from animal viruses, plant viruses or bacteriophages and are usually constructed by inserting through genetic engineering a short heterologous peptide on a surface-exposed loop of a capsid protein. Alternatively, heterologous peptides or proteins can be fused to the exposed N- or C-terminus of a capsid protein, as in the original phage display concept. In contrast to phage display, viral chimeras are most frequently based on nucleic acid-free natural capsids or, especially, viral-like particles (VLPs); virions are used for certain applications, such as gene therapy (see below). Virus chimeras have found many different uses. A potentially very important application is the development of novel or improved vaccines against viral or bacterial diseases, especially in those cases where the use of the pathogens themselves for vaccination is unfeasible or unadvisable for technical or safety reasons. Most virus chimeras constructed to date have been the result of a rational or semi-rational design based on some

Engineering viruses for studying structure function relationships

A large number of virions and viral capsids have been subjected to fundamental structure function studies using sitedirected mutagenesis or, to a much lesser extent, directed evolution methods. The many results obtained in those studies, which are beyond the scope of this review, have led to a greatly improved understanding of several stages of the virus life cycle; some have had already important direct consequences for the ght against infectious diseases.

Engineering bacteriophages for peptide and protein display, and selection of variants with desired properties
One of the rst approaches to virus engineering for direct applications was the development of phage display (Smith, 1985; Huse et al., 1989; Scott and Smith, 1990). This 54

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knowledge of the structure of the viral particle and the determinants of its relevant properties and functions. One of the difculties found for engineering virus chimeras is that even insertion of short peptides at the tips of the most exposed capsid surface loops may impair assembly or at least destabilize the virus particle (e.g. Carreira et al., 2004). Another difculty is that a heterologous epitope inserted in any protein scaffold may be constrained to adopt non-native conformation(s), which may severely affect its activity (e.g. Benito et al., 1995). Despite these potential shortcomings, several experimental viral chimeras that carry heterologous immunogenic epitopes have been successfully engineered and tested in model systems. In recent years, the engineering of virus chimeras has expanded to include also other peptides, and even large proteins without disrupting capsid assembly (e.g. Muratori et al., 2010), in order to confer new functionalities for fundamental studies or novel applications. As an example, a VLP derived from ock house virus was engineered to carry a receptor domain exposed on its surface and proved to be active as an anthrax antitoxin as well as a vaccine (Manayani et al., 2007). Different capsids labeled with uorescent marker proteins have been used to investigate virus host interactions (e.g. Asokan et al., 2008). Also, viral particles that incorporate heterologous cell receptor recognition sites to alter their cellular tropism are being used in experimental gene therapy approaches (see next).

In addition to modied cell tropism, if AAV and other virus candidates are to be used as vectors for human gene therapy (or drug delivery, see below), they should be provided with the ability to evade immune recognition in the patients. This is being attempted either by chemical means, such as coating with polyetylenglycol (e.g. Raja et al., 2003) or other compounds, or by protein engineering using directed evolution approaches (Mahesri et al., 2006; Perabo et al., 2006; Schaffer et al., 2008; Zaiss and Muruve, 2008).

Engineering virus particles for targeted drug delivery

The possibility to specically deliver not only genes but also drugs and other molecules into target cells has recently triggered intense research and development on a host of inorganic, organic, biological or hybrid nanoparticles for diagnosis and/or therapeutic treatment of many pathologies. The feasibility of engineering viral particles to convert them into nanocarriers or nanocontainers for targeted drug delivery is already supported by a remarkable number of experimental studies (Arora and Kirshenbaum, 2004; Garcea and Gissmann, 2004; Douglas and Young, 2006; Saini et al., 2006; Uchida et al., 2007; Steinmetz and Evans, 2007; Young et al., 2008; Steinmetz et al., 2009; Flenniken et al., 2009). Some important aspects of the approaches used to address particular aspects of targeted drug delivery are mentioned next. Targeting of thiols, amines, carboxylates or other functional groups in viral capsids, either naturally occurring or introduced in them by protein engineering, has allowed the chemical coupling of small molecules to the capsid (e.g. Wang et al., 2003; Soto et al., 2006; Lewis et al., 2006). In nearly all cases, the genetic modication and/or chemical functionalization have been carried out on amino acid residues located at the capsid outer surface, leading to nanocarriers but not nanocontainers. One potential general problem of this approach is that carrying the drug non-concealed on the outer surface may not prevent its degradation or clearance in the organism, or any toxic effect of the drug during circulation in the body. In a few cases, the functionalization of the capsid inner surface has been attempted (e.g. Wang et al., 2002; Hooker et al., 2004). But in such a case, the drug to be carried must be adequately internalized, stored within and, once inside the target cell, adequately released. There are very few studies on the feasibility of modifying viral capsids by protein engineering to allow these processes to occur. However, in an early study, several positively charged residues at the N-terminus of the capsid protein of cowpea chlorotic mottle virus (CCMV) were replaced by negatively charged residues using protein engineering. The modied CCMV capsids were assembled in vitro in the presence of positively charged ferrous ions, and shown to be able to load and oxidize thousands of Fe ions per capsid, leading to mineralized particles (Douglas et al., 2002). This work provided a proof of principle that viral capsids can be rationally engineered as nanocontainers to store unnatural cargos with dened properties. However, many chemotherapeutic agents are small organic molecules, and their mere internalization in viral nanocontainers without strong binding to the capsid inner wall may not be adequate for targeted delivery. As an example, an 55

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Engineering virus vectors for gene therapy

Adenovirus, retrovirus, adeno-associated virus (AAV) and other animal viruses carrying modied genomes have been used for many years in experimental protocols to specically deliver potentially therapeutic genes into target cells involved in pathological processes (Verma and Weitzman, 2005). More recently, the need to improve some properties or modify some functionalities of the natural virions used as vectors for gene therapy has been clearly realized (Wu et al., 2006; Schaffer et al., 2008). The modication of the cellular tropism of the virus vector for targeting different medically relevant cells provides an important example of virus engineering for gene therapy. One of the simplest ways attempted to alter viral tropism is the engineering of virus chimeras that carry heterologous peptides containing recognition sites for receptors present on the target cell membrane (e.g. Asokan et al., 2010). In addition, random peptide libraries have been engineered and displayed on AAV and other virus vectors to directly select for virus mutants with the desired tropism conferred by the heterologous variant peptide they carry on their surface (e.g. Muller et al., 2003). Unfortunately, in many instances, recognition sites and other molecular determinants of cellular tropism in virions cannot be mimicked by short peptides. Rational protein engineering of these and other functionalities in viral particles is still severely impaired by the limited knowledge available on structure function relationships in viruses and proteins in general. As a consequence combinatorial, directed evolution approaches, such as error-prone PCR and DNA shufing, are being increasingly applied to the engineering of viral vectors with altered tropism (Soong et al., 2000; Wu et al., 2000, 2006; Mahesri et al., 2006; Schaffer et al., 2008).

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unmodied VLP from Hibiscus chlorotic ringspot virus was able to encapsulate small polyacids during its assembly but, due to poor retention, these molecules readily diffused out through capsid pores (Ren et al., 2006). In contrast, in a subsequent study, the same authors encapsulated a high molecular weight (200 kDa) polyacid in the same VLPs and no leaching was observed. The polyacid served to electrostatically retain inside the viral particle a small-size anticancer drug, doxorubicin. In addition, folic acid was conjugated to those VLPs for targeting to tumor cells. The complex viral nanoparticles thus engineered were able to improve the uptake and cytotoxicity of doxorubicin in ovarian cancer cells (Ren et al., 2007). There is a growing number of studies in which a marker or bioactive molecule carried by an engineered viral particle is successfully delivered into target cells (e.g. Yu et al., 2005; Singh et al., 2006a,b; Inoue et al., 2008). As an example, lamentous phages were engineered to display a heterologous antibody-binding ZZ domain, and antibodies specic for receptors on cancer cell membranes were bound to the phages through the ZZ domains. The capsid surface was also functionalized with a large payload of a cytotoxic drug, either hygromycin or doxorubicin. Hygromycin was chemically conjugated to the capsid via an amide bond; doxorubicin was conjugated through engineered cathepsin A sites on the capsid. The phages thus genetically and chemically modied were targeted to cancer cells via the antibodies bound to the ZZ domains, which resulted in growth inhibition of the cells with a 1000-fold potentiation factor over the free drug (Bar et al., 2008). These and other model studies carry the promise for a future use in biomedicine of engineered viral particles for targeted drug delivery and specic chemotherapy. Chemical and/or genetic modication of virus particles may also serve to convert them into in situ activable therapeutic agents. For example, modied adenoviruses with cancer cell tropism and carrying gold nanoparticles could be used for specic, hyperthermia-mediated tumor cell death (Everts et al., 2006; Saini et al., 2008).

the way for the use of paramagnetic viral nanoparticles with a high relaxivity as target-specic high-contrast agents for MRI.

Engineering virus particles for new technological materials and nanostructures

There is already a plethora of novel nanomaterials and nanostructures that are being developed for multiple biomedical, mechanical, electrical, electronic, optical or other nanotechnological applications. Some novel hybrid nanostructures include engineered viral particles (Douglas and Young, 1999; Knez and Gosele, 2006; Fischlechner and Donath, 2007; Steinmetz and Evans, 2007); a few examples will be mentioned to provide a avor of this vast interdisciplinary eld. One important goal in materials science is the development of methods for the spatially constrained formation of inorganic nanoparticles or organic polymers. Viral capsids provide ready-made templates or scaffolds with a central cavity and are proving particularly suitable to achieve these goals. In an early study, mineralization of negatively charged paratungstate and decavanadate polyions was achieved inside natural capsids of CCMV (Douglas and Young, 1998). Moreover, the modication of the net charge in the capsid interior by protein engineering allowed the mineralization of positively charged iron oxide nanoparticles (Douglas et al., 2002). Engineered virus particles are being used also as nanometric-sized building blocks for the hierarchical assembly of hybrid materials with distinct one-, two- or threedimensional structures (Fischlechner and Donath, 2007). In some instances, suitable amino acid residues on the viral particle surface, either naturally occurring or introduced by protein engineering, were functionalized with an appropriate chemical group. In other instances, the viral particle was engineered to display a peptide able to bind a non-biologic ligand, including inorganic materials. The peptides were chosen because of their binding specicity (e.g. for a metal), or selected by molecular display (Kriplani and Kay, 2005). These modications are intended to nucleate and control the organization and growth of the structure. The nanostructured materials thus formed may be further functionalized and combined with other components for the eventual construction of nanodevices. Some examples follow. A medically important application of the strategy just outlined is the development of tissue-regenerating materials. In one example, the long rod-shaped phage M13 was engineered to display cell signaling peptides, which allowed their self-assembly in an organized, three-dimensional array of viral nanobers; these scaffolds were able to support cell proliferation and differentiation, as well as oriented cell growth in three dimensions (Merzlyak et al., 2009). Another promising application is the development of nanostructures that could be used as components of future electronic devices (Knez and Gosele, 2006; Ross, 2006). For example, M13 phages were engineered to display goldbinding motifs on the capsid and streptavidin-binding moieties at one end, and used to assemble Au and CdSe nanocrystals into ordered monodimensional arrays and more complex nanoarchitectures (Huang et al., 2005). Such monodimensional arrays could be used to nucleate electrically conductive nanowires. Engineered M13 phages were also

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Engineering virus particles as target-specic contrast agents for medical imaging

In vivo imaging is now widely used in medicine for diagnosis and/or evaluation of therapeutic treatments. Any kind of molecular imaging requires accumulation of a suitable contrast agent in the target site. Thus, many different nanoparticles, including natural or engineered viral particles able to target specic cells and tissues, are being further modied to act as selective contrast agents in order to increase the specicity and sensitivity of current in vivo imaging techniques (Manchester and Singh, 2006; Debagge and Jaschke, 2008; Cormode et al., 2010). One relevant example will be mentioned. Contrast agents in magnetic resonance imaging (MRI) include metals such as Gd3 ions. In several recent studies, large numbers of Gd3 ions were bound to the capsid of viruses such as CCMV and phage MS2, either by chemical means (direct chelation to metal-binding sites in the CCMV capsid, or conjugation of chelating groups) or by protein engineering using a metal binding peptide that was fused to the N-terminus of the CCMV capsid protein (Allen et al., 2005; Anderson et al., 2006; Liepold et al., 2007). These studies have opened up 56

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used for the self-assembly of gold-cobalt oxide nanowires that could be used as electrodes to fabricate thin, exible Li-ion batteries (Nam et al., 2006, 2008). In another example, M13 phages engineered to have specic, peptidebased recognition sites for ZnS, CdS, CoPt or FePt were used as templates for ordering quantum dots (nanometricsized semiconductor crystals that can act as inorganic uorophores) in self-assembled, semi-conducting or magnetic nanowires (Lee et al., 2002; Mao et al., 2003, 2004). Engineered cowpea mosaic virus (CPMV) has been also used as a scaffold to build electrically conductive molecular networks. Cysteine residues were introduced by protein engineering at dened positions of the capsid surface, which allowed the anchoring of gold particles that were subsequently interconnected by molecular wires, to create a three-dimensional conducting network on each viral particle. The nanoparticles were then interconnected using thiolterminated organic molecules. Different structures were constructed, including voltage-controlled switchable networks that could be used in memory nanocircuits (Blum et al., 2005, 2007). In another example, tobacco mosaic viruses (TMV) with platinum nanoparticles attached behaved as memory elements that could be also switched on and off electronically (Tseng et al., 2006; Knez and Gosele, 2006). Still another, materials-related application of engineered viral particles is the development of nanobiosensors (Jain, 2005; Donath, 2009; Mao et al., 2009). Nanobiosensors are nanometric-sized sensors based on immobilized biomolecules that specically bind a target ligand (analyte). As one example, virus particles were engineered by site-directed mutagenesis to bind both nickel nanohairs and antibodies against troponin I, a protein marker found in patients with a higher risk of heart attacks. In this way, a regular threedimensional array with a very high density of anti-troponin I antibodies could be formed, and was used as an ultrasensitive biosensor to detect extremely low levels of troponin I (Park et al., 2009). Engineering virus particles for improved physical stability Many virions and their capsids show a remarkable degree of conformational instability and/or metastability (e.g. Filman et al., 1989; Speir et al., 1995; Casjens, 1997; Chow et al., 1997; Rossmann et al., 1997; Belnap et al., 2000; Smyth and Martin, 2002; Hogle, 2002; Johnson, 2003, 2010; Liepold et al., 2005; Cotmore and Tattersall, 2007; Johnson, 2010). These characteristics may facilitate the delivery of the viral genome inside the host cell through a structural rearrangement of the capsid, with or without capsid dissociation into subunits. The activation energy barrier for the required conformational change can be lowered in vivo by specic physiological factors, including virus binding to cell receptors, capsid binding or unbinding of metal ions or organic molecules, or acidication in the endosomes. On the other hand, limited stability and/or signicant metastability may also facilitate unproductive conformational rearrangements and/or dissociation of the capsid: in the extracellular environment, non-specic physicochemical agents can lower the activation energy barrier, leading in this case to inactivation of the virus particle. Thus, each virus may have evolved an optimum compromise between stability

and instability, as well as a degree of metastability, that would depend on its ecological niche and the molecular mechanisms underlying its life cycle. From a nanobiotechnological point of view, conformational metastability and/or instability of a viral particle may be either desirable or undesirable, depending on the application. Engineered virions for gene therapy and empty capsids or VLPs for targeted drug delivery should generally preserve some degree of instability/metastability, as they should be still capable of dynamic rearrangements or disassembly to deliver their cargo into target cells. In contrast, for many other applications (e.g. vaccines, contrast agents, diagnostic agents, building blocks for some nanomaterials etc.), viral particles may not need to be conformationally dynamic. In fact, they may encounter relatively harsh physical and/or chemical conditions during purication, fabrication processes, storage and/ or use. High temperatures, acidic or dehydrating conditions, high hydrostatic pressure, centrifugal forces or shear forces causing mechanical stress, organic solvents or chemical reagents are known to lead to dissociation or conformational change of different viral particles. Structural rearrangements, with or without particle disintegration, may lead to loss of function for different reasons, including the untimely release of the cargo, distortion of active moieties on the capsid surface, or changes in the relative position of functional components. In such cases, the highest possible physical stability may be desirable, or even critical. Some possible solutions to the virus stability problem in biotechnology do not require virus engineering. These include: (i) the use of hyperstable viral particles isolated from extreme natural environments (Tsuboi et al., 2005; Flenniken et al., 2009); some viral particles derived from less extreme environments are also remarkably resistant to high temperatures, non-neutral pH, desiccation and/or other harsh conditions (e.g. Hernando et al., 2000; Ashcroft et al., 2005; Mani et al., 2006; Carrasco et al., 2009); (ii) natural virus mutants of increased stability obtained by selection, especially from highly heterogeneous viral populations (Domingo, 2006); (iii) stabilization of viral particles by physical conditions such as low temperatures (or high pressure, Ferreira et al., 2009), or chemical additives (Croyle et al., 2001; Kissmann et al., 2008a,b). All those alternatives may prove useful in specic cases, but they may also present important limitations. For example, viruses that are both hyperstable and adequate for the intended functional modications may be difcult to nd. Stable virus mutants may be very rare or not present in natural populations due to low tness or other biological factors (e.g. compare results in Mateo et al., 2007 with those in Martn-Acebes et al., 2010). The molecular mechanism responsible for resistance of natural mutants to inactivation of infectivity (the phenotype usually selected) has been elucidated in a few cases (e.g. Speir et al., 2006), but not in many others; however, for biotechnological applications, it may be extremely important to differentiate if a virus particle is stabilized against its dissociation into subunits, or against a conformational rearrangement without the loss of integrity. Finally, chemical additives, either alone or combined with very low temperatures, have sometimes improved the longterm storage or transportation of viruses and virus-based vaccines; however, they may be frequently incompatible with the intended use of the viral particles. 57

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Chemical modication and combinatorial approaches and rational protein engineering using genetic techniques have begun to be applied for improving the intrinsic physical stability of viral particles. Some of the very few studies yet on this subject are reviewed in this section.

Stabilization of viral particles by chemical procedures

Irreversible (bio)chemical modication is used by many natural virions during their maturation (Johnson 2003, 2010). For example, chemical cross-linking is involved in the natural stabilization of phage HK97 (Ross et al., 2005). Likewise, in vitro cross-linking with glutaraldehide may provide a rather nonspecic approach to capsid stabilization (e.g. Uchida et al., 2007). Phage MS2 solubilized in organic solvents showed also an increased resistance against thermal inactivation (Johnson et al., 2007). A dramatic stabilization of a hollow, capsid-like oligomeric protein to 1208C was achieved by means of a cross-linked branched polymer synthesized in the protein central cavity (Abedin et al., 2009). Unfortunately, stabilization of a viral particle using a chemical procedure may be a rather specic, complex and expensive process. In many cases, it may be much more desirable to obtain genetically altered viral particles of intrinsically higher stability.

Stabilization of viruses by combinatorial/directed evolution approaches

The use of genetic methods such as error-prone PCR and DNA shufing on viral particles allows the exploration of vast areas of sequence space not represented in natural viral populations. Up to date, only a few studies have used combinatorial/directed evolution techniques for the selection of variant viruses of increased physical stability. Murine leukemia virus (MLV), an enveloped virus, is readily inactivated under ultracentrifugation conditions, or at 378C, which severely limits its usefulness as a vector for gene therapy. This instability has been attributed to a labile interaction between the transmembrane and surface (SU) subunits of the envelope glycoprotein; at moderate temperatures or in response to physical forces, SU would be shed from the virus, leading to the loss of infectivity. In an early study on directed evolution for viral stabilization, six MLV strains were bred by DNA shufing of the genomic region corresponding to the envelope protein. Several complex chimeric viruses were obtained and found to be much more stable than the parental viruses against inactivation by ultracentrifugation (Powell et al., 2000). In another example, the use of a mutator strain was used to generate further variation along the entire MLV genome; mutants were selected for infectivity after prolonged incubation at 378C. A single mutation in the viral protease (PR), a non-structural protein, was identied as responsible for the enhanced stability. Other substitutions at the same position led to the same effect that was increased by site-directed mutation of other PR residues (Vu et al., 2008). The authors suggested that the mutations could have an effect on the stability of the PR dimer that would indirectly affect virus assembly and/or maturation.

rearrangements of the virion (with or without capsid disassembly) that are triggered by local changes of pH. Thus, the conformation and/or integrity of many viral particles are naturally quite sensitive to acidication or alkalinization. Many nanobiotechnological applications could benet from the development of rational procedures to engineer viral particles able to withstand pH extremes. A few examples of rational virus engineering for increased stability in acidic or alkaline conditions are available. A study on TMV provided an early example. Infection by TMV involves a physical destabilization of the virion at the relatively high pH inside the host plant cells that results in exposure of a part of the viral genome. This pH-dependent instability of the TMV virion is due to increased electrostatic repulsions between carboxylate groups of a number of Asp and Glu residues in the capsid. By rationally mutating some of these residues, TMV variants with increased resistance to dissociation at alkaline pH were obtained (Culver et al., 1995). A second example is provided by a study on foot-andmouth disease virus (FMDV). In the process of infection by FMDV, acidication in the endosomes triggers the dissociation of the capsid into pentameric subunits, leading to the uncoating of the viral genome. The pKa of FMDV dissociation corresponds approximately to that of histidine. This observation, inspection of the three-dimensional structure of FMDV and electrostatic potential calculations suggested that the acid sensitivity of FMDV could be due to protonation of histidines located close to the interpentamer interfaces. Specically, it was proposed that intersubunit repulsions between positively charged H142 and a helix dipole would facilitate dissociation (Acharya et al., 1989; Curry et al., 1995; Twomey et al., 1995). Based on this hypothesis, an inversion-of-charge mutation H142D was engineered on the FMDV capsid. As expected, the modied capsid was more resistant than the non-mutated capsid against acid-induced dissociation (Ellard et al., 1999). It must be emphasized that, for some specic applications such as gene or drug delivery, acid sensitivity of a viral particle may be in fact a desirable feature. This viral property has even inspired the engineering of analogous pH-dependent molecular switches in non-viral assemblies. In a recent study, a pyruvate dehydrogenase complex that was originally stable in neutral and acidic solutions was made sensitive to dissociation by acidication at pH 5 by genetically manipulating the intersubunit interfaces (Dalmau et al., 2009).

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Modifying the thermal stability of virus particles by rational engineering of intersubunit disulde bonds
Naturally occurring disulde bonds are involved in stabilizing the capsids of several viruses in the non-reducing extracellular environment, where high conformational stability may be a denite selective advantage (e.g. Zhou and Standring, 1992; Li et al., 1998; Ashcroft et al., 2005; Caldeira and Peabody, 2007). CCMV does not usually contain disulde bonds; however, a natural mutant acquired a Cys mutation that allowed the formation of intersubunit disulde bonds, leading to increased resistance to dissociation (Fox et al., 1997). These observations support the engineering of disulde bonds between capsid subunits as an attractive approach for the conformational stabilization of viral particles, even if these are to be used in gene therapy or

Modifying the pH-dependent stability of virus particles by rational protein engineering

For several animal and plant viruses, uncoating of the genetic material within the host cell involves conformational 58

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targeted drug delivery: hyperstable disulde-bonded capsids would still be able to normally deliver their cargo, because their increased stability would be lost in the reducing intracellular environment. The rational, homology-based engineering of disulde bonds in the MS2 capsid (Ashcroft et al., 2005) has provided a proof of principle for this approach. The capsids of phages Qb and MS2 have extensive sequence identity and are structurally nearly identical, but have dramatically different thermal stabilities that were correlated with the presence in Qb of a putative intersubunit disulde bond between Cys74 and Cys80 (Golmohammadi et al., 1996). In a recent study, the remarkable stability of the Qb capsid was shown to be dependent on the formation of intersubunit disuldes. In the same study, cysteines were introduced in the MS2 capsid, in positions roughly equivalent to those of Cys74 and Cys80 in Qb. Under non-reducing conditions, some engineered MS2 capsids showed as expected a substantially increased thermostability relative to the non-mutated capsid (Ashcroft et al., 2005). Homology-based introduction of disulde bonds has been used also to successfully stabilize non-viral oligomeric proteins (Heikoop et al., 1997). Unfortunately, this homology approach may have a very limited applicability, as most protein oligomers and viruses have no adequate disuldebonded homologs. Rationally engineered non-homologous intermolecular disuldes in small protein oligomers have proven either stabilizing or destabilizing. The outcome probably depends in part on the delicate energetic balance between actual formation of the disulde and the local conformational strain that this reaction may impose on the protein. A recent attempt at the stabilization of the FMDV virion against thermal dissociation by engineering nonhomologous disulde bonds between capsid subunits has failed (Mateo et al., 2008). An additional design limitation in this case was the need to preserve the infectivity of the virion because of the intended application (see below). This precluded the choice of Cys mutations for disuldes that were predicted to be energetically more favorable than the ones actually introduced. The severe constraints imposed by the need to preserve each of the multiple functions that are needed for infectivity (e.g. Mateu, 1995; Chiu et al., 1997; Mateo et al., 2003) may signicantly limit the possibilities for engineering infectious virions; fortunately, some of those constraints do not apply to non-infectious empty capsids and VLPs.

A proof of principle for this approach is provided by a recent study using FMDV. Current foot-and-mouth disease (FMD) vaccines are formulated using chemically inactivated FMDV virions. These are intrinsically highly thermolabile and readily dissociate into pentamers, which are much less immunogenic than whole viral particles. As a consequence, transportation of the vaccine requires a strict and expensive cold chain, which is prone to failures in many countries where this economically very important disease is prevalent. Thus, there is a clearly perceived need to develop FMD vaccines that are less dependent on a faultless cold chain (Hegde et al., 2009; Paton et al., 2009). As a step in that direction, the FMDV virion has been rationally engineered to increase its stability against thermal dissociation into pentameric subunits, without disrupting any of the many biological functions needed for its infectivity. Normal infectivity was required because the virus must be grown during vaccine production. A systematic alaninescanning analysis of the interpentamer interfaces in the FMDV virion was rst undertaken to try and nd functionally tolerant sites for mutation. The vast majority of the interfacial residues were found to be required for infectivity (Mateo et al., 2003), which severely limited the choices. These results and the three-dimensional structure of the FMDV virion (Lea et al., 1994) were then used in the design of potentially stabilizing mutations. Some of the few amino acid side chains located at or near the interpentamer interfaces, and either predicted or found to be dispensable for infectivity, were replaced by others that had the potential to establish either disulde bonds or new electrostatic interactions between pentamers. Two of the ve electrostatically modied virions were normally infectious, genetically stable and antigenically indistinguishable from the natural virus, but showed as intended a dramatically increased thermal stability against dissociation into pentamers, relative to the non-mutated virion (Mateo et al., 2008). Electrostatic inter actions do mediate this stabilizing effect (V.Rincon and M.G.M., unpublished results).

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Modifying the mechanical stability of virus particles by rational protein engineering

The increasing use of virus particles in materials science and nanotechnology has led to a renewed interest in viruses from the physicists point of view. Several groups have recently started to theoretically or experimentally investigate the mechanical properties of viral particles, considered as physical objects of nanometric size. In a pioneering study, the mechanical exibility of a virus ( phage f29) was determined by atomic force microscopy (AFM) (Ivanovska et al., 2004). The mechanical exibility of several other phage, animal virus and plant virus particles have also begun to be explored by AFM (Carrasco et al., 2006, 2008, 2009; Michel et al., 2006; Kol et al., 2006, 2007; Ivanovska et al., 2007; Uetrecht et al., 2008; Roos et al., 2009; reviewed in Roos and Wuite, 2009). In this approach to virus mechanics, viral particles are physically deformed (indented) by applying a force through the extremely ne tip of the cantilever in an atomic force microscope. From the data, the spring constant k of the viral particle can be determined. This parameter provides a direct measure of the mechanical stiffness/rigidity (or an inverse measure of the mechanical elasticity/exibility) of the particle along the direction of the applied force. 59

Modifying the thermal stability of virus particles by rational engineering of intersubunit electrostatic interactions
A number of studies on very different viruses have shown that electrostatic interactions involving charged residues can play important roles in capsid assembly, stability and/or disassembly (e.g. Culver et al., 1995; Ellard et al., 1999; Mateo et al., 2003; Reguera et al., 2004; Douglas et al., 2004; del Alamo and Mateu, 2005; Mateu, 2009). Thus, the rational engineering of additional electrostatic interactions between capsid subunits could provide a general approach for the thermal stabilization of viral particles. Compared with the introduction of disulde bonds, introduction of electrostatic and especially Coulombic interactions may have the advantage of being much less stereochemically demanding.

M.G.Mateu

Different viruses have been shown to differ in mechanical stiffness. For MVM, CCMV and phage l, the nucleic acidlled virion is more rigid than the empty capsid and, for MVM at least, the increase in stiffness is anisotropic (Carrasco et al., 2006; Michel et al., 2006; Ivanovska et al., 2007). A natural mutation in the CCMV capsid that increases the stability in high salt conditions also increases virion stiffness (Michel et al., 2006). These and other studies have started to provide some hints on the molecular basis for the mechanical properties of viruses. From a nanobiotechnological point of view, viral particles with increased mechanical stability may be useful for applications in which they may be subjected to high mechanical stress. They could be useful for many other applications as well, as they may withstand better the action of heat or other physical or chemical agents without losing function (see next). Very recently, a mechanical property of viral particles has been rationally manipulated by protein engineering for the rst time (Carrasco et al., 2006, 2008; M.Castellanos, R.Perez and M.G.M., unpublished results). Crystallographic studies of MVM and other parvoviruses had shown that ordered segments of the genomic single-stranded DNA interact with DNA-binding sites located at equivalent positions in the capsid inner wall (Tsao et al., 1991; Agbandje-McKenna et al., 1998). A mutational analysis by alanine scanning showed that those capsid-DNA interactions contribute to stabilize the MVM virion against thermal inactivation without particle dissociation (Reguera et al., 2005). It was then hypothesized that (for variants of a same virus species) there could be a direct relationship between thermal stability and mechanical rigidity, because an increase in capsid stiffness could impair the spatial displacements of atoms and functional groups associated with a thermally induced change of conformation. In such a case, the capsid DNA interactions that thermally stabilize MVM should also contribute to increase its mechanical rigidity, and the DNA-containing virion would be mechanically more rigid than the DNA-free capsid. This prediction was experimentally conrmed using AFM (Carrasco et al., 2006). As the MVM capsid is structurally nearly unchanged (even at the atomic level) in the presence or absence of the genomic DNA (Kontou et al., 2005), the difference in stiffness could be clearly traced to the DNA. Unexpectedly, for MVM the DNA-mediated increase in stiffness was anisotropic, with the virion being stiffer than the capsid along the threefold and, especially, 2-fold symmetry axes, but not along the 5-fold axes. The predictions of a very simple nite element model supported the proposal that the observed DNA-mediated anisotropic increase in stiffness would be specically due to the capsid-bound DNA patches (Carrasco et al., 2006). This prediction was experimentally tested by removing in the virion-specic capsid DNA interactions by site-directed mutation of capsid residues at the DNA binding site. AFM measurements showed that the mutations did not affect the stiffness of the empty capsid, but they did signicantly reduce as predicted the difference in stiffness between the DNA-lled virion and the empty capsid (Carrasco et al., 2008). From previous knowledge on MVM and the above results, it has been proposed that the DNA-mediated, anisotropic mechanical rigidication of MVM may have arisen from a 60

balance between two selection pressures: on the one hand, the biological advantage of a higher mechanical rigidity that would impair the observed thermally induced inactivation of the virion (Reguera et al., 2005); on the other hand, the biological advantage of a higher mechanical exibility around the pores located at the capsid 5-fold axes that would allow the conformational rearrangements needed for DNA translocation and externalization of signal-containing N-terminal segments of capsid subunits through the pores, events that are required for MVM infection (Lombardo et al., 2002; Maroto et al., 2004; Valle et al., 2005; Riolobos et al., 2006; Cotmore and Tattersall, 2007). By evolving in the MVM capsid DNA-binding sites closer to the 2-fold and 3-fold axes and farther from the 5-fold axes pores, the genomic DNA may have been recruited to act as an architectural element that mechanically and thermally reinforces the MVM virion, while still allowing the local conformational rearrangements needed for infectivity (Carrasco et al., 2008). This model has in turn led to the prediction that mutations of certain capsid residues that impair infectivity would increase the mechanical stiffness, relative to that of the natural capsid; this prediction has been now experimentally conrmed by protein engineering (M.Castellanos, R.Perez and M.G.M., unpublished results). In summary, from a biotechnological point of view, this series of studies has provided: (i) genetically modied virions of increased mechanical exibility, by removing DNA capsid interactions; (ii) genetically modied capsids of increased mechanical rigidity, by engineering amino acid substitutions at predicted capsid locations; (iii) a proof of principle that it is possible to rationally manipulate the mechanical properties of viral particles by protein engineering. Conclusions Many virus particles are being chemically and/or genetically modied for their use in biomedicine, biotechnology or nanotechnology. The chemical approach has generally involved the functionalization of the viral particle surface (or inner wall) through a chemically reactive group, either naturally present or genetically engineered. The protein engineering approach has relied in most cases on knowledge-based genetic manipulation of the viral particle to: (i) present heterologous peptides or proteins on the capsid surface (or include them inside the capsid cavity), by incorporating the ( poly)peptides as an extension of a free terminal end of a capsid protein; (ii) present heterologous peptides on the capsid surface by insertion in exposed loops; (iii) substitute in a capsid protein one or a few dispensable residues, to provide new sites for covalent cross-linking or non-covalent binding of heterologous inorganic, organic or biological components or, much less frequently, to modify some intrinsic property or functionality of the viral particle itself. Thanks to the inbuilt features and versatility of the proteinmade virus capsids, the above (bio)chemical and genetic approaches have led to a vast collection of modied viral nanoparticles with remarkable and diverse properties and functions. More complex genetic modications of the intrinsic structure and embedded properties and functions of the viral particles themselves (and not only for the attachment of heterologous functional elements) may have to be attempted

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Virus engineering

for many applications. As for proteins in general, increasing the use of semi-rational combinatorial/directed evolution approaches for relatively large jumps in sequence space may prove extremely useful. For virions, where phenotype and genotype are physically coupled, this approach can very productive. However, for empty capsids and VLPs in general, at least some combinatorial approaches may be technically difcult to implement. Rational protein engineering is and will remain a fundamental approach for the modication of viral particles. Structure function studies on the self-assembly, physical properties and the molecular mechanisms underlying the complex functions of viral particles are providing part of the knowledge needed for emerging virus engineering projects, including the physical stabilization of viral particles. In turn, the exploration of uncharted areas of sequence and conformational space of viruses and their protein capsids with applied goals in mind is also throwing new light on the properties and biological function of viruses, and suggesting new approaches in the ght against viral disease. Acknowledgements
Virus engineering work in the authors laboratory greatly beneted from a 2-year sabbatical stay at the MRC Centre for Protein Engineering, Cambridge, UK. I am indebted to Sir A.R.Fersht and associates for their help and support there. I also gratefully acknowledge recent past and present members of my group (M. del Alamo, R.Bocanegra, A.Carreira, M.Castellanos, M.A.Fuertes, I.Lopez, E.Luna, R.Mateo, R.Perez, P.J.Perez, J.Reguera, V.Rincon and A.Rodrguez-Huete) and our collaborators in the studies mentioned here (J.M.Almendral and associates, C.Carrasco, P.J.dePablo, J.Gomez-Herrero and M.Menendez). M.G.M. is an associate member of the Institute for Biocomputation and Physics of Complex Systems, Zaragoza, Spain.

Funding Work in the authors laboratory is supported by grants from Ministerio de Ciencia e Innovacion (BIO2009-10092), Comunidad Autonoma de Madrid (S-2009/MAT/1467) and FIPSE (36557/06), and by an institutional grant from Fundacion Ramon Areces. References
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