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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 information and services can be found at:

    These include:REFERENCES This article cites 48 articles, 22 of which can be accessed free

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  • JOURNAL OF VIROLOGY, Oct. 2011, p. 99749983 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 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 withand, in the case of the amino terminusof VP1, tether particles toartificial 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:

    ! Published ahead of print on 20 July 2011.


    on February 21, 2012 by Albert R. Mann Library

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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.


    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 50C 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