Manipulation of the mechanical properties of a virusby protein engineeringCarolina Carrasco*, Milagros Castellanos†, Pedro J. de Pablo*‡, and Mauricio G. Mateu†‡

*Departamento de Fısica de la Materia Condensada C-III and †Centro de Biologıa Molecular Severo Ochoa (Consejo Superior de Investigaciones Cientıficasand Universidad Autonoma de Madrid), Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid, Spain

Edited by Alan R. Fersht, University of Cambridge, Cambridge, United Kingdom, and approved January 5, 2008 (received for review August 24, 2007)

In a previous study, we showed that the DNA molecule within aspherical virus (the minute virus of mice) plays an architectural roleby anisotropically increasing the mechanical stiffness of the virus.A finite element model predicted that this mechanical reinforce-ment is a consequence of the interaction between crystallographi-cally visible, short DNA patches and the inner capsid wall. We havenow tested this model by using protein engineering. Selectedamino acid side chains have been truncated to specifically removemajor interactions between the capsid and the visible DNApatches, and the effect of the mutations on the stiffness of virusparticles has been measured using atomic force microscopy. Themutations do not affect the stiffness of the empty capsid; however,they significantly reduce the difference in stiffness between theDNA-filled virion and the empty capsid. The results (i) reveal thatintermolecular interactions between individual chemical groupscontribute to the mechanical properties of a supramolecular as-sembly and (ii) identify specific protein–DNA interactions as theorigin of the anisotropic increase in the rigidity of a virus. Thisstudy also demonstrates that it is possible to control the mechan-ical properties of a protein nanoparticle by the rational applicationof protein engineering based on a mechanical model.

atomic force microscopy � nanomechanics � protein–DNA interactions

V iruses are extremely successful biological entities with anubiquitous presence in the biosphere (1). The extracellular

form of any virus (the virion) is a self-assembled nucleoproteincomplex, with or without a lipid envelope. These supramolecularassemblies have evolved the ability to withstand the physico-chemical aggressions they may encounter during organism-to-organism propagation (2). Some viruses can resist extremes oftemperature, pH, radiation, or dehydration (3, 4). In addition,recent results indicate that virions may be also subjected tosubstantial mechanical stress (5–13). Investigation of the struc-tural determinants that allow virus particles to deal with phys-icochemical extremes may be important for a better understand-ing of viruses as evolving biological machines and may benefitnanobiotechnological applications, such as the development ofrobust nanocontainers (14).

Very recently, the mechanical properties of virus particleshave begun to be experimentally investigated. The protein shells,or capsids, of �29 and � bacteriophages have been shown towithstand a very high internal pressure exerted by the encapsi-dated DNA (5, 9, 13). The stiffness/elasticity of a few viruseshave been analyzed by atomic force microscopy (AFM), inexperiments that involve the application of indentation forces.Nonenveloped virus particles, including those of phages �29(15) and � (13), cowpea chlorotic mottle virus (CCMV) (16), andthe minute virus of mice [MVM, a single-stranded DNA(ssDNA) virus] (17), are mechanically robust, while possessingremarkable elastic properties. Interestingly, these properties canbe biologically modulated. In two enveloped retroviruses (Molo-ney murine leukemia virus and HIV), the immature virion isrelatively stiff, whereas the mature, infectious virion is consid-erably softer (18, 19).

Some molecular determinants of those mechanical propertieshave already been identified. The different stiffness of theimmature and mature HIV virions is mediated by the cytoplas-mic domain of the gp120 envelope protein (19). A variant,salt-stable CCMV virion whose capsid differed in a single aminoacid residue per subunit was also stiffer (16). Comparison of themechanical properties of the nucleic acid-filled virion of MVMwith those of the empty capsid (devoid of DNA) showed that theenclosed nucleic acid molecule can play a structural role byincreasing the stiffness of the particle (17). This mechanicalreinforcement was anisotropic [i.e., higher when the force wasapplied along a capsid 2-fold (S2) symmetry axis, lower along a3-fold (S3) axis, and insignificant along a 5-fold (S5) axis]. Anucleic acid-mediated overall increase in stiffness has also beenalso detected for phage � (13) and CCMV (16).

The MVM capsid is formed by 60 structurally equivalentprotein subunits arranged in a simple (T � 1) icosahedralsymmetry (20–22). In the crystal structures of many icosahedralviruses, all, or a large part, of the nucleic acid is invisible becauseit is oriented randomly within the particles in the crystal.However, in MVM (as in other viruses; see refs. 23–28), somesegments of the viral nucleic acid molecule adopt very similarconformations at symmetrically equivalent positions inside thecapsid and have been crystallographically visualized. Each ofthese DNA patches is noncovalently bound to several amino acidresidues located at one of 60 equivalent sites on the inner capsidwall (refs. 21 and 22, and see Fig. 1). Use of a finite elementmodeling approach allowed us to predict that the observedDNA-mediated mechanical reinforcement of MVM may bespecifically attributed to those capsid-bound DNA patches (17).However, this approach was necessarily based on highly simpli-fying assumptions. The adequateness of any theoretical ap-proach to predict whether, and how, the mechanical propertiesof a virus or supramolecular assembly are modified whenintermolecular interactions are introduced or removed remainedto be experimentally validated.

The present study provides experimental proof that, in theMVM virion, the DNA exerts its mechanical reinforcementeffect mainly through interactions between the capsid-boundDNA patches and specific amino acid residues. It also providesproof of principle that the mechanical properties of a supramo-lecular biological assembly can be rationally modified by usingprotein engineering.

ResultsIn the refined crystallographic model of MVM (22), the visiblessDNA at each equivalent site includes two short stretches (of 11

Author contributions: C.C. and M.C. contributed equally to this work; P.J.d.P. and M.G.M.designed research; C.C. and M.C. performed research; C.C., M.C., P.J.d.P., and M.G.M.analyzed data; and M.G.M. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

‡To whom correspondence may be addressed: E-mail: mgarcia@cbm.uam.es orp.j.depablo@uam.es.

This article contains supporting information online at www.pnas.org/cgi/content/full/0708017105/DC1.

© 2008 by The National Academy of Sciences of the USA

4150–4155 � PNAS � March 18, 2008 � vol. 105 � no. 11 www.pnas.org�cgi�doi�10.1073�pnas.0708017105

Dow

nloa

ded

by g

uest

on

Nov

embe

r 20

, 202

0

and 8 nucleotides). This DNA patch adopts a wedge-like shapethat penetrates a concavity of the inner capsid wall and estab-lishes noncovalent interactions with several amino acid residues(refs. 21, 22, 29; and see Fig. 1). To determine whether thesespecific interactions are responsible for the DNA-mediatedmechanical reinforcement of the virion, we have analyzed byAFM the effect of mutation of capsid residues on the mechanicalstiffness of MVM particles. Two single mutations—Asn183Alaand Asp58Ala—were aimed at removing some of the majorinteractions between the capsid and the bound DNA patches,without compromising intracapsid interactions. Mutation to Alainvolved simply the removal of either the amide group ofAsn-183 or the carboxylate group of Asp-58 and was chosen todisrupt native interactions without introducing new interactionsand because it had a very low probability of altering theconformation of the polypeptide backbone (30, 31). In therefined model of the MVM virion (22), the amide of Asn-183 andthe carboxylate of Asp-58, respectively, are involved in hydrogenbonds with the sugar–phosphate backbone and with a purinebase of the longer capsid-bound DNA stretch (Fig. 1b); they alsoestablish several van der Waals contacts with the DNA. Similarinteractions were observed between the DNA and the structur-ally equivalent capsid residues Asn-180 and Asn-56 in the refinedstructure of the homologous canine parvovirus (29, 32, 33). Thisfinding provided further support for the presence and functionalrelevance of these interactions. None of the atoms that wereremoved by either mutation were found to be involved inintracapsid interactions.

An infectious DNA clone of MVM carrying either mutation,and the nonmutated clone as a control, were used to transfectsusceptible cells. The virus particles produced consisted of bothempty capsids and DNA-filled, infectious virions, which werepurified and separated from each other based on their differentbuoyant densities. Detectable contamination of empty capsids byinfectious virions was excluded by titration; significant contam-ination of infectious virions by empty capsids was equallyexcluded by recentrifuging the virion preparation and verifyingthe absence of MVM particles with a density corresponding tothat of empty capsids (data not shown). The virion-free empty-capsid preparations, and the empty-capsid-free virion prepara-tions obtained after the second density gradient, were used in theexperiments described below.

The nonmutated and mutant empty capsids and DNA-filledvirions were visualized by transmission electron microscopy [seesupporting information (SI) Figs. 6 and 7] and by AFM (Fig. 2).All of the particles were indistinguishable in dimensions, shape,and topography. AFM images of many individual particlesclearly showed the expected topographic features, including thespikes located at the particle S3 axes and the cylindrical protru-sions at the S5 axes. These features served to identify the specificorientation of virus particles that were positioned with a capsidS5, S3, or S2 axis on top (Fig. 2).

To quantitatively determine the mechanical stiffness of themodified MVM particles, we performed nanoindentations on

Fig. 3. Indentation curves (a) and deflection curves (b) for the nonmutatedvirion probed along an S2, S3, or S5 axis. Each indentation graph is the averageof five curves carried out with the same cantilever to probe particles alongeach type of symmetry axis in a same experimental session. Each deflectioncurve shown is a typical curve obtained in one of these five measurements. Thethin line in b corresponds to the deflection curve when the force was appliedon the glass surface.

Fig. 1. DNA–capsid interfaces in the MVM virion. (a) Cutaway section acrystallographic model of the MVM virion (22). The visible DNA stretches (red)are bound to amino acid residues (green, except Asn-183, blue) at equivalentsites on the inner capsid wall. The rest of the DNA molecule and the internalN-terminal segment of each capsid protein subunit are crystallographicallyinvisible. For orientation, one of the capsid S5 axes is located at the center(black spot) of the model. (b) Close-up view of one of the equivalent DNA–capsid interfaces. The two ssDNA stretches that form each capsid-bound patchare shown as sticks models. The capsid residues that establish interactions withthe DNA (except Asp-58 and Asn-183) are shown as cyan, green, or yellowspacefilling models, according to the subunit involved. The two amino acidresidues we mutated (Asp-58 and Asn-183) are shown in violet and magenta,respectively, with the hydrogen bonds they establish with the DNA repre-sented as white lines.

Fig. 2. MVM virions imaged by AFM and viewed along an S5 (Left), S3(Center), or S2 (Right) symmetry axis. (a) Molecular surface model of thenonmutated MVM virion derived from crystallographic data. (b–d) AFMimages corresponding to nonmutated virion (b), Asn183Ala mutant (c), andAsp58Ala mutant (d). The MVM topography appears laterally expanded be-cause of the usual tip-sample dilation effects. Only those particles with asymmetry axis at, or very close to, the top (center of the image) were selectedfor indentation. Most particles showed a symmetry axis clearly off-center, orno recognizable symmetry, and were discarded.

Carrasco et al. PNAS � March 18, 2008 � vol. 105 � no. 11 � 4151

BIO

PHYS

ICS

Dow

nloa

ded

by g

uest

on

Nov

embe

r 20

, 202

0

individual intact particles in a physiological buffer, with the forceapplied along an S5, S3, or S2 symmetry axis (17). Fig. 3 showsexamples of averaged indentation curves for MVM particleswhen probed along the three axis types, together with typicalcantilever deflection curves. Examples for each virus particletype and symmetry axis are given as SI Figs. 8 and 9. The springconstants, k, obtained for each orientation were represented ina histogram, and a Gaussian fit of the histogram yielded theaverage spring constant of the particle when measured along thatorientation (Fig. 4). Larger deviations of the k values wereobtained for the virions relative to the empty capsids, especiallywhen probed along an S2 axis (Fig. 4 and see SI Table 1). If thedeviations were due solely to experimental error, those obtainedby using empty capsids or virions should have been similar, atleast when similar average k values were compared. Thus, thelarger deviations probably reflect a broader Gaussian distribu-tion of actual stiffness values. Likely causes are inaccuracies inthe exact orientation of the viral particle being indented (the axiswas not always exactly on top), as well as slight shifts in the

orientations of both tip and particle after each indentation,which were clearly detected by imaging. Because the stiffness ofthe MVM virion (but not the empty capsid) is not isotropic (17),indentation at slightly different, off-axis positions could lead tosmall differences in the k value obtained in each indentation,even when the same individual particle was used. In addition, theintrinsic anisotropy of the single DNA molecule would make the60 n-fold (n � 5, 3, or 2) axes not strictly equivalent in the virion(but not in the DNA-free capsid), potentially leading to slightlydifferent k values, depending on the specific S2 (or S3, or S5) axisprobed. Because the capsid-bound DNA segments are locatedcloser to the S2 axes and farther from the S5 axes (Fig. 1a), thiseffect could be greater when the virions are probed along an S2axis, as was actually observed.

To validate a comparative analysis, it was important to ascer-tain to what approximation the quantitative value of k could bereproduced. The results from using nonmutated capsids andvirions in independent experiments showed an excellent agree-ment in the average k values (see SI Table 1). This reproduc-

Fig. 4. Comparison of the mechanical properties of nonmutated and mutant MVM particles. (a) Comparison of the mechanical properties of the nonmutatedand mutant empty capsids of MVM. Histograms depict the stiffness (k value) obtained for individual empty capsids along different symmetry axes. Red,nonmutated; blue, Asn183Ala mutant; green, Asp58Ala mutant. The associated tables indicate the average k values and standard deviations obtained fromGaussian fits for indentations along an S5 (Left), S3 (Center), or S2 (Right) axis. (b) Comparison of the mechanical properties of nonmutated and mutant DNA-filledvirions of MVM. The corresponding histograms and tables for the virions are depicted as in a. The vertical black bar in each histogram indicates the k value forthe nonmutated empty capsid (to facilitate comparison between virions and empty capsids); the mutant empty capsids yielded k values that were indistin-guishable from this value (compare a). A Student t test for samples having an unequal variance reveals, with �99% confidence, that the mean k values obtainedfor the nonmutated virions and for either mutant along the 2-fold or 3-fold axes do not overlap.

4152 � www.pnas.org�cgi�doi�10.1073�pnas.0708017105 Carrasco et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 20

, 202

0

ibility, observed even in those cases where a broad Gaussiandistribution occurred, experimentally validated any statisticallysignificant difference that could be obtained when comparingthe stiffness of different particles under the same experimentalconditions.

We then compared the stiffness of the mutants vs. nonmutatedvirus particles. Any difference found in the stiffness of themutant virions relative to the nonmutated virion could be due,in principle, to the removal of interactions between the truncatedgroups and other capsid residues and/or the bound DNA seg-ments. The MVM empty capsid and virion are structurallyalmost indistinguishable, even at atomic resolution (22). Thus, ifa mutation affects the stiffness of the virion because of thedisruption of intracapsid interactions, it would equally affect thestiffness of the empty capsid. We carried out indentations onseveral individual intact empty capsids carrying no mutations, orincluding either the Asn183Ala or Asp58Ala mutations (Fig. 4aand see SI Table 2). Gaussian fits of the histograms yielded kvalues along the S5, S3, and S2 axes that were indistinguishablefrom each other. The k values were also indistinguishable whenthe two mutant empty capsids and the nonmutated empty capsidwere compared (Fig. 4a). To confirm that the number ofmeasurements carried out was sufficient to obtain good fittingvalues, in one arbitrary case (mutant Asp58Ala oriented alongthe 3-fold axis) much larger numbers of force-vs.-distance curvesand individual particles were subsequently used in the calcula-tion; no significant differences in the fitting k value, or in thedistribution of individual measurements, were obtained. Thesame was observed for other particle or symmetry axis typeswhen the number of particles indented and the total number ofindentations was increased (data not shown). The above resultsindicate that any effect of these mutations on the stiffness of theMVM virion would be due to the removal of DNA–capsidinteractions and not to removal of intracapsid interactions.

We then performed many indentations on several DNA-filledvirions carrying no mutations or including either the Asn183Alaor Asp58Ala mutations (Fig. 4b and see SI Table 2). Remark-ably, introduction of either mutation substantially reduced theincrease in the k value that was observed for the nonmutatedvirion along the S3 axes and, to a greater extent, the S2 axes,relative to the empty capsid (Fig. 4b and Fig. 5). The presenceof the DNA in the nonmutated particle led to increases of �60%and 110% when probed along S3 and S2 axes, respectively.Mutations Asn183Ala or Asp58Ala reduced those values to�30% and 40% or 30% and 30%, respectively. A Student t testrevealed that the average k values obtained along the 2-fold axisfor the mutant virions and the nonmutated virion are signifi-cantly different (with a 99% confidence). The reduced k valuescannot be due to a change in the particle surface electrostaticsbecause the mutations removed only neutral groups from inter-nal residues of the capsid and did not affect any of the chargedgroups of the capsid or the DNA, which remained unmodified.We conclude that removal of major noncovalent interactionsbetween specific amino acid residues located at the inner wall ofthe MVM capsid, and the capsid-bound DNA patches, led to avery substantial reduction in the DNA-mediated mechanicalreinforcement of the virus particle.

DiscussionA finite element calculation allowed us to propose that theDNA-mediated mechanical reinforcement of the MVM virioncould be specifically due to small DNA patches intimately boundto the inner capsid wall. These DNA patches would effectivelyincrease the local thickness of the capsid wall, acting as molec-ular buttresses (17). The results of the present study experimen-tally confirm such prediction and show that specific noncovalentinteractions holding the capsid and those DNA segments to-gether are directly responsible for the observed reinforcement.

In the Asn183Ala and Asp58Ala mutant virions, the absence ofsome of the major capsid–DNA interactions could cause thoseDNA segments to be more loosely bound to their recognitionsites in the capsid wall, thus reducing their reinforcing (buttress)effect. In either mutant, only some of the major interactionsbetween the capsid and each DNA patch were removed and,consequently, the reinforcing effect of the DNA was not ex-pected to be completely prevented. However, the stiffness of theN183A virion was reduced to the point of making it statisticallyindistinguishable from that of the empty capsid; the stiffness ofthe D58A virion was also substantially reduced, although it wasstill significantly higher than that of the capsid, according to aStudent t test (99% confidence). Loss of cooperative effectsbetween different intermolecular interactions at each equivalentDNA–capsid interface may explain such a large mechanicalaction, in the same way that it explains equally drastic reductionsin binding affinity caused by single-residue mutations in manyprotein–ligand interfaces. In addition, a single amino acid re-placement will remove in the MVM virion equivalent sets ofinteractions between the only DNA molecule and up to 60 sitesin the capsid wall. Cooperative effects due to the loss ofinteractions at so many sites may also amplify the effect of eachmutation (34).

The physicochemical cause for a nucleic acid-mediated mechan-ical reinforcement may depend on the virus and nucleic acidinvolved. A recently developed analytical model indicated that thedouble-stranded DNA-mediated mechanical reinforcement ofphage � is due to the osmotic pressure generated by DNA-hydratingwater molecules (13). The ssDNA inside MVM is also denselypacked, and the presence of a substantial internal pressure in MVMis a definite, albeit unproven, possibility (17). However, when theinteraction between the bound DNA segments and the MVMcapsid was debilitated through mutation, the stiffness of the virionwas reduced to a value similar to that of the empty capsid, eventhough the complete, unmodified DNA molecule was still inside thevirus particle. Thus, the ssDNA inside MVM may not generatesubstantial internal pressure. Alternatively, if a DNA-mediatedinternal pressure exists in MVM, it may not significantly contributeto the mechanical stiffness of this virus.

It may be important to relate the observed mechanical effects tothe biology of MVM. We had shown previously (29) that capsidresidues involved in interactions with DNA patches, including N183and D58 involved in the DNA-mediated stiffening effect, contrib-ute to virion stability against thermal inactivation and to virusinfectivity, which was reduced �10-fold by truncation of individual

Fig. 5. Schematic representation of the increase in stiffness of the DNA-filledvirion, relative to the empty capsid, along the 5-fold (S5), 3-fold (S3), or 2-fold(S2) symmetry axes. Arrows refer to unmodified MVM (red), Asn183Ala mu-tant (blue), or Asp58Ala mutant (green). Arrow length is proportional to �k,the difference in k value between the virion and the empty capsid. If �k is notsignificantly different from zero according to a Student t test, an arrowheadis used.

Carrasco et al. PNAS � March 18, 2008 � vol. 105 � no. 11 � 4153

BIO

PHYS

ICS

Dow

nloa

ded

by g

uest

on

Nov

embe

r 20

, 202

0

side chains interacting with the DNA. Also, each visible DNA patchbinds the inner capsid wall by penetrating and filling a concavity andestablishing multiple interactions with residues from three neigh-boring capsid subunits, contributing to cementing together of thesesubunits. Thus, one possibility is that the bound DNA serves tomechanically and thermically reinforce the capsid, allowing moreextracellular virions to remain infectious until they reach their hostcells. A nonexclusive possibility is that binding of the DNA patchescould have a role in freezing the capsid in a stiffer, mechanicallymore stable conformation and prevent it from undergoing unpro-ductive conformational changes during the infection cycle. Asimilar situation may occur in bean pod mottle virus (BPMV).Biophysical evidence has suggested that the BPMV empty capsidhas a highly dynamic structure, as opposed to the ssRNA-filledvirion, and the presence of nucleic acid in the BPMV virion wasshown to stabilize the capsid against thermal denaturation (35, 36).

In the above scenario, the absence of a DNA-mediatedincrease in the stiffness of the MVM virion around the S5 axescould also be explained in biological terms. The pores located atthe S5 axes are used for externalization of capsid polypeptideN-terminal segments and for entry and/or exit of the viral DNA.These events are critically necessary for infection (21, 22, 37–41)and depend on the occurrence of local conformational rear-rangements modulated by some capsid residues lining the pores(39, 41–43), as well as by the size and shape of capsid cavitieslocated nearby (44). Those conformational changes may requirea certain mechanical f lexibility in the regions around the pores,precisely the only large areas of the capsid where bound DNA isnot observed (Fig. 1a). Thus, it is tempting to suggest the actionon MVM of a selective pressure to keep the capsid regions thatare closer to the 5-fold axis pores free from binding, stiffeningDNA segments and thus flexible enough to allow those biolog-ically critical translocation events. In short, MVM and otherparvoviruses may have evolved a functionally compatible, mul-tiple-site interaction between their DNA genomes and the innercapsid wall, leading to improved mechanical and thermal sta-bility and contributing to the biological success of these viruses.

From a nanobiotechnological perspective, the present studydemonstrates that the mechanical properties of a biomolecularcomplex can be rationally manipulated using a protein engineer-ing approach. The predictions of a simple mechanical model ledus to obtain, by site-directed mutagenesis, a modified virusparticle that is mechanically softer than the natural virus. Wepropose that protein engineering may be a suitable approach totailoring the mechanical properties of protein nanoparticles.

Materials and MethodsRecombinant Plasmids and Mutagenesis. Site-directed mutagenesis of the VP1/VP2 gene of MVM (strain p) was carried out using the QuikChange system(Stratagene) on recombinant plasmid pSVtk-VP1/2 originally provided by J. M.Almendral (Centro de Biologıa Molecular, Madrid, Spain) (45). The mutationswere introduced by subcloning in an MVMp infectious clone originally providedby P. Tattersall (Yale University Medical School, New Haven, CT) (46) and thenmodified (45). The presence of the mutations was confirmed by sequencing.

Electroporation of Mammalian Cells and Infectivity Assays. NB324K cells wereelectroporated with the MVM infectious plasmid carrying the appropriatemutations (47). Virions were recovered from transfected monolayers andtitrated in plaque assays.

Production and Purification of MVM Empty Capsids and Virions. MVM emptycapsids and virions were obtained as described in ref. 17, with some modifi-cations. Briefly, MVM particles were produced by infection of NB324K cells ata low multiplicity of infection. After adsorption of the virus and incubation at37°C, the cells were suspended in culture medium, plated at low density, andincubated at 37°C until complete cytopathic effect. The empty capsids andvirions obtained from the remaining infected cells and those obtained fromthe supernatant of infection were mixed, supplemented with SDS to 0.5%,deposited on layers containing 20% sucrose in TE buffer (50 mM Tris�HCl pH8.0, 0.5 mM EDTA) plus 0.1 M NaCl and centrifuged for 5.5 h at 35,000 rpm inan SW40 rotor (Beckman) at 10°C. The sediment was thoroughly resuspendedin TE buffer containing 0.2% Sarkosyl, and the suspension was centrifuged ina cesium chloride gradient in the same buffer for 24 h at 50,000 rpm in a TFT75.13 rotor (Kontron) at 10°C. The gradient was fractionated, and the aliquotswere analyzed for the presence of empty capsids (buoyant density 1.363) orvirions (density 1.373) by assaying their hemagglutination activity. The frac-tions of interest were extensively dialyzed against PBS (pH 7.2). To exclude anycross-contamination of virions and capsids, only the central fractions of wellresolved peaks were used. Titration in plaque assays was used to ascertain theabsence of virions in the empty-capsid preparation. To exclude empty capsidsfrom the virion preparation, the latter was layered on a second cesiumchloride gradient, recentrifuged as above, and extensively dialyzed again. Thepurity, integrity, and concentration of MVM particles were assessed by elec-tron microscopy.

AFM of Viral Particles. AFM experiments were carried out essentially asdescribed in ref. 17. Briefly, one drop (20 �l) of diluted stock of purified emptycapsids or virions in PBS was deposited on a sylanized glass surface. The dropwas left on the surface for 30 min and then rinsed twice with 20 �l of PBS. Thetip was also prewetted with 20 �l of PBS. The atomic force microscope(Nanotec Electronica) was operated in jumping mode (48) in liquid. We usedrectangular cantilevers (RC800PSA; Olympus) with spring constant of 0.05 �0.01 N/m. The maximum normal force during AFM imaging was always �100pN. Dynamic mode could not be used because the softer cantilevers requiredfor reproducible imaging using this mode would have led to increased errorsin the k values. AFM images were processed by using WSxM software (49).

To determine the stiffness of empty capsids and virions once individualparticles were located on the surface, the lateral piezo scan was stopped whenthe tip was on top of the equatorial area of the particle. Then, force-vs.-distance curves were obtained by elongating the z-piezo until the tip estab-lished mechanical contact with the virus particle, and a nanoindentation wasperformed. To avoid particle damage, the maximum applied force was limitedto 0.9 nN, with typical indentations of �2 nm. We observed that, after a fewcontact events, the force-vs.-distance curve exhibited marked steps, whichcorresponded to an irreversible modification of the virus particle. In this case,we moved to another particle. The curves were processed assuming thecantilever and the virus to be two springs in series, to obtain the stiffness(spring constant, k) of the virus particle along the direction of the appliedforce (15). Each cantilever was calibrated as described in ref. 50 and asimplemented online at www.ampc.ms.unimelb.edu.au/afm/theory.html#normal.

Molecular Graphics and Structural Analyses. The refined PDB coordinates of theof MVMi virion (1Z1C) and of the MVMp empty capsid (1Z14) (22), as well asthe software programs Insight II (Biosym Technologies), RasMol (51), WHAT IF(52), and PyMOL (DeLano Scientific), were used.

ACKNOWLEDGMENTS. We thank J. Gomez-Herrero, P. A.Serena, and I. A. T.Schaap for fruitful discussions and critical reading of the manuscript; P.Tattersall, J. M. Almendral, and J. Reguera (Centro Nacional de Biotecnologıa,Madrid, Spain) for providing MVM plasmids; and L. Riolobos and E. Grueso fortechnical advice. M.C. is a predoctoral fellow from Comunidad de Madrid(CM). This work was supported by CM Grants S-0505/MAT/0303 (to M.G.M. andP.J.P.), Ministerio de Educacion y Ciencia Grant BIO2006-00793 (to M.G.M.),CM Grant GR/MAT/0254/2004 (to P.J.P.), and an institutional grant fromFundacion Ramon Areces to the Centro de Biologıa Molecular. M.G.M. is anassociate member of the Centro de Biocomputacion y Fısica de los SistemasComplejos, Zaragoza, Spain.

1. Suttle CA (2005) Nature 437:356–361.2. Chiu W, Burnett R, Garcea RL, eds (1997) Structural Biology of Viruses (Oxford Univ

Press, Oxford).3. Prigent M, Leroy M, Confalonieri F, Dutertre M, DuBow MS (2005) Extremophiles

9:289–296.4. Hernando E, Llamas-Saiz AL, Foces-Foces C, McKenna R, Portman L, Agbandje-

McKenna M, Almendral JM (2000) Virology 267:299–309.5. SmithDE,TansSJ,SmithSB,GrimesS,AndersonDL,BustamanteC(2001)Nature413:748–752.

6. Kindt J, Tzlil S, Ben-Shaul A, Gelbart WM (2001) Proc Natl Acad Sci USA 98:13671–13674.

7. Purohit PK, Kondev J, Phillips R (2003) Proc Natl Acad Sci USA 100:3173–3178.8. Cordova A, Deserno M, Gelbart WM, Ben-Shaul A (2003) Biophys J 85:70–74.9. Evilevitch A, Lavelle L, Knobler CM, Raspaud E, Gelbart WM (2003) Proc Natl Acad Sci

USA 100:9292–9295.10. Zandi R, Reguera D, Bruinsma RF, Gelbart WM, Rudnick J (2004) Proc Natl Acad Sci USA

101:15556–15560.

4154 � www.pnas.org�cgi�doi�10.1073�pnas.0708017105 Carrasco et al.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 20

, 202

0

11. Zandi R, Reguera D (2005) Phys Rev E Stat Nonlin Soft Matter Phys 72:021917.12. van der Schoot P, Bruinsma R (2005) Phys Rev E Stat Nonlin Soft Matter Phys 71:061928.13. Ivanovska I, Wuite G, Jonsson B, Evilevitch A (2007) Proc Natl Acad Sci USA 104:9603–

9608.14. Douglas T, Young M (2006) Science 312:873–875.15. Ivanovska IL, de Pablo PJ, Ibarra B, Sgalari G, MacKintosh FC, Carrascosa JL, Schmidt CF,

Wuite GJL (2004) Proc Natl Acad Sci USA 101:7600–7605.16. Michel JP, Ivanovska IL, Gibbons MM, Klug WS, Knobler CM, Wuite GJL, Schmidt CF

(2006) Proc Natl Acad Sci USA 103:6184–6189.17. Carrasco C, Carreira A, Schaap IAT, Serena PA, Gomez-Herrero J, Mateu MG, de Pablo

PJ (2006) Proc Natl Acad Sci USA 103:13706–13711.18. Kol N, Gladnikoff M, Barlam D, Shneck RZ, Rein A, Rousso I (2006) Biophys J 91:767–774.19. Kol N, Tsvitov M, Barlam D, Shneck RZ, Kay MS, Rousso I (2007) Biophys J 92:1777–1783.20. Llamas-Saiz AL, Agbandje-McKenna M, Wikoff WR, Bratton J, Tattersall P, Rossmann

MG (1997) Acta Crystallogr D 53:93–102.21. Agbandje-McKenna M, Llamas-Saiz AL, Wang F, Tattersall P, Rossmann MG (1998)

Structure 6:1369–1381.22. KontouM,GovindsamyL,NamHJ,BryantN,Llamas-SaizAL,Foces-FocesC,HernandoE,Rubio

MP, McKenna R, Almendral JM, Agbandje-McKenna M (2005) J Virol 79:10931–10943.23. Chen Z, Stauffacher C, Li Y, Schmidt T, Bomu W, Kamer G, Shanks M, Lomonosoff G,

Johnson JE (1989) Science 245:154–159.24. McKenna R, Xia D, Willingmann P, Ilag LL, Krishnaswamy S, Rossmann MG, Olson NH,

Baker TS, Incardona NL (1992) Nature 355:137–143.25. Fisher AJ, Johnson JE (1993) Nature 361:176–179.26. Larson SB, Koszelak S, Day J, Greenwood A, Dodds JA, McPherson A (1993) Nature

361:179–182.27. Tang L, Johnson KN, Ball A, Lin T, Yeager M, Johnson JE (2001) Nat Struct Biol 8:77–83.28. Blink HHJ, Pleij CWA (2002) Arch Virol 147:2261–2279.29. Reguera J, Grueso E, Carreira A, Sanchez-Martinez C, Almendral JM, Mateu MG (2005)

J Biol Chem 280:17969–17977.

30. Cunningham BC, Wells JA (1989) Science 244:1081–1085.31. Lau FT, Fersht AR (1989) Biochemistry 28:6841–6847.32. Tsao J, Chapman MS, Agbandje M, Keller W, Smith K, Wu H, Luo M, Smith TJ, Rossmann

MG, Compans RW, Parrish CR (1991) Science 251:1456–1464.33. Chapman MS, Rossmann MG (1995) Structure 3:151–162.34. Zlotnick A (1994) J Mol Biol 241:59–67.35. Li T, Chen Z, Johnson JE, Thomas GJ, Jr (1992) Biochemistry 31:6673–6682.36. Da Poian AT, Johnson JE, Silva JL (2002) J Biol Chem 277:47596–47602.37. Cotmore SF, D’Abramo AM, Ticknor CM, Tattersall P (1999) Virology 254:169–181.38. Maroto B, Valle N, Saffrich R, Almendral JM (2004) J Virol 78:10685–10694.39. Reguera J, Carreira A, Riolobos L, Almendral JM, Mateu MG (2004) Proc Natl Acad Sci

USA 101:2724–2729.40. Valle N, Riolobos L, Almendral JM (2005) in Parvoviruses, eds Kerr JR, Cotmore SF,

Bloom ME, Linden RM, Parrish CR (Arnold, London), pp 291–304.41. Farr GA, Cotmore SF, Tattersall P (2006) J Virol 80:161–171.42. Carreira A, Menendez M, Reguera J, Almendral JM, Mateu MG (2004) J Biol Chem

279:6517–6525.43. Farr GA, Tattersall P (2004) Virology 323:243–256.44. Carreira A, Mateu MG (2006) J Mol Biol 360:1081–1093.45. Ramırez JC, Santaren JF, Almendral JM (1995) Virology 206:57–68.46. Gardiner EM, Tattersall P (1988) J Virol 62:2605–2613.47. Lombardo E, Ramırez JC, Garcıa J, Almendral JM (2002) J Virol 76:7049–7059.48. Moreno-Herrero F, de Pablo PJ, Fernandez-Sanchez R, Colchero J, Gomez-Herrero J,

Baro AM (2002) Appl Phys Lett 81:2620–2622.49. Horcas I, Fernandez R, Gomez-Rodriguez JM, Colchero J, Gomez-Herrero J, Baro AM

(2007) Rev Sci Instrum 78:013705–013708.50. Sader JE, Chon JWM, Mulvaney P (1999) Rev Sci Instrum 70:3967–3969.51. Sayle RA, Milner-White EJ (1995) Trends Biochem Sci 20:374–376.52. Vriend G (1990) J Mol Graphics 8:52–56.

Carrasco et al. PNAS � March 18, 2008 � vol. 105 � no. 11 � 4155

BIO

PHYS

ICS

Dow

nloa

ded

by g

uest

on

Nov

embe

r 20

, 202

0