JOURNAL OF VIROLOGY, Nov. 2008, p. 10556–10566 Vol. 82, No. 210022-538X/08/$08.00�0 doi:10.1128/JVI.00907-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Foot-and-Mouth Disease Virus, but Not Bovine Enterovirus, Targetsthe Host Cell Cytoskeleton via the Nonstructural Protein 3Cpro�

Hannah Armer,1†‡ Katy Moffat,1† Thomas Wileman,1,2 Graham J. Belsham,1,3 Terry Jackson,1W. Paul Duprex,4 Martin Ryan,5 and Paul Monaghan1*

Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, United Kingdom1; School of Medicine, Institute of Health,University of East Anglia, Norwich, Norfolk NR4 TJU, United Kingdom2; National Veterinary Institute, Technical University of

Denmark, Lindholm, 4771 Kalvehave, Denmark3; School of Biomedical Sciences, The Queen’s University of Belfast,97 Lisburn Road, Belfast, Northern Ireland BT9 7BL, United Kingdom4; and Division of Cell and Molecular Biology,

University of St. Andrews, Irvine Building, North Street, St. Andrews, Fife KY16 9AL, United Kingdom5

Received 1 May 2008/Accepted 13 August 2008

Foot-and-mouth disease virus (FMDV), a member of the Picornaviridae, is a pathogen of cloven-hoofedanimals and causes a disease of major economic importance. Picornavirus-infected cells show changes in cellmorphology and rearrangement of cytoplasmic membranes, which are a consequence of virus replication. Weshow here, by confocal immunofluorescence and electron microscopy, that the changes in morphology ofFMDV-infected cells involve changes in the distribution of microtubule and intermediate filament componentsduring infection. Despite the continued presence of centrosomes in infected cells, there is a loss of tethering ofmicrotubules to the microtubule organizing center (MTOC) region. Loss of labeling for �-tubulin, but notpericentrin, from the MTOC suggests a targeting of �-tubulin (or associated proteins) rather than a totalbreakdown in MTOC structure. The identity of the FMDV protein(s) responsible was determined by theexpression of individual viral nonstructural proteins and their precursors in uninfected cells. We report thatthe only viral nonstructural protein able to reproduce the loss of �-tubulin from the MTOC and the loss ofintegrity of the microtubule system is FMDV 3Cpro. In contrast, infection of cells with another picornavirus,bovine enterovirus, did not affect �-tubulin distribution, and the microtubule network remained relativelyunaffected.

Infection of cells with most picornaviruses leads to a cyto-pathic effect (CPE) characterized by dramatic changes in cellshape and the production of large numbers of membrane ves-icles within the cytoplasm. Early studies focused on the CPEproduced by poliovirus (PV) and coxsackievirus (16, 33, 70)and revealed crystalline arrays of virus in the cytoplasm andviruses associated with, and contained within, membrane ves-icles (7, 10, 43). The origin of vesicles induced by PV has beenthe subject of several studies with various conclusions. High-pressure freezing preparation for electron microscopy showeddouble-membrane vesicles in cells infected with PV (71). Thevesicles are thought to be derived from the endoplasmic retic-ulum (ER), arising from either COPII-coated vesicles or au-tophagic vesicles (6, 32, 63, 66, 71, 73, 74). However, the pres-ence of other organelle markers associated with PV vesicles, atlater time points of infection, suggests that the ER is not thesole contributor of these membranes (66).

In a previous publication (51), we reported that while foot-and-mouth disease virus (FMDV) induced the formation ofmembrane vesicles in infected cells, as observed in PV-infectedcells, there were a number of differences between the effects

observed with these two viruses. FMDV-induced vesicles pre-dominantly had a single membrane and were not found inclusters as reported for PV. Further differences between PVand FMDV replication are seen in studies of virus sensitivity tothe fungal metabolite, brefeldin A, which inhibits the functionof Arf1-GTPase. Brefeldin A completely inhibits PV replica-tion, whereas FMDV replication appears unaffected (15, 22,30, 48, 51, 58). Differences between PV and FMDV are alsoseen in some of the functions of the nonstructural proteins.Studies examining the effects of FMDV, and its nonstructuralproteins, on the host-cell protein secretory pathway revealedthat, unlike the PV 3A protein, FMDV 3A was unable to slowprotein secretion (12, 49). Interestingly, although both FMDV2BC and PV 2BC are able to slow protein trafficking thoughthe secretory pathway, they appear to act at different sites. PV2BC is thought to slow the processing of secretory proteins atthe Golgi body, whereas FMDV 2BC expression leads to re-tention of secretory proteins within the ER (2, 19, 49, 50, 64).Taken together, these observations indicate significant differ-ences in the virus–host cell interactions of PV (an enterovirus)and FMDV (an aphthovirus).

In contrast to the work on membrane vesicles, relativelylittle work has been reported on the effect of picornavirusinfection on the cytoskeleton. However, it is becoming increas-ingly clear that many of these viruses use and modify thecytoskeleton for cell entry, replication, and egress. For exam-ple, Theiler’s virus (a cardiovirus) has been reported to asso-ciate with intermediate filaments (55). PV has been reported toassociate with the cytoskeleton and intermediate filaments (42,

* Corresponding author. Mailing address: Institute for Animal Health,Ash Road, Pirbright, Woking, Surrey GU24 0NF, United Kingdom.Phone: 44 (0)1483 232441. Fax: 44 (0)1483 232448. E-mail: paul.monaghan@bbsrc.ac.uk.

† H.A. and K.M. contributed equally to this study.‡ Present address: London Research Institute, Cancer Research

UK, 44 Lincoln’s Inn Fields, London WC2A 3PX, United Kingdom.� Published ahead of print on 27 August 2008.

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75), and the 3Cpro protein of PV cleaves microtubule-associ-ated protein 4 (34, 35). Coxsackievirus B4 infection inducescleavage of the intermediate filament cytokeratin 8 via the 2Aprotease, and this occurs during the onset of CPE (67). Inaddition, it has been reported that FMDV proteins may asso-ciate with intermediate filaments (23). Rearrangement of in-termediate filaments has been reported in cells infected withother viruses around sites of virus assembly and also actinprojections have been implicated in virus release and cell-to-cell spread (28, 36–38, 54, 59, 61, 83).

We have now sought to determine the changes in the cy-toskeleton that underlie the FMDV-induced modifications incell morphology. Most interesting is the observation thatFMDV infection leads to a lack of tethering of the microtu-bules to the microtubule organizing center (MTOC) withininfected cells. This appeared to result from virus-induced lossof �-tubulin (but not pericentrin) from the MTOC. We soughtto identify the viral protein(s) responsible through the expres-sion of individual FMDV nonstructural proteins within unin-fected cells and assessing their effect upon the �-tubulin lo-cated at the MTOC. The FMDV nonstructural protein 3Cpro

was identified as the causative protein from this screeningprocess. Comparison, within the same cell type, of the FMDV-induced changes to those induced by bovine enterovirus (BEV)which (like PV), is an enterovirus, showed substantial differ-ences between the cellular effects of these two virus infections.

MATERIALS AND METHODS

Cells and viruses. BHK-38 and BHK-21 (ECACC 85011433) cells were ob-tained from ECACC, and BSR T7/5 cells (which express the T7 RNA polymer-ase) were kindly provided by K. Conzelmann (8). Cells were cultured in growthmedium (Dulbecco modified Eagle medium supplemented with 10% fetal calfserum [FCS], 2 mM glutamine, penicillin [100 SI U/ml], and streptomycin [100�g/ml]) at 37°C in 5% CO2. BSR cells were supplemented with G418 (0.5 mg/ml)(Sigma). Cells were seeded for 16 h before use onto 13-mm glass coverslips (AgarScientific, Ltd., Stansted, United Kingdom) for immunofluorescence and onto13-mm Thermanox coverslips (Science Services, London, United Kingdom) forelectron microscopy.

Stocks of FMDV O1BFS and BEV were prepared, and virus titers weredetermined using BHK-38 cells as described previously (31) (except that forBEV, horse serum was used in place of FCS). In all subsequent experimentsinvolving BEV, pig serum was used in place of FCS. The multiplicity of infection(MOI) was based on the virus titer on BHK-38 cells. The modified vaccinia virusAnkara (MVA-T7), a modified, host-range-restricted, vaccinia virus strain thatexpresses the bacteriophage T7 RNA polymerase gene (72), was kindly providedby Gerd Sutter (Institut fur Molekulare Virologie, Oberschleissrheim, Ger-many).

Virus infections. Cells prepared on coverslips as described above were washedwith cold phosphate-buffered saline (PBS) containing 1 mM CaCl2 and 1 mMMgCl2 and then incubated with virus at an MOI of between 10 and 100 PFU/cellfor 30 min at 4°C. The cells were washed with PBS to remove unattached virus,cell culture medium was added, and the cells were incubated at 37°C.

Antibodies. Mouse anti-FMDV 2C (1C8; 1:1,000) and mouse anti-FMDV 3C(4E9, isotype immunoglobulin G2b [IgG2b]; 1:10) were kindly provided by E.Brocchi (IZSLE, Brescia, Italy). Rabbit anti-FMDV capsid (1:100) was providedby IAH, and rabbit anti-BEV capsid antibody (1:100) was from the The Queen’sUniversity of Belfast. Mouse anti-V5 (isotype IgG2a; 1:1,000) was purchasedfrom Invitrogen (Paisley, United Kingdom). Mouse anti-�-tubulin (1:1,000; or1:4,000 for Western blot), mouse anti-vimentin (1:200), and mouse anti-�-tubulin(isotype IgG1) (1:200; or 1:5,000 for Western blot) were purchased from Sigma(Poole, United Kingdom). Mouse anti-pericentrin (1:200) was purchased fromCovance (Cambridge Bioscience, Cambridge, United Kingdom). Actin was de-tected with phalloidin-Alexa488 conjugate diluted 1:25 in PBS (Invitrogen).

Plasmids. Plasmids, based on the vector pcDNA3.1 (Invitrogen), which ex-press the FMDV nonstructural proteins 2BV5 (which contains a V5 C-terminaltag), 2BC, 2C, 3A, and 3AB have been described previously (49). The plasmid

pSKRH3C (5) expresses the FMDV 3Cpro under the control of the T7 RNApolymerase promoter; therefore, some experiments used BSR T7/5 cells, whichconstitutively express the T7 RNA polymerase (8).

Transfection. BHK-21 cells or BSR T7/5 were prepared as described aboveand then transfected with plasmid DNA (0.8 �g/well in 24-well plates) by usingLipofectamine 2000 (Invitrogen) in HEPES-buffered Dulbecco modified Eaglemedium for 3 h at 37°C. Cells were incubated for a further 16 h, at 37°C, ingrowth medium essentially as described previously (49).

Characterization of changes in cell morphology observed with live-cell imag-ing. Cells were seeded onto tissue culture dishes with an optically clear glasscoverslip cemented into the base (Iwaki; SLS, Nottingham, United Kingdom).After 16 h the cells were infected with FMDV as described above and thentransferred to a climate-controlled chamber on a Leica SP2 confocal microscope.Cells were imaged every 2 min for 3 h postinfection (hpi) by using differentialinterference contrast (DIC) optics.

Indirect immunofluorescence. Coverslips with infected and/or transfected cellsplus uninfected control cells were fixed for 30 min in 4% paraformaldehyde inPBS at 2 hpi for FMDV, 4 hpi for BEV; time points were selected as theestimated “midpoint” in infection (51) or 20 h posttransfection with FMDVplasmids. Fixed coverslips were washed in PBS and stored at 4°C. Immediatelybefore �-tubulin staining, cells were incubated in methanol for 4 min and washedin PBS. Cells used for immunolabeling were permeabilized in 0.05% TritonX-100 in PBS for 15 min, followed by a wash in PBS. Nonspecific labeling wasblocked with a 30-min incubation in PBS with 0.5% bovine serum albumin(PBS/BSA; Sigma). Primary antibodies were diluted in PBS/BSA and incubatedfor 1 h at room temperature. After three 5-min PBS washes, the primary anti-bodies were detected with Alexa488- or Alexa568-conjugated species-specificantibodies (Molecular Probes/Invitrogen) diluted 1:200 in PBS/BSA for 1 h.After three 5-min washes, the coverslips were incubated in DAPI (Sigma) diluted1:10,000 in water for 5 min to label the cellular DNA. Labeled coverslips weremounted in Vectashield (Vector Laboratories, Peterborough, United Kingdom)and sealed with clear nail varnish. Control uninfected cells were labeled as forinfected cells. The cells were imaged with a Leica TCS SP2 confocal microscope.Most images are from single confocal image planes, but in order to illustrateMTOC labeling in several cells, some images are Z-stacks. This means that insome images the MTOC will appear to be “in” the nucleus, whereas it is actuallyabove (or below) the nucleus.

Metabolic labeling and immunoprecipitation. BSR cells grown to ca. 90%confluence in 175-cm2 flasks were incubated for 0, 1, 2, 3, and 4 h with cyclo-heximide at 100 �g/ml. Cells were treated with trypsin, starved in methionine andcysteine-free Eagle medium for 10 min, and then labeled with 10 MBq of35S-Promix (Amersham Life Sciences, Chalfont St Giles, United Kingdom) perml in the same medium for 10 min. Cells were washed and lysed on ice inimmunoprecipitation buffer (10 mM Tris [pH 7.5]; 150 mM NaCl; 10 mMiodoacetamide; 1 mM EDTA; 1 mM phenylmethylsulfonyl fluoride; and 1 mg/mleach of leupeptin, pepstatin, chymostatin, and antipain) containing 1% NonidetP-40. Alpha-tubulin was immunoprecipitated by overnight incubation with anti-bodies immobilized with protein G coupled to Sepharose 4B. Proteins wereresolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by autoradiography.

Western blotting. Proteins were resolved by SDS-PAGE and transferred ontoa Protran BA85 cellulose nitrate membrane (Schleicher & Schuell, Dassel, Ger-many). The membrane was blocked for 1 h in 5% Marvel, 10% normal goatserum, and PBS-Tween 20; incubated with the primary mouse antibody; washedin PBS-Tween 20; and further incubated with goat anti-mouse antibody conju-gated to horseradish peroxidase (Southern Biotechnology Associates, Inc., Bir-mingham, AL); antibodies were diluted in 10% normal goat serum-5% MarvelPBS-Tween 20. After the washes, detection was achieved by using enhancedchemiluminescence reagents (Amersham Life Sciences, Buckinghamshire,United Kingdom).

Electron microscopy. At 2 hpi, control uninfected and FMDV-infected cellsgrowing on Thermanox coverslips cultured as for the glass coverslips were fixedin situ in 2% glutaraldehyde in 0.05 M phosphate buffer (pH 7.2 to 7.4, osmoticpressure adjusted to 350 mosmol with sucrose) for 2 h. Cells were postfixed in 1%osmium tetroxide in phosphate buffer for 4 h. They were dehydrated through anethanol series and embedded in Agar 100 resin (Agar Scientific, Stansted, UnitedKingdom). The resin was polymerized at 60°C, but before the resin had fullypolymerized, the Thermanox coverslips were peeled from the resin block, leavingthe cells embedded in the resin. Sections were cut parallel to the coverslipsurface with a Diatome diamond knife (Leica Microsystems, Milton Keynes,United Kingdom) and contrasted with uranyl acetate and lead citrate (EMStain;Leica). They were imaged in a FEI Tecnai 12 transmission electron microscopeat 100 kV.

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RESULTS

FMDV-induced CPE. Infection by positive-strand RNA vi-ruses results in CPE, which is characterized by cell roundingand subsequent detachment from the substratum. Majorchanges in cell shape are generally associated with changes inthe cytoskeleton. Here we have undertaken a detailed investi-gation of the effects of FMDV and BEV on the cytoskeleton byimmunofluorescence microscopy. These viruses were chosenbecause they are representatives of the aphthovirus and en-terovirus genera of the Picornaviridae which replicate in thesame cell types. In Fig. 1, a series of images are shown from alive-cell recording at increasing times after infection of cellswith FMDV (see supplemental information at http://www.iah.ac.uk/research/bioimaging/media/bioimg_vid1.shtml). The cellswere viewed by DIC optics to monitor their outline and surfaceproperties. During the first hour after infection the cells re-mained flat, indicating secure attachment to the coverslip, andmany cells were motile and produced cellular extensions.Changes in cell shape were observed from between 1 and 2 hafter infection. After this time, the number of cellular exten-sions produced was greatly reduced, and the cells began toround up and detach from the coverslip. CPE was also seen inBEV-infected cells except that the cell rounding occurred be-tween 2.5 and 4 hpi (data not shown).

Infections by FMDV and BEV have different effects on thecytoskeleton. In the following experiments (unless otherwisestated) FMDV infections are shown at 2 hpi and BEV at 4 hpidue to the BEV replication cycle being slower than that ofFMDV. At these times, the cells were still adherent to thecoverslip but were beginning to show changes in morphology.Other time points were also examined for both viruses, and theobservations reported here were consistent and not specific toany particular time point. FMDV-infected cells were detectedby labeling with the rabbit anti-capsid antibody, which identi-fies the virus replication site generally located in a perinuclearregion. BEV-infected cells were detected with a rabbit anti-serum raised against whole virus. The cells were infected at anMOI that allowed infected and uninfected cells to be seen inthe same image. Antibodies against �-tubulin and vimentinwere used to identify microtubules and intermediate filaments,respectively, while phalloidin-Alexa488 was used to image fil-amentous actin (F-actin).

The effects of viral infection on vimentin were examined to

compare the distribution of intermediate filaments in cellsinfected with FMDV (Fig. 2a to c) or BEV (Fig. 2d to f) to thatseen in uninfected cells (Fig. 2g). Although the levels of vi-mentin expression in uninfected cells appeared to vary be-tween cells, it was arranged in a filamentous network runningthroughout the cytoplasm (Fig. 2g). In cells infected withFMDV or BEV, vimentin was found redistributed to sites ofviral replication. Interestingly, FMDV and BEV had differenteffects on vimentin distribution. In FMDV-infected cells, vi-mentin was rearranged into a ring surrounding the area oflabeling for viral proteins (Fig. 2a to c), while BEV caused astriking concentration of the majority of the cellular vimentininto a ball of filaments in the same area of the cell occupied byBEV proteins (Fig. 2d to f).

Examining the appearance of microtubules in infected cellsalso revealed differences between the effects of FMDV andBEV (Fig. 3). In control cells, the microtubules (visualizedwith antibodies recognizing �-tubulin) were arranged in a fil-amentous network running throughout the cytoplasm. How-ever, the tubulin pattern differed from the distribution ofvimentin in that it showed a radial distribution, with microtu-bules originating from the MTOC (Fig. 3g). The microtubulesin FMDV-infected cells (Fig. 3a to c) still extended to theplasma membrane; however, the peripheral microtubules weredisorganized and lacked the radial distribution seen in controlcells. Most interesting was the exclusion of most of the micro-tubules from the area of the cell containing FMDV proteins(Fig. 3a and c). In contrast, in BEV-infected cells the micro-tubule distribution retained a radial pattern (Fig. 3d to f).

The effects of viral infection on F-actin in cells were exam-ined using fluorescently labeled phalloidin (data not shown). Incontrol cells, F-actin was predominantly found in “stress fi-bers” in the region of the cell adjacent to the coverslip surface.FMDV infection led to a loss of actin stress fibers; however,some actin labeling was still visible at the plasma membrane.This suggested that FMDV infection leads to depolymerizationof F-actin. BEV infection also induced the loss of F-actinbundles from the basal region of the cell. However, in contrastto FMDV, actin labeling at the plasma membrane was notdetectable in BEV-infected cells.

FMDV infection disrupts �-tubulin localization but notpericentrin or centrioles. As shown above, FMDV infectionleads to microtubule disruption throughout the cell. The cen-

FIG. 1. CPE in FMDV-infected cells. Still images from a live-cell experiment in which BHK-21 cells were infected with FMDV and imaged ina confocal microscope by DIC optics are shown. The three still images are from the cells at 0 (a), 76 (b), and 152 min (c) postinfection. Scale bar(for all three panels), 20 �m.

10558 ARMER ET AL. J. VIROL.

trosome consists of pericentrin and a matrix of large proteincomplexes containing �-tubulin and associated proteins. Thismatrix is thought to be the site of microtubule nucleation.FMDV-induced disorganization of microtubules (comprised ofheterodimers of � and � tubulin) suggested that the virus maybe affecting the function of the centrosome. In control cells, wefound that 99 of 100 cells examined showed strong staining atthe MTOC for �-tubulin and pericentrin. We therefore exam-ined the distribution of �-tubulin in cells infected with FMDV.Figure 4a to c shows the analysis of cells at 3 hpi with FMDV.Labeling for FMDV was with an antibody recognizing non-

structural FMDV 3A protein. In these images, two cellsshowed strong labeling for FMDV, while a third cell was un-infected. Significantly, �-tubulin staining (red) was absent fromthe cells with high levels of FMDV protein, while it remainedpresent in uninfected cells (Fig. 4a to c). The image is repre-sentative of 100 FMDV-infected cells examined at 3 hpi. Wefound that in 85% of cases FMDV infection led to a loss of�-tubulin at the MTOC. In the remaining 15% of cases, �-tu-bulin at the MTOC was detectable, but staining was weakerthan that observed in control cells. Interestingly, this corre-sponded with a lower level of FMDV infection. Our results

FIG. 2. Effect of FMDV and BEV infection on host cell vimentin. BHK-21 cells were infected with FMDV and fixed with paraformaldehyde at 2 hpi(panels a and b, merged in panel c) and BEV fixed at 4 hpi (panels d and e, merged in panel f). An uninfected cell is shown in panel g. The cells wereimmunolabeled for the presence of viral structural proteins detected with anti-rabbit antibody-Alexa488 (green) and mouse anti-vimentin detected withanti-mouse antibody-Alexa568 conjugate (red). Nuclei were labeled with DAPI (blue). Scale bars: a to c, 10 �m; d to f, 20 �m; g, 10 �m.

VOL. 82, 2008 FMDV CYTOPATHIC EFFECT 10559

indicated that FMDV infection led to a loss of �-tubulin fromthe MTOC with a concomitant untethering of the microtu-bules. This is consistent with the disorganization of microtu-bules observed in previous experiments (see Fig. 3). This effectwas also seen at earlier time points. Figure 4d to f shows thatBEV infection had no noticeable effect on �-tubulin distribu-tion in cells, which remained similar to that observed in controlcells. Labeling for �-tubulin was clearly visible in cells express-ing high levels of viral proteins.

The loss of �-tubulin labeling from the MTOC of cells in-

fected with FMDV could either reflect a specific loss of �-tu-bulin from the centrosomes or a more generalized disruptionof the organelle. Either scenario could lead to microtubuledisorganization. In order to determine which of these possibil-ities was occurring, we analyzed FMDV-infected cells for thepresence of another centrosome component: pericentrin. Thisprotein is a structural component of the centrosome and in-teracts with �-tubulin (17) to form a lattice required for theregulation of microtubule formation. Pericentrin also associ-ates with the motor protein dynein and this enables pericen-

FIG. 3. Effect of FMDV and BEV infection on host cell microtubules. BHK-21 cells were infected with FMDV and fixed with paraformal-dehyde at 2 hpi (panels a and b, merged in panel c) and BEV fixed at 4 hpi (panels d and e, merged in panel f). An uninfected cell is shown inpanel g. The cells were immunolabeled for the presence of FMDV and BEV structural proteins, which were detected with anti-rabbit antibody-Alexa488 conjugate (green), and the microtubule cytoskeleton was immunolabeled with mouse anti-�-tubulin, which was detected with anti-mouseantibody-Alexa568 conjugate (red). Nuclei were labeled with DAPI (blue). Scale bars: a to f, 10 �m; g, 10 �m.

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trin–�-tubulin complexes to be transported along microtubulesto the centrosome (60, 82). Using an antibody specific forpericentrin, we examined the effects of FMDV infection on thecentrosomal structure. In uninfected cells, the pattern of �-tu-bulin (Fig. 5a) and pericentrin (Fig. 5b) labeling is similar, withsmall dots often close to the nucleus (Fig. 5a and b). In cellsinfected with FMDV the characteristic �-tubulin labeling seenat the perinuclear location in control cells was lost (Fig. 5d),whereas pericentrin labeling was unaffected (Fig. 5e). Theseresults suggested that the MTOC may remain intact, but itsinability to organize microtubules in cells infected with FMDVwas caused by loss of the tethering protein, �-tubulin. Cellsinfected with FMDV were therefore examined by electronmicroscopy for evidence of intact centrioles. Infection of cellswith FMDV led to an accumulation of membrane vesicles inthe cytoplasm, and these were used to identify infected cells inelectron micrographs. Figure 5c shows a control cell with theMTOC present in the center of the image, and Fig. 5f shows acell infected with FMDV at 2 hpi. The cytoplasm containsFMDV-induced vesicles, and the main structural componentof the centrioles of the MTOC was still present and is locatedin the center of the image. Centrioles consist of two sets ofshort microtubules at 90° to each other and may have differentappearances depending on the angle of sectioning.

FMDV nonstructural protein 3Cpro induces loss of �-tubu-lin. We then sought to identify the cause of FMDV-induced�-tubulin loss from the MTOC and hypothesized that a viralnonstructural protein or proteins (rather than structural pro-teins) would be a likely candidate. We undertook a series oftransfections of BHK cells with plasmids that express theFMDV nonstructural proteins or their precursors. Plasmids,transfected into cells, encoded FMDV 2B, 2BC 2C, 3A, 3AB,or 3C. Transiently transfected cells were fixed after 20 h andlabeled for expression of the V5 tag (FMDV 2B) or FMDVnonstructural protein (2BC 2C, 3A, 3AB, and 3Cpro) usingspecific antibodies and for the presence of �-tubulin. In allcases, except for cells that expressed FMDV 3Cpro (see below),�-tubulin was unaffected. As an example, Fig. 5g shows a cellexpressing FMDV 2B, where the viral protein is labeled redand �-tubulin is green.

Transient expression of FMDV 3Cpro can be achieved fromplasmid pSKRH3C in BHK cells after infection with the re-combinant vaccinia viruses vTF7-3 or MVA-T7, which expressthe T7 RNA polymerase (44). Preliminary experiments usingthe MVA-T7 system, to drive transcription of the plasmid,indicated that the expression of 3Cpro disrupted the �-tubulinlabeling pattern in a similar way to FMDV infection. However,a previous study with vaccinia virus has shown a loss of �-tu-

FIG. 4. FMDV, but not BEV, induces loss of �-tubulin from the MTOC. BHK-21 cells were infected with FMDV and fixed with paraformal-dehyde at 2 hpi (panels a and b, merged in panel c) and BEV fixed at 4 hpi (panels d and e merged in panel f). The cells were immunolabeledfor the presence of viral structural proteins, which were detected with anti-rabbit antibody-Alexa488 conjugate (green), and �-tubulin, which wasdetected with anti-mouse antibody-Alexa568 conjugate to show the location of the MTOC (red). Nuclei were labeled with DAPI (blue). Scale bar,10 �m.

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bulin (59) during virus infection. Although changes in �-tubu-lin distribution in the MVA-T7-infected cells were not ob-served, we performed transfection experiments with BSR T7/5cells to exclude totally the possibility that the virus infectionwas modifying the result. These cells constitutively express T7RNA polymerase (8), thus removing the need for the vacciniavirus to drive FMDV 3Cpro expression. The results clearly

showed that transient expression of FMDV 3Cpro alone in cells(in the absence of vaccinia virus) led to a depletion of �-tubulinlabeling at the MTOC (Fig. 5h and i). This image is represen-tative of 50 3C transfected cells examined. In 90% of trans-fected cells examined, the �-tubulin at the MTOC was notdetectable. The remaining 10% of transfected cells showedsome �-tubulin labeling, and this staining was weaker that that

FIG. 5. Effect of FMDV infection or nonstructural protein 3Cpro expression on the MTOC. (a to c) Uninfected BHK-21 cells fixed inparaformaldehyde and labeled for �-tubulin (a) and pericentrin (b) (green; arrows) or for TEM showing centrioles (c). (d and e) BHK21 cellsinfected with FMDV and fixed in paraformaldehyde at 2 hpi and labeled for FMDV structural proteins detected with anti-rabbit antibody-Alexa568conjugate (red) and either �-tubulin (d) or pericentrin (e) (green). (f) BHK-21 cells infected with FMDV and fixed for TEM at 2 hpi. The cellcytoplasm contains typical FMDV-induced vesicles, but the centrioles are visible in the center of the image. (g) BHK-21 cells transfected with 2BV5and immunolabeled with anti-V5 tag detected by anti-mouse IgG2a-Alexa568 (red) and �-tubulin detected by anti-mouse IgG1-Alexa488 (green).(h and i) BSR cells transfected with pSKRH3C, which expresses the FMDV 3Cpro, and immunolabeled for 3Cpro (antibody 4E9) detected byanti-mouse IgG2b-Alexa568 (red) and with mouse anti-�-tubulin (green). Nuclei were labeled with DAPI (blue). Scale bars: a, b, d, e, g, h, andi, 10 �m; c, 500 nm; f, 400 nm.

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seen in control cells. These cells expressed much lower quan-tities of the FMDV 3C protease. Similarly, as with FMDVinfection, transient expression of FMDV 3Cpro in cells did notaffect the presence of pericentrin at the MTOC. Of 50 cellsexamined, 96% showed labeling for pericentrin.

To determine whether FMDV was targeting �-tubulin fordegradation, the steady-state level of �-tubulin was examinedover the course of an FMDV infection. Infected cells werelysed at 1-h intervals starting from 2 hpi and analyzed byWestern blotting (Fig. 6a). FMDV protein expression was de-tectable (using an antibody against the nonstructural protein2C) by 3 hpi, and the levels of 2C increased with time. With alonger exposure of the blot, virus infection was also clearlydetectable at 2 hpi (data not shown). Both �- and �-tubulin

were detectable throughout the virus infection at constant lev-els, indicating that while �-tubulin was being displaced fromthe MTOC, it was not targeted for degradation.

FMDV 3Cpro has been shown to modify the host cell proteinsynthesis machinery and induces cleavage of eIF4A andeIF4GI (5). This may contribute to the loss of protein synthesisat late times within infected cells (but the initial loss of hostprotein synthesis is mediated through the action of the FMDVleader [L] protease). To examine whether host protein synthe-sis shutoff alone leads to disruption of �-tubulin labeling at theMTOC, BSR cells were incubated with cycloheximide (cyclo-heximide blocks peptidyl transferase activity in eukaryotic ri-bosomes, leading to a block in protein synthesis), and cellswere examined at hourly intervals over 4 h. To detect protein

FIG. 6. Effects of FMDV and cycloheximide on �- and �-tubulin. (a) BHK-21 cells were infected with FMDV 01BFS strain. Samples were lysedat 0, 2, 3, and 4 hpi. Proteins were separated by SDS-PAGE, blotted, and analyzed for the presence of FMDV 2C, �-tubulin, and �-tubulin. (b)Cycloheximide was added to cells at a concentration of 100 �g/ml. Cells were examined over the subsequent 4 h. The level of �-tubulin synthesisin cells was examined at 0, 1, 2, 3, and 4 h after the addition of cycloheximide, by radiolabeling with 35S followed by immunoprecipitation. (c)�-Tubulin levels at the MTOC were examined by confocal microscopy at the same time points as in panel b. (d) BSR cells transfected withpSKRH3C, which expresses the FMDV 3Cpro, and immunolabeled for 3Cpro (antibody 4E9) detected by anti-mouse IgG2b-Alexa488 (green) andwith mouse anti-�-tubulin detected with Alexa568 (red). Scale bars, 10 �m.

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synthesis, a portion of the cells were labeled with [35S]methi-onine for 10 min. Immunoprecipitation of the cellular hostprotein �-tubulin revealed that cycloheximide had shut off hostprotein synthesis within the first hour, as expected. [35S]methi-onine-labeled �-tubulin was not detectable after the additionof cycloheximide (Fig. 6b). The remaining cells were labeledfor the presence of �-tubulin (Fig. 6c). The levels and locationof �-tubulin in cycloheximide treated cells were similar to thoseof untreated cells, indicating that the effects triggered by hostprotein synthesis shutoff did not lead to disruption of the�-tubulin at the MTOC.

FMDV nonstructural protein 3Cpro induces dissociation ofmicrotubules from the MTOC. BSR cells transiently express-ing FMDV 3Cpro were further examined to determine theeffects of the loss of �-tubulin on the microtubule network.Staining for �-tubulin in cells expressing FMDV 3Cpro (Fig.6d) revealed that the majority of �-tubulin has relocated to theedges of the cell, thus closely mimicking the effect of FMDVinfection and confirming that a major contributor to thechange in cell morphology is due to the dissociation of micro-tubules from the MTOC.

DISCUSSION

FMDV. Infection of BHK cells with FMDV causes dramaticchanges in cell morphology.

The cytoskeleton shows dramatic rearrangements in many ofits components. The network of actin filaments is disruptedfrom the early stage of infection, and this is reported to be acommon response in cells to viral infection (4, 68). However,taking the other observations reported here it is likely thatchanges in other components of the cytoskeleton, and micro-tubules in particular, may play a key role in this process. Thechanges in the microtubule network are also clear from theearly stage in FMDV infection and result in the FMDV rep-lication area being devoid of microtubules. Early signs ofFMDV infection (as detected by labeling for nonstructuralproteins) were seen in the area of the Golgi body (39), and thisorganelle is often closely associated with the MTOC. Changeshave also been detected in the buoyant density of Golgi mem-branes during FMDV infection (50), suggesting Golgi disrup-tion or modification. The protein �-tubulin forms a ringaround the centrosome and acts in anchoring the “�” ends ofmicrotubules to the MTOC. The loss of �-tubulin labeling inFMDV-infected cells gave a possible mechanism for the dis-ruption of the microtubule structure. However, surprisingly,the MTOC protein pericentrin appeared to be unaffected, asdid the basic physical structure of the centrioles as seen inFMDV-infected cells by TEM. Thus, a specific targeting of thetethering of microtubules to the MTOC seemed possible.

We hypothesized that a nonstructural (rather than a struc-tural) FMDV protein was most likely to be responsible for thiseffect. The expression of individual and precursor forms ofnonstructural FMDV proteins in uninfected cells showed that3Cpro alone induced the loss of �-tubulin labeling from theMTOC region of the cell. This effect was not seen in cells thatexpressed any of the other nonstructural proteins or their pre-cursors. Furthermore, the microtubule pattern in cells express-ing 3Cpro alone closely resembled that seen in FMDV-infectedcells, in that the microtubules accumulated in a peripheral

location. Exactly how FMDV 3Cpro exerts its effect on �-tubu-lin remains unclear since the �-tubulin does not appear to betargeted for degradation, and we did not detect any cleavage ofit during virus infection (Fig. 6). Microtubule nucleation at thecentrosome is mediated by the �-tubulin ring complex, and inhuman cells this complex is made up of �-tubulin and the�-tubulin complex proteins (GCP): 2-6 (21, 52, 53) andGCP-WD (or NEDD1) (24, 45). Interestingly, depletion ofGCP-WD (a �-tubulin targeting factor) results in a depletionof �-tubulin, at the MTOC, but not pericentrin (45), and thelevels of �-tubulin in the cell detected by Western blottingremained constant. Since �-tubulin does not appear to be adirect target of the FMDV 3Cpro, it may be that FMDV 3Cpro

targets a different member of the �-tubulin ring complex,which indirectly leads to �-tubulin depletion at the centro-some.

The effects on the host cell MTOC that we have detectedand attributed to FMDV protein 3Cpro have not been reportedfor any other picornavirus. The functions of the PV nonstruc-tural proteins on host cells have been investigated by transfec-tion of each protein individually and in combination. Theseexperiments indicate that PV 2C and 2BC induce the forma-tion of smooth membrane vesicles (11) and that 2B inducesdisassembly of the Golgi apparatus (64) and causes plasmamembrane permeabilization (1, 19, 41, 46). PV 3A slows pro-tein movement through the secretory pathway (12, 18, 19) andcauses swelling of ER membranes (18). The PV 3Cpro has anumber of reported functions on host cells, and these includeinhibition of host cell RNA synthesis through proteolytic cleav-age, and thus the inactivation, of specific transcription factors(13, 14, 76, 79, 80), induction of apoptosis (3, 9), suppression ofNF-�� through proteolytic cleavage of the p65-RelA compo-nent (57), and contribution to translation shutoff via inactiva-tion of ribosome-associated PABP (40). However, no directeffect on the host cell microtubules or the MTOC has beenreported, although cleavage of microtubule-associated protein4 (MAP4) by 3Cpro has been demonstrated (35).

The redistribution of microtubules may well explain the pat-tern of vimentin seen around the FMDV replication area. Themechanisms that control the expression and distribution ofvimentin filaments in uninfected cells are not clear. In unin-fected cells vimentin forms a network spread through the cy-toplasm with no clear focal point. In FMDV-infected cells thevimentin filaments are rearranged into a network surroundingthe replication area. The formation of a vimentin networkaround areas of viral replication has recently been reported forother viruses (25, 26, 56, 62, 65, 69, 77, 78), and the similaritybetween this pattern and the structure of aggresomes is tanta-lizing. It is possible to speculate that the vimentin “cage”around the FMDV area of replication is actually a host cellresponse to the infection rather than a virus induced-changethat enhances viral replication.

Comparison of cells infected by FMDV and BEV. Althoughthere is some consistency in the overall pattern of cellularresponses to infection by FMDV and BEV, there are a numberof key differences. Infection of cells with BEV does not lead tothe loss of �-tubulin or pericentrin labeling at the MTOC, andthere was no resultant dramatic change in the overall patternof microtubules. From its major role in anchoring componentsin normal cells, it could be predicted that there should be less

10564 ARMER ET AL. J. VIROL.

disruption of the cellular components in BEV-infected cells.This was not actually the case. Although vimentin filaments aredrawn around the FMDV replication area, in BEV-infectedcells, the vimentin is drawn into the virus replication area andmay play a similar role for other viruses (56, 77, 78). Interest-ingly, the 3Cpro protein of PV has been shown to cleave MAP4(35), and also observed in PV-infected cells is the rearrange-ment of intermediate filaments into a perinuclear region (42).MAP4 is thought to bind microtubules and stimulate theirpolymerization and increase their stability (27, 47). A trun-cated form of MAP4 has also been shown to increase micro-tubule stability (81); however, MAP4 detachment from micro-tubules leads to an increased instability of the microtubulenetwork (29). A breakdown of the vimentin network has alsobeen observed with detachment of MAP4 from the microtu-bules (20). It may be that in BEV-infected cells, cleavage ofMAP4 could leave a truncated version on the microtubules,retaining microtubule stability, while also leading to vimentindetachment from the microtubules and allowing it to move intothe virus replication complex.

One additional aspect of this study is the role played in theCPE by the host cell response to infection. BEV-infected cellsshow clear signs of apoptosis after around 5 hpi and, by 6 hpi,all of the cells showed peripheral clumping of nuclear hetero-chromatin visible by both light and electron microscopy, whichis highly characteristic of apoptosis. Thus, some of the virus-induced effects may be masked by the cellular apoptosischanges. There were no overt signs of apoptosis in any of theFMDV-infected cells.

ACKNOWLEDGMENTS

This study was supported by BBSRC grants 49/CO7867, 49/C14570,and 201/S14654 to T.W., M.R., and G.J.B.

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