Genome-Released Poliovirus Particles between Two Distinct Sites ...

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Published Ahead of Print 20 July 2011. 2011, 85(19):9974. DOI: 10.1128/JVI.05013-11. J. Virol. Alasdair C. Steven, James M. Hogle and David M. Belnap Jun Lin, Naiqian Cheng, Marie Chow, David J. Filman, Genome-Released Poliovirus Particles between Two Distinct Sites on An Externalized Polypeptide Partitions http://jvi.asm.org/content/85/19/9974 Updated information and services can be found at: These include: REFERENCES http://jvi.asm.org/content/85/19/9974#ref-list-1 at: This article cites 48 articles, 22 of which can be accessed free CONTENT ALERTS more» articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new http://jvi.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders: http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to: on February 21, 2012 by Albert R. Mann Library http://jvi.asm.org/ Downloaded from

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Published Ahead of Print 20 July 2011. 2011, 85(19):9974. DOI: 10.1128/JVI.05013-11. J. Virol.

Alasdair C. Steven, James M. Hogle and David M. BelnapJun Lin, Naiqian Cheng, Marie Chow, David J. Filman, Genome-Released Poliovirus Particlesbetween Two Distinct Sites on An Externalized Polypeptide Partitions

http://jvi.asm.org/content/85/19/9974Updated information and services can be found at:

These include:REFERENCES

http://jvi.asm.org/content/85/19/9974#ref-list-1at: This article cites 48 articles, 22 of which can be accessed free

CONTENT ALERTS more»articles cite this article),

Receive: RSS Feeds, eTOCs, free email alerts (when new

http://jvi.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders: http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:

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JOURNAL OF VIROLOGY, Oct. 2011, p. 9974–9983 Vol. 85, No. 190022-538X/11/$12.00 doi:10.1128/JVI.05013-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

An Externalized Polypeptide Partitions between Two Distinct Sites onGenome-Released Poliovirus Particles!

Jun Lin,1 Naiqian Cheng,2 Marie Chow,3 David J. Filman,4 Alasdair C. Steven,2James M. Hogle,4 and David M. Belnap1,2*

Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 846021; Laboratory of Structural Biology Research,National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 208922;

Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock,Arkansas 722053; and Department of Biological Chemistry and Molecular Pharmacology,

Harvard Medical School, Boston, Massachusetts 021154

Received 3 May 2011/Accepted 11 July 2011

During cell entry, native poliovirus (160S) converts to a cell-entry intermediate (135S) particle, resulting inthe externalization of capsid proteins VP4 and the amino terminus of VP1 (residues 1 to 53). Externalizationof these entities is followed by release of the RNA genome (uncoating), leaving an empty (80S) particle. Theantigen-binding fragment (Fab) of a monospecific peptide 1 (P1) antibody, which was raised against a peptidecorresponding to amino-terminal residues 24 to 40 of VP1, was utilized to track the location of the aminoterminus of VP1 in the 135S and 80S states of poliovirus particles via cryogenic electron microscopy (cryo-EM)and three-dimensional image reconstruction. On 135S, P1 Fabs bind to a prominent feature on the externalsurface known as the “propeller tip.” In contrast, our initial 80S-P1 reconstruction showed P1 Fabs alsobinding to a second site, at least 50 Å distant, at the icosahedral 2-fold axes. Further analysis showed that theoverall population of 80S-P1 particles consisted of three kinds of capsids: those with P1 Fabs bound only at thepropeller tips, P1 Fabs bound only at the 2-fold axes, or P1 Fabs simultaneously bound at both positions. Ourresults indicate that, in 80S particles, a significant fraction of VP1 can deviate from icosahedral symmetry.Hence, this portion of VP1 does not change conformation synchronously when switching from the 135S state.These conclusions are compatible with previous observations of multiple conformations of the 80S state andsuggest that movement of the amino terminus of VP1 has a role in uncoating. Similar deviations fromicosahedral symmetry may be biologically significant during other viral transitions.

For a successful virus infection, a virus must breach theplasma or endosomal membrane and deposit its genome in theappropriate intracellular compartment. Enveloped viruses,such as HIV and influenza virus, cross the membrane via mem-brane fusion. However, nonenveloped viruses, such as picor-naviruses, must enter the cell by a different mechanism. Thismechanism is thought to involve either endosomal lysis or poreformation (33, 41, 44).

Poliovirus, the prototypic member of the picornavirus fam-ily, is one of the major model systems to study nonenvelopedcell-entry mechanisms. Both the virus and its cellular receptor,poliovirus receptor (Pvr; also known as CD155), are exten-sively characterized and structurally defined (2, 4, 17, 21, 27,28, 48, 49). Like other picornaviruses, the poliovirus capsid iscomposed of 60 copies of four capsid proteins (VP1, VP2, VP3,and VP4), arranged with icosahedral symmetry (Fig. 1). VP1,VP2, and VP3 have a !-jellyroll topology and form prominentfeatures known as the “mesa,” “canyon,” and “propeller” onthe outer surface of the capsid (Fig. 1). VP4 lies in an extendedconformation on the inner surface of the capsid shell, as do theamino-terminal segments of VP1, VP2, and VP3. The capsid

surrounds an approximately 7,500-nucleotide, single-strandedRNA genome.

During cell entry, the binding of native virus (sedimentationcoefficient, 160S) to CD155 initiates conformational rear-rangements that lead to formation of the genome-containingcell-entry intermediate (135S) particle (15, 27, 30). After un-coating (release of RNA into the host cell), the resultant emptycapsid shell sediments at 80S.

The conformational rearrangements that form the 135S in-termediate involve irreversible externalization of VP4 (which ismyristoylated at its amino terminus [10]) and the amino-ter-minal extension of VP1 (which is predicted to form an amphi-pathic helix [18]) from the capsid interior; externalization ofthese viral polypeptide sequences appears to facilitate poliovi-rus cell entry (2, 7, 8, 16, 18, 29, 33, 44). These exposed se-quences interact with—and, in the case of the amino terminusof VP1, tether particles to—artificial membranes (18, 43). Theexposed sequences also insert into cellular membranes duringinfection (16). Analyses of VP4 mutants show that VP4 plays akey role in the formation and properties of virus ion channelsand in uncoating (16). For VP1, residues 1 to 53 were pre-dicted to be exposed on the capsid surface, with residues 54 to71 linking the externalized portion with the inner capsid sur-face; specifically, according to the model, the amino terminusof VP1 extends through a small opening between adjacent VP1subunits, bridges the canyon, and emerges at the propeller tip(Fig. 1) (8). The amino-terminal 31 residues were proposed tobe mobile or disordered but located near the propeller tip, far

* Corresponding author. Mailing address: Department of Chemistryand Biochemistry, Brigham Young University, C100 BNSN, Provo, UT84602. Phone: (801) 422-9163. Fax: (801) 422-0153. E-mail: [email protected].

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from the 5-fold axis (8). Uncoating is accompanied by addi-tional conformational changes in the virus particle that arereflected by the presence of at least three forms of the emptycapsid, or 80S particle (6, 34).

Cryogenic electron microscopy (cryo-EM) of antibody-la-beled macromolecular complexes provides a way to mapepitopes, (e.g., see references 5, 13, 31, 42, and 45). Antibodiesare mixed with the complex, and the resulting combination isimaged, followed by three-dimensional (3D) reconstruction.Monoclonal or monospecific antibodies are preferred becausethey provide specificity for the selected epitope, which givesconsistency in the 3D reconstruction. The use of antigen-bind-

ing fragments (Fabs) is preferred to avoid formation of aggre-gates (e.g., immunoprecipitation) and to avoid large masses ofdisordered protein that add noise and interfere with orienta-tion finding (from antibodies binding to only one epitope).After a 3D reconstruction of the Fab-labeled complex is com-puted, atomic coordinates of the antigen and Fab are fittedinto the 3D density. (Usually, coordinates of any Fab are suf-ficient.) In this way, an epitope can be localized on the surfaceof the complex. Conformational changes can be inferred if theepitope in question is exposed in one state but not another orif the epitope changes position.

To understand the structural rearrangements associatedwith the amino-terminal residues of VP1, we determined 3Dreconstructions from cryo-EM images of peptide 1 (P1) Fab-labeled 135S and 80S particles. Polyclonal antibodies raisedagainst P1 recognize amino acid residues 24 to 40 of VP1 (11).The epitope monospecificity of these antibodies allows thelocation of the P1 epitope to be tracked as the virus particleundergoes the conformational rearrangements associated withcell entry and genome uncoating. We found that the P1epitope is located on the propeller tip of the 135S particle,which is consistent with the previously modeled position of theamino terminus of VP1 (8). The P1 Fab binds to two sites onthe 80S particle (one at the propeller tip, one at the 2-foldaxis), and individual particles were found to have Fabs boundto one of the two sites or to both sites.

MATERIALS AND METHODS

Preparation of viruses, 135S particles, 80S particles, P1 Fabs, and virus-Fabcomplexes. Poliovirus virions (160S particles) were grown in a suspension ofHeLa cells, harvested by centrifugation and freeze-thawing, and purified by CsCldensity gradient centrifugation as described previously (15, 39). Altered 135Sparticles were prepared via heat treatment (8, 15). Briefly, a solution of 160Sparticles was diluted 5-fold in prewarmed low-salt buffer (20 mM Tris, 2 mMCaCl2, pH 7.5) and was incubated at 50°C for 3 min (the sample was placed inice water after incubation). Antiserum raised against the VP1 P1 peptide wasaffinity purified to remove antibodies against keyhole limpet hemocyanin (KLH),the carrier protein to which the peptides were conjugated for the immunizations(11). P1 Fab was prepared from the affinity-purified antibodies by dialysis in 20mM phosphate buffer and 10 mM EDTA (pH 7.0) and the ImmunoPure Fabpreparation kit (Pierce, Thermo Fisher Scientific, Rockford, IL).

In our experiments, we used heat to induce the change from the native (160S)particle to cell-entry intermediate (135S) and RNA-released (80S) particles.135S particles produced by heat or obtained from infected HeLa cells (i) infectcells normally nonpermissive for poliovirus infection by 160S particles, (ii) havelost VP4, (iii) are similarly sensitive to proteolysis, and (iv) similarly react withantibodies to the amino terminus of VP1 (15). 80S particles produced by heatshare antigenicity, sedimentation properties, and protease sensitivity with emptyparticles that accumulate within cells (after genome uncoating during cell entry)and, like those particles, have externalized both VP4 and genomic RNA.

Solutions of 135S particles (0.6 mg/ml) and P1 Fab (0.8 mg/ml) were mixed atroom temperature with an antibody-to-virus ratio of 600:1 (mole:mole). Thirty-four percent of the imaged particles in the 135S sample were 80S particles (cf.reference 15). 135S and 80S particles were readily distinguished by the presenceand absence of density corresponding to the viral RNA, respectively. These 80Sparticles were used to solve the structure of the 80S-P1 complex.

Electron microscopy. Thin films containing poliovirus complexed with P1 Fabswere suspended over holey carbon films, vitrified, and imaged as describedpreviously (50). A CM200 electron microscope (FEI, Hillsboro, OR) equippedwith a Gatan 626 cryogenic transfer holder (Pleasanton, CA) was used to recordfocal pairs of micrographs at magnifications of "38,000 at 120 kV.

3D reconstruction. Particle images were extracted, processed, and normalizedas described previously (2). Focal settings ranged from 0.81 to 2.24 #m under-focus. Focal-pair images were computationally combined for orientation deter-mination but used separately for origin determination and computation of the3D reconstruction. A previous reconstruction (2) or a model determined via

FIG. 1. Structural features of 160S and 135S particles. (Top) Prom-inent structural features on the exterior of the 160S poliovirus particle(2). Fivefold (pentagon), 3-fold (triangle), and 2-fold (oval) symmetryaxes are labeled. A second 3-fold axis is labeled to show an asymmetricunit, which is the triangle formed with the 5-fold and two 3-fold axesas vertices. (The line connecting 3-fold axes passes through the 2-foldaxis.) The asymmetric unit is the unique portion of the structure. Therest of the structure (59 other equivalent portions) is made by sym-metry operations. (Bottom) Close-up view of four prominent struc-tural features on the exterior of the 135S poliovirus particle (8), withone symmetry-related copy of the “propeller tip” and “bridge” labeled.The predicted helices in the canyon are residues 42 to 52 from theamino terminus of VP1 (black wire diagram). The gray net is acryo-EM reconstruction. For both 160S and 135S particles, the mesa isformed solely by VP1, and the canyon and propeller are formed byVP1, VP2, and VP3. The propeller tip is formed by the EF loop (loopbetween E and F !-strands) of VP2 and flanking polypeptide se-quences from VP1 and VP3. Each mesa is centered on a 5-fold sym-metry axis, and each propeller is centered on a 3-fold symmetry axis.

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common lines (19) was used to begin iterative model-based determination oforientations and origins. Origins and orientations of the extracted particles weredetermined via the model-based technique with the PFT2 program (1), whichwas adapted to use phase and amplitude information in orientation selection(40). 3D reconstructions were calculated via the EM3DR2 program (14, 19).Icosahedral symmetry was applied during the reconstruction computations andorientation searches. Bsoft routines were employed to assess and correct forcontrast transfer function (CTF) effects (25) by use of the algorithm by Conwayand Steven (12), but images within a focal pair were not combined during CTFcorrection. Spherically averaged density plots were used to calibrate size (3, 25)against previously calibrated structures (2) and a poliovirus X-ray crystal struc-ture (46). 3D reconstructions were displayed at contour levels designated by thenumber of standard deviations ($) above the average map density.

Classification of particles. Our initial 80S-P1 reconstruction showed two P1Fab-binding sites, and we sought to separate particles by binding site. Using theUniversity of California, San Francisco (UCSF), Chimera package (37), wedeleted Fabs via the volume-eraser function, leaving structures with Fabs in onlyone type of site. The modified maps served as the starting models for multiple-model-based classification, using a method similar to that used by Heymann et al.(26). Each particle image was compared to a model projection from each state.For a particle to be selected, a minimum correlation coefficient value of 0.30 (forhigh- and low-pass Fourier filters of 90%1 Å%1 and 21%1 to 16%1 Å%1, respec-tively) was used, as was a minimum difference (range, 0.005 to 0.010) between thecorrelation coefficients obtained from each form; i.e., a selected particle had tohave a correlation coefficient value of 0.30 or greater, and that value had to be0.005 to 0.010 higher than the value for the other 80S-P1 state. Fifty percent ofthe total number of particles was assigned to one of the two forms. By using themultiple-model-based classification method, we also attempted to classify the80S particles into three groups: one with Fab bound at the propeller tip, one withFab bound at the 2-fold axis, and one with Fab bound to both binding sites.

The Fab density in the 135S-P1 reconstruction was very low. We attribute thisto a combination of low occupancy due to the relatively low affinity of the Fab forthe 135S particle and to a reduction of density in the icosahedrally averagedreconstructions due to the flexibility of the epitope in the 135S particle (8). A135S map (2) and the original 135S-P1 map were applied to classify the 135Sparticles in the same manner as that used for the two forms of 80S-P1 (describedin the previous paragraph). However, in this case, the classification values wereused only to determine which particles from the previous (weak P1 Fab) setwould be used to compute the final reconstruction. Orientations and originsdetermined previously—without classification—were applied.

One-particle reconstruction. To assess Fab binding to individual particles,one-particle reconstructions (e.g., see reference 9) were computed to a limit of40-Å resolution. (A close-to-focus and far-from-focus image pair was used tocompute each one-particle reconstruction.) Icosahedral symmetry was appliedduring computation of the one-particle reconstructions, as it was in the multiple-particle reconstructions. Central slices of these reconstructions were observed toassess whether Fabs were present and, if so, where the Fabs were bound.

Modeling. C3 Fab coordinates (47) were used to model the virus-P1 interac-tion. Coordinates were fitted by eye into 135S-P1 and 80S-P1 maps via the UCSFChimera package (37).

RESULTS

We used antipeptide antibodies to the amino terminus ofVP1 to locate this peptide in two key cell-entry intermediates.Purified virions were heated at 50°C to produce a mixture of135S and 80S particles, and the particles were mixed with Fabfragments of a monospecific antibody raised against a peptide(P1) corresponding to residues 24 to 40 of VP1 (11).

Micrographs (i.e., same fields of view) contained complexesof the Fab with both 135S and 80S particles. These two parti-cles were easily distinguished by the lack of internal densitycorresponding to the viral RNA in the 80S particles. The twoforms were separated during particle image extraction (“box-ing”) and used to compute separate 3D reconstructions.Therefore, the imaging conditions for the 135S-P1 and 80S-P1complexes are identical and are not responsible for the ob-served differences between the complexes.

We computed the 3D structures of the 135S-P1 and 80S-P1

complexes by cryo-EM and 3D image reconstruction (Table 1;Fig. 2 to 4). Estimates for the resolutions of the two structuresbased on Fourier shell coefficient analysis (using a 0.5 cutoff)were 12 Å for the 135S-P1 complex and 13 Å for the initial80S-P1 complex (two other 80S-P1 reconstructions were cal-culated to 18-Å resolution; see section “Two P1 Fab-bindingpositions in 80S-P1 complex” below). The density correspond-ing to the Fab was weak in all reconstructions. However, con-tinual density corresponding to the Fab extends outwards fromthe capsid with no evidence of a contrast fringe (e.g., the darkregion immediately outside the capsid in Fig. 4A), which oc-curs at contrast edges in underfocused images at low resolu-tion. For comparison, we note that a reconstruction of thevirus-receptor complex, where the receptor is known to bebound at high occupancy, also has continuous density extend-ing from the virus particle into the receptor (Fig. 4D). Wetherefore conclude that the Fab fragments are bound and thatthe weak density can be attributed to low occupancy, flexibilityof the peptide epitope, overlapping monospecific Fabs, or acombination of these factors. To make Fab density easier tosee, we also computed 3D reconstructions for both complexesat lower resolution (26 Å for the 135S-P1 complex, 21 Å for theinitial 80S-P1 complex).

Because of conformational dynamics or “breathing”, theamino terminus of VP1 (as well as that of VP4) is transientlyand reversibly exposed in 160S particles, and this exposure istemperature dependent (35, 38). P1 Fab binding to 160S viri-ons is, therefore, also temperature dependent—occurring to asignificant extent at 37°C but not 25°C (11, 18, 35). In contrast,P1 antibody readily binds to 135S and 80S particles over a widetemperature range, indicating the exposure and accessibility ofthis sequence on these particle surfaces (11, 18).

TABLE 1. Microscopy and image reconstruction data

Sample Particleimagesa

Resolutionb

(Å)

EMDataBank

identifier

135S-P1 2,110c 26d 528210,160c 12 5280

80S-P1 (both binding sites) 5,256 21d 528413 5283

80S-P1 (propeller tip only) 1,132 18 528680S-P1 (2-fold only) 1,814 18 5285

a Total number of images; divide by two for the number of image pairs (focalpairs). These images were extracted from seven focal-pair micrographs, wherethe first image in the pair was taken closer to focus and the second image wastaken farther from focus. The pixel size for the reconstructions was 1.83 Å. Forthe orientation and origin determination, images were corrected only for phase-flipping effects of the CTF. For the final, published reconstruction, images werefully deconvoluted for CTF effects and a signal-decay correction was also applied(see Materials and Methods section).

b Determined by the point at which the Fourier shell correlation value reaches0.5. For the Fourier shell correlation test, images were corrected only for thephase-flipping effects of the CTF.

c The difference in the number of particles used is attributable to classificationof particles that matched best with a model containing an Fab on the propellertip (2,110) against a model without an Fab bound (see Materials and Methods).The other reconstruction (10,160) was from iterative model-based orientationand origin refinement against all 135S particles in the data set. In the latter, allparticles that matched well were included.

d Lower-resolution versions of the 12-Å 135S-P1 and 13-Å 80S-P1 maps werecomputed so that the bound Fabs could be seen more easily.

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One P1 Fab-binding position in 135S-P1 complex. On the135S particle, P1 Fab binds to a single position within theasymmetric unit (Fig. 1 and 2A and B). Fab density connectsfrom the propeller tip outwards. In addition to having lowoccupancy (&16%; Fig. 4C), the reduced size of the Fab do-mains at this contour level (Fig. 2A and B) suggests that res-idues 24 to 40 of VP1 are flexible in the 135S particle or thatmonospecific Fabs bind to slightly different amino acid resi-dues (the section “Fab epitope flexibility and Fab occupancy”in the Discussion explains factors contributing to the small sizeand low density of the Fabs).

Two P1 Fab-binding positions in 80S-P1 complex. Inspec-tion of the initial 80S-P1 map showed that P1 Fabs bound totwo distinct areas on the capsid (Fig. 3A and 4A). The P1 Fabat the propeller tip in the 80S-P1 structure bound to the samesite as in the 135S-P1 complex. A second Fab bound at the80S-P1 2-fold axis. We used classification methods to deter-mine whether the two states were found on the same particlesor if particles were in one state or the other.

We found that 80S-P1 particles with Fabs bound to thepropeller tip or 2-fold axis could be separated by the positionof the bound Fab (Fig. 3B, C, and D and 4B). Therefore, thepopulation of 80S-P1 particles exists in at least two states, withthe P1 epitope located at one of the two binding sites: thepropeller tip (Fig. 3B; 3D, left; and 4B, left) or the 2-fold axis

(Fig. 3C; 3D, right; and 4B, right). Although we cannot rule outthe possibility that some “cross-contamination” in the propel-ler tip and 2-fold axis data sets exists, the Fab density for eachwas clearly dominant in one form or the other (Fig. 3B and C).

In the particles with Fab at the 2-fold axes, the Fab-relateddensity shows a direct connection to the 2-fold symmetry axesof the capsid (Fig. 3D). The density for the Fab at the 2-foldaxes is 2-fold symmetric and is closer to the size of an Fab thanis the density at the propeller tips of either the 135S-P1 or the80S-P1 structures. Note, however, that the density for the con-stant domain is larger than needed, indicating either that thereis flexibility in the epitope being bound, that there are over-lapping monospecific antibodies, or that the two copies of theepitope at this site (which could be contributed by either of two2-fold-axis-related copies of VP1) are oriented slightly differ-ently.

To determine whether P1 epitopes can simultaneously oc-cupy both locations on the same particle, we attempted to usecorrelation-based classification to separate the 80S-P1 particlesinto three classes: propeller-tip-bound Fab, 2-fold-axis-boundFab, and Fab bound to both. However, the number of Fabsbound per particle was relatively low (Fig. 4A and B), whichgave little contribution from bound Fab in the noisy particleimages and thus prevented clear discrimination among thesethree classes. (Note that the reconstruction showing Fabs

FIG. 2. Stereo views of reconstructed 135S and 135S-P1 complex. (A) 135S-P1 complex (resolution, 26 Å) reconstruction at a contour level of0$. Fab coordinates (cyan) are shown with the variable domain fitted into the P1 density. The fitting here provides only an estimate of the averageFab position. In the Fab variable domain, the volume occupied by the coordinates is significantly larger than the cryo-EM density, indicating thatthe epitope is flexible or that polyclonal Fabs overlap. (B and C) Coordinates (wire diagram) of the 135S model (8) (Protein Data Bank accessionnumber 1XYR) fitted in the 135S-P1 (B) and 135S (C) (8) maps (fine mesh). Coordinates for VP1 (blue), VP2 (yellow), VP3 (red), and residues42 to 52 of the amino terminus of VP1 (green) are shown.

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bound to both sites [Fig. 3A and 4A] was an average structureof all selected 80S particles prior to classification.)

Because the classification experiment failed to give a con-clusive answer, we decided to compute one-particle recon-structions. One-particle reconstructions are 3D reconstruc-tions computed from one image of a particle. Although 3Dreconstructions require multiple views of an object, one-parti-cle reconstructions are possible for objects with icosahedralsymmetry because one view has 59 other symmetry-relatedviews. We computed one-particle reconstructions to only 40-Åresolution because of the high noise level that arises from thelimited averaging implicit in one-particle reconstructions. Sev-eral reconstructions showed Fabs bound to both the propellertips and 2-fold axes of 80S particles (Fig. 5C). Other structures

showed Fabs bound to either the propeller tips or 2-fold axes(Fig. 5A and B). Thus, these reconstructions demonstrated theexistence of 80S particles where P1 epitopes are located atboth the propeller tips and 2-fold axes simultaneously.

DISCUSSION

In part because crystallization of picornavirus cell-entry in-termediates has been unsuccessful, cryo-EM studies provideunique potential for structurally characterizing the entry pro-cess (e.g., see references 2, 4, 6, 8, 21–24, 32, 34, 36, 48, 49).During viral entry, receptor-mediated conformational changesresult in the externalization of the myristoylated protein VP4and the amino-terminal extension of VP1 (18), both of which

FIG. 3. Views of reconstructed 80S-P1 complexes. (A to C) Stereo views of 80S-P1 complex reconstructions. P1 Fabs bind 80S capsid (white)at the propeller tips (dark gray) and 2-fold axes (light gray). (A) The unclassified 80S-P1 (resolution, 21 Å) reconstruction at a contour level of0.2$. (B and C) Reconstructions of 80S-P1 complexes (resolution, 18 Å) with Fab bound to the propeller tip (B) and with Fab bound at the 2-foldaxis (C), both at a contour level of 0.5$. (D) Fab coordinates (47) (ribbon structures) fitted into the two different binding sites in the 80S-P1 map(mesh). The Fab bound to the propeller tip is shown in the left panel. The Fab bound at the 2-fold axis is shown in the right panel. In both cases,the density in the antigen-binding domain (of the Fab) was not complete. The fitting shown here provides only an estimate of where the Fabs mightbe positioned. The constant (non-antigen-binding) domain of the Fab bound at the 2-fold axis is larger than the volume occupied by the ribbonmodel. The extra density likely results from 2-fold positioning of the epitope (but only one Fab can bind at a time), the monospecific nature ofthe antibody, or the flexibility of the constant domain.

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become associated with cellular membranes (16, 18, 43). Insubsequent steps, unknown triggers result in the externaliza-tion of the viral RNA and its translocation across a membraneto gain access to the cytoplasm for replication (see references27, 33, and 44).

Because the externalized peptides are thought to play a rolein internalization, genome release, and translocation of thegenome across membranes (27, 33), knowledge of the locationof the externalized portion of VP1 is key to the refinement ofmodels for the entry process. A previously published study ofthe 135S particle and a 135S particle in which the first 31residues of VP1 were removed by proteolysis suggested thatthe amino terminus of VP1 is located at the propeller tip (8).Three-dimensional reconstructions of the 80S particle also sug-gested that the amino terminus of VP1 is located at the tips ofthe propeller in some but perhaps not all particles (34) (see thesection “Amino terminus of VP1 makes conformationalchanges during uncoating” below). However, in the previousstudies, the 135S and 80S reconstructions lacked density forthe amino terminus of VP1 (presumably because it is flexiblytethered to the virion). In this study, we took advantage of theavailability of antibodies against the amino-terminal extensionof VP1 to visualize its location in the 135S and 80S particles.The results here are consistent with those of the previousstudies, indicating that P1 epitopes (and, hence, the aminotermini of VP1) are located at the propeller tips of 135S and80S particles. However, our results show that this peptide canalso be located at 2-fold axes in the 80S particles. Surprisingly,these results also indicate that copies of the same peptidesequences in some cases can occupy two different positions inthe same 80S particle, which represents a significant breakfrom icosahedral symmetry for these particles.

Specificity of P1 antibody. To infer the locations of the P1peptide on the basis of the sites of Fab binding, the mono-specificity of the P1 antisera is essential. In other words, anti-bodies raised against peptide P1 must recognize only aminoacid residues 24 to 40 of VP1 and not bind nonspecifically toother regions of the 80S or 135S particles. In a previous char-acterization of the P1 antibody used in the present study,anti-P1 sera recognized the P1 peptide (VP1 residues 24 to 40)and capsid protein VP1; other peptides from VP1 and theproteins VP2 and VP3 were not recognized (11). Recognitionby anti-P1 sera was blocked specifically by competition withsoluble P1 and not other VP1 peptides (11). In addition, beforeits use in the present study, the antiserum was affinity purifiedwith the P1 peptide (see Materials and Methods). Although wedo not know whether Fab binding at the propeller tip and2-fold axes of the 80S particle involves identical amino acidswithin the P1 peptide, the characterized reactivities of the P1antisera indicate that the Fabs bind only to P1 peptide se-quences. Thus, we strongly favor the interpretation that theFab-80S complexes reported here represent Fab binding to P1sequences located in two different sites (the propeller tips and2-fold axes).

Fab epitope flexibility and Fab occupancy. Density corre-sponding to the amino terminus of VP1 was not apparent inthe earlier 135S (8) and 80S (34) density maps, presumablybecause the domain has a flexible, i.e., inconsistent, structure.If true, then signs of domain flexibility may be evident if Fabsare bound. Several characteristics of the 135S and 80S density

FIG. 4. Binding of P1 Fab to poliovirus. Central slices from densitymaps of P1 Fab complexed with 80S particle (A and B) and 135Sparticle (C) and poliovirus (160S form) complexed with poliovirusreceptor (Pvr) (4) (D). The highest densities are white. Arrows indi-cate P1 Fab-related density: Fabs bound at the propeller tip (unfilledarrowhead) or 2-fold axis (filled arrowhead). The 80S-P1 complexmaps were computed from unclassified (A) and classified (B) particles.(A) Left, resolution of 21 Å; right, resolution of 13 Å. (B) Left, Fabbound to the propeller tip; right, Fab bound at the 2-fold axis (bothstructures at 18-Å resolution). (C) 135S-P1. Left, resolution of 26 Å(from classified particles); right, resolution of 12 Å (from unclassifiedparticles). For relative density estimates, a sphere with a radius of 3pixels was centered on the designated spots (A, right) and the averagedensity was computed. In the unclassified 80S-P1 map at a resolutionof 21 Å (A, left), P1 Fab at the propeller tip (unfilled arrowhead) has15% of capsid density and P1 Fab at the 2-fold axis (filled arrowhead)has 24%. In the classified 80S-P1 maps at a resolution of 18 Å (B), P1Fab bound to the propeller tip (unfilled arrowhead) has 25% of capsiddensity (B, left) and P1 Fab bound at the 2-fold axis (filled arrowhead)has 23% (B, right). In the 135S-P1 map at a resolution of 21 Å (C, left),P1 Fab has 16% of capsid density. (Insets) Smaller, unlabeled versionsof each panel, given to allow the faint Fab density to be seen moreeasily. (D) Control image showing Pvr attached to poliovirus 160Sparticle (4). Bars, longer bar for larger images, shorter bar for insetimages. (Note that a faint bit of density appears on the 2-fold axes inpanel B, left. This faint density does not appear in the surface render-ing in Fig. 3B and therefore is much lower than the density observedat the propeller tip. We attribute this faint density to errors in classi-fication and to artifacts that often appear along symmetry axes inreconstructions.).

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maps shown here provide clues suggesting that the amino-terminal domain of VP1 is indeed a flexible domain.

Because 3D reconstructions of freestanding particles areaverages of multiple particles that are assumed to be similar,lower average density within subvolumes must indicate loweroccupancy, flexible domains, or both. If the low-density sub-volume has the full shape of a protein domain, then the do-main must be rigidly positioned but have lower occupancy (i.e.,not all positions in all particles have the domain). If the lower-density region is smaller or larger than the full shape of thedomain, then the domain must be flexible or have multiplerigid conformations. Overlapping regions will have the highestdensities. The higher the occupancy, the larger a flexible do-main will appear. Hence, observation of a small low-densityportion of the Fab volume at the propeller tip in 135S-P1 (Fig.2A) or 80S-P1 (Fig. 3A, B, and D, left) indicates not only lowoccupancy by the Fab but also that the P1 Fab at the propellertip is located in multiple positions in the 135S and 80S parti-cles.

If some parts of a structure have higher resolution thanother parts, then the lower resolution is another indication offlexibility. We observed this pattern for the 135S-P1 and80S-P1 maps. Sections show a well-resolved capsid with fuzzy,poorly resolved density outside the capsid where Fab is bound(Fig. 4A and C, right panels). “Local” resolution tests (reso-lution of small subvolumes of the reconstruction) confirmedthe observation of higher resolution in the capsid and lowerresolution in the Fab regions (data not shown).

Each voxel (pixel volume) in a 3D reconstruction shows theaverage density (at the given x-y-z position) for the particlesused. Therefore, density averages will be more diffuse for flex-ible components than for rigid components. Furthermore, thehigher the resolution of a 3D map, the more unlikely that

FIG. 5. One- or eight-particle reconstructions of 80S-P1 com-plexes. (A to C) Central slices of one-particle reconstructions of80S-P1 complex with P1 Fab bound at the propeller tip (A), at the2-fold axis (B), and at both binding sites (propeller tip and 2-fold axis)(C). Arrowheads, Fab bound at the propeller tip; asterisks, Fab boundat the 2-fold axis. (D) Central slices of eight-particle reconstructions of80S-P1. The same particle images that were used to make the one-particle reconstructions in panels A to C were combined to give thestructures seen here. From left to right, eight-particle reconstruction of80S-P1 with P1 Fab bound at the propeller tip, eight-particle recon-struction of 80S-P1 with P1 Fab bound at the 2-fold axis, mixed eight-particle reconstruction (four particles of 80S-P1 with P1 Fab bound atthe propeller tip and four particles of 80S-P1 with P1 Fab bound at the2-fold axis), and eight-particle reconstruction of 80S-P1 with P1 Fabbound at both binding sites. (E) Control structures. Central slices ofone-particle reconstructions from a different poliovirus-antibody (C3)complex (top row) (J. Lin et al., unpublished data) and from thepoliovirus-Pvr complex (bottom row) (4). The left three panels in eachrow are one-particle reconstructions, and the rightmost panel is aneight-particle reconstruction. The one-particle reconstructions showbound Fab (top row) or bound receptor (bottom row). The C3 anti-body binds very tightly to poliovirus 160S, 135S, and 80S particles (18),and Fabs were found to bind to the tips of the mesa (Lin et al.,unpublished). Pvr binds to one side of the propeller tip and bridges thecanyon (4). The monotone gray circular center seen in some slices wasadded artificially to mask very high positive or negative density valuesat the center of the 3D reconstruction. Extreme values at the centerare common artifacts in one-particle, icosahedral reconstructions.

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flexible regions will be observed. If the 3D reconstruction iscomputed at lower resolution, flexible regions will be moreapparent. This was the case with our 135S-P1 and 80S-P1maps; Fab density was more easily observed at 26- and 21-Åresolutions, respectively, than at 12- and 13-Å resolutions, re-spectively (Fig. 4A and C). Even at the lower resolutions, Fabdensity was seen at relatively low contour levels in surfacerenderings (Fig. 2A and B and 3A) and faintly in densitysections (Fig. 4A and C).

Because the Fab density is weak, are Fabs actually bound oris the weak density an artifact? Continual density extendingoutwards from the capsid is additional evidence that Fabs arebound (Fig. 4). In many places, a dark region is located im-mediately outside the capsid density (e.g., Fig. 4A). This re-gion is an effect of defocusing (specifically, underfocusing)that is routinely performed during cryo-EM to enhance con-trast. In incompletely corrected two-dimensional images and3D image reconstructions, defocusing causes a fringe ofintensity of opposite contrast at edges. No fringe is found atthe Fab-binding sites, indicating the presence of continuousdensity. A similar connection was observed for receptor-bound particles (Fig. 4D).

Monoclonal versus monospecific antibodies. Although thedensity characteristics of the structures shown here are consis-tent with those of a flexible amino-terminal domain (of VP1),the appearance of flexible epitopes could also be explained bythe use of monospecific, not monoclonal, antibodies. The pop-ulation of Fabs binding to the P1 region from the monospecificresponse could recognize slightly different epitope subsets ofthe P1 sequence. In this situation, the average structure ob-served in the reconstructions would then have the appearanceof a flexible epitope that had a monoclonal antibody bound.

Our observation of two binding sites in 80S particles suggeststhat monospecific antibodies—to a short peptide—may bevaluable probes for assessing conformational changes. For 3Dcryo-EM reconstructions, monoclonal antibodies are usuallypreferred over monospecific antibodies because monoclonalantibody binding would unambiguously identify the epitopelocation. However, because antibody binding is also dependenton the epitope conformation, a monoclonal antibody thatbinds to an epitope in one conformation may not recognize thesame amino acid residues in a different conformation. In thissituation, one might erroneously conclude that the epitope wasnot exposed on the particle surface. The polyclonal nature ofmonospecific antibodies raised against short peptides allowsrecognition of the peptide epitope in a variety of conforma-tions and increases the probability that the epitope can betracked as a virus particle transitions through different confor-mational states. Thus, monospecific antibodies to short pep-tides may recognize conformational differences better thanmonoclonal antibodies and still provide enough specificity forstructurally consistent 3D density maps. Conversely, because ofoverlapping binding regions, the appearance of a flexibleepitope may always result from the use of monospecific anti-bodies.

Amino terminus of VP1 is located near the propeller tip in135S and 80S particles. The 3D reconstructions of 135S-P1and 80S-P1 (Fig. 2A and B, 3A and B, and 3D, left) show thatP1 Fab binds to the propeller tip in 135S and 80S particles, withP1 Fab bound at the propeller tip alone or at both binding

sites. These observations are consistent with existing models of135S (8) and 80S (34) and with a difference map of 135S minus135S'N31 (8), which placed the P1 epitope (residues 24 to 40[11]) near the propeller tip. For example, residues 42 to 52 ofVP1 (modeled as an ( helix) are close to the propeller tip (Fig.1, bottom, and Fig. 2B and C), and difference density wasobserved at the propeller tip (8).

Nonsynchronous changes in 80S particles. As with manyother nonenveloped viruses, poliovirus capsid proteins are or-ganized to form an icosahedrally symmetric shell. An impor-tant question in icosahedral virus assembly and disassembly iswhether all copies of pertinent protein domains participatesynchronously in the conformational changes associated withthese processes and whether significant deviations from icosa-hedral symmetry can exist within an individual particle duringconformational transitions. Because cryo-EM structures areaverages of many particle images, structural features that areconveyed at lower resolution than the rest of the structureare likely caused by conformational variability among the pop-ulation of particles. But, for symmetric particles such as polio-virus, these differences might be attributable to variations be-tween particles that, individually, are symmetric. Here, weprovide direct evidence that significant deviations from icosa-hedral symmetry—for portions of capsid proteins—do existwithin the same particle. Such deviations may be biologicallysignificant during viral transitions.

The observation of epitopes in two concurrent positionsshows that significant portions of the capsid proteins within theicosahedral shell may have deviations from icosahedral sym-metry and, hence, do not change conformation synchronously.80S-P1 particles with both Fab-binding sites (Fig. 5C and D,right) show that the amino termini of VP1 are simultaneouslylocated at two different sites (the propeller tips and 2-foldaxes) on the same particle. This 80S state may indicate thatduring uncoating, the corresponding conformational change inthe capsid is a gradual process. In other words, for an individ-ual particle, the 60 copies of the amino terminus of VP1 arenot required to change their conformations at the same time orin the same way.

As stated, the Fabs used in this experiment were monospe-cific and not monoclonal. Therefore, although all P1 Fabs bindwithin residues 24 to 40 of VP1, the Fabs bound at the pro-peller tips and 2-fold axes may recognize different amino acidresidues within the same P1 epitope. However, the straight-linedistance between antigen-binding sites on the propeller tip andthe 2-fold axis is 52 ()1) or 62 ()0.4) Å for the two closest2-fold axes (measurement is based on placement of Fab coor-dinates into the Fab density; e.g., Fig. 3D). Assuming that (i)each residue occupies a linear distance of 3.8 Å along thepolypeptide chain, (ii) residues 24 to 40 are not in a straight-line conformation, and (iii) each Fab binds several residueswithin the P1 peptide, P1 Fab binding to the propeller tips and2-fold axes likely cannot result from two Fabs binding to dif-ferent ends of the P1 epitope from one VP1 subunit. Theseemingly simultaneous binding of P1 Fabs to different sites onthe same 80S particle (in the icosahedrally averaged structure)most likely occurs because P1 epitopes from different VP1subunits lie at a 2-fold axis or at a propeller tip.

We hypothesize that deviations from icosahedral symmetryare more likely to occur in extensions of the polypeptide chains

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(e.g., amino termini, carboxy termini, or large loops) that arenot critical to interactions within the core shell structure. Forexample, the 53-residue amino terminus of VP1 could lie indifferent conformations on the capsid surface without neces-sarily disturbing interactions between the !-jellyroll cores ofVP1, VP2, and VP3, which form the shell. In contrast, if in-teractions of cores in one portion of the capsid are altered, wehypothesize that these changes would be more likely to prop-agate rapidly throughout the capsid shell. In our case, thesymmetry-averaged cores of the VP1 to VP3 subunits wereresolved at 13 Å or better, indicating that the core structures of80S particles were well-ordered.

Amino terminus of VP1 makes conformational changes dur-ing uncoating. The transition of VP1 residues 24 to 40 from thepropeller tip to the 2-fold axis appears to be a consequence ofthe uncoating process. Besides having the same binding site asin the 135S-P1 complex, P1 Fabs bind to a second site in80S-P1, at the 2-fold axis, indicating either that conformationalrearrangements occur during uncoating or that an additionalvariable, characteristic of the multiple-form 80S particle (6,34), exists. Because only one P1 Fab at a time can bind toresidues 24 to 40 of VP1 (see previous section), our resultsshow that 80S particles contain the epitope in one or both oftwo locations.

The observation that peptide 1 (P1) changes position andperhaps conformation during the 135S-to-80S transition is con-sistent with previously published immunoprecipitation experi-ments that showed a change in the amino terminus of VP1between these two states. For 80S particles, titers of P1 mono-specific and monoclonal antibodies and a monospecific anti-body against peptide 0 (P0; residues 7 to 24) differed signifi-cantly from the corresponding titers for 135S particles (18).The transition from the 135S particle to the 80S particle alsoinvolves the externalization of the RNA genome of the parti-cle, and, interestingly, the new position for the P1 peptide isvery close to the position where RNA was shown to be releasedfrom the particle (6), suggesting that the movement of thepeptide may play a role in RNA release (often referred to as“uncoating”) during infection.

Previous studies have shown at least three distinct forms ofheat-derived 80S particles (6, 34). In these studies, the 80Sparticles from preparations heated at 56°C for shorter timeshad increased numbers of particles in the conformation la-beled the “early” form (80S.e). Conversely, samples heated forlonger times showed increased numbers of the “late” form(80S.l). Because both 80S.e and 80S.l were seen by Levy et al.(34) at all time points, even their 4-min incubation, the 80Sparticles in this study are also likely in these two states. Oursample was prepared at a lower temperature (50°C) for arelatively short time (3 min). These conditions are typicallyused to produce 135S particles, but even this mild treatmenttypically produces a significant number of 80S particles (15).

We compared the 80S.e and 80S.l maps (34) to our 80Sstructures but were unable to make satisfactory distinctions;i.e., the correlation coefficients were nearly identical. Never-theless, Levy et al. (34) showed differences between 80S.e and80S.l in a density feature that is ascribed to a predicted helix,spanning VP1 residues 41 to 53 in the 135S structure (8). If thissequence assignment is accurate, it would place the residuescorresponding to the P1 epitope at the tip of the propeller, as

observed in this study (Fig. 2 and 3) and a previous study (8).Density for the helix is weaker in the 80S.e reconstruction thanhelix density in the 80S.l or 135S reconstructions, implying thatsome fraction of the helices (and perhaps the P1 epitopes aswell) have moved in the 80S.e particles. The moved segmentsmay be relocated at the 2-fold axes and, therefore, would beresponsible for the Fab binding at the 2-fold axes that weobserved. Both 80S.e and 80S.l reconstructions had holes inthe shell that suggested that the viral RNA may be releasednear a 2-fold axis (34). A third 80S intermediate actuallyshowed RNA leaving the capsid near the 2-fold axis at a sitethat corresponded to these holes (6). Because our experimentwas carried out under conditions different from those used byLevy et al. (34), we cannot rule out the possibility that ourresults represent additional conformations of 80S particles notseen in the previous studies (6, 34).

Canyon-exit model. During cell entry, native poliovirus(160S) binds to its receptor, which stimulates conversion to the135S cell-entry intermediate particle (18). During this transi-tion, the membrane-binding entities, VP4 and the amino ter-minus of VP1, are externalized. Externalization of these pro-teins requires conformational adjustments in the capsid. Onthe basis of hypothesized parallels between the uncoating pro-cesses of human rhinoviruses and poliovirus, one model pro-poses that receptor binding induces formation of a channelalong the 5-fold axes and that VP4 and the amino terminus ofVP1 exit the capsid through this channel (20, 22, 32). In analternative (canyon-exit) model, VP4 and the amino terminusof VP1 exit the capsid at the base of the canyon (2, 8). Ourstructures of virus-antibody complexes are consistent with thelatter model.

The 135S-P1 reconstruction reported here supports the“canyon-exit” model by providing more evidence that at least aportion of the externalized amino terminus of VP1 is located atthe propeller tip. The alpha helix proposed for residues 42 to52 of VP1 is very close to the P1 Fab-binding site at thepropeller tip, close to the canyon and the 3-fold axis (Fig. 1 and2B), which is consistent with the P1 epitope (residues 24 to 40)being located nearby and, hence, with the suggestion that theamino terminus of VP1 is at the propeller tip, as modeled byBubeck et al. (8) for 135S particles. Our results are also con-sistent with a difference-density map between V8-protease-cleaved 135S and unmodified 135S particles that showed adifference density at the tip of the propeller (8).

P1 Fab was also bound at the propeller tip in 80S-P1 parti-cles. This is consistent with the model of the 80S structure byLevy et al. (34). The Fab density bound at the propeller tip washighest near the propeller tip (Fig. 2A and B and 3A, B, and D,left), indicating that P1 Fab was most constrained where it isbound to the capsid. The density volume of the P1 Fab boundat the propeller tip is smaller than that of the P1 Fab bound atthe 2-fold axis (Fig. 2A and B and 3), which indicates that theamino terminus of VP1 has more flexibility at the propeller tipthan at the 2-fold axis. The increased flexibility at the propellertip may be necessary for the amino terminus of VP1 to becomeembedded in the membrane to prepare the virus for uncoating.The more rigid 2-fold-axis position may be required for or bea consequence of RNA release.

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ACKNOWLEDGMENTS

This work was supported by BYU institutional funds (to D.M.B.)and by the National Institute of Arthritis and Musculoskeletal and SkinDiseases of the National Institutes of Health (to D.M.B., N.C., andA.C.S.). Jun Lin was partially supported by grant R15AI084085 fromthe National Institute of Allergy and Infectious Diseases (to D.M.B.)and a fellowship from the BYU Department of Chemistry and Bio-chemistry. This work was also supported by NIH grant R01AI020566(to J.M.H.) and grants R01 AI022627 and AI042390 (to M.C.).

We thank Eduardo Sanz-Garcia and Bernard Heymann for com-puter help; Peter Shen, Hazel Levy, and Mihnea Bostina for helpfuldiscussions; Simon Tsang, Norman Watts, and Mario Cerritelli forhelp with Fab production; and Linda Rodriquez for image-processingwork.

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