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Virology 282, 237–244 (2000)doi:10.1006/viro.2000.0728, available online at http://www.idealibrary.com on

Association of Bovine Papillomavirus Type 1 with Microtubules

Wen-Jun Liu, Ying-Mei Qi, Kong-Nan Zhao, Yue-Hua Liu, Xiao-Song Liu, and Ian H. Frazer1

Centre for Immunology and Cancer Research, Princess Alexandra Hospital, University of Queensland, Woolloongabba, Queensland 4102, Australia

Received August 17, 2000; returned to author for revision September 27, 2000; accepted October 24, 2000

Transport of BPV-1 virus from the cell membrane to the nucleus was studied in vitro in CV-1 cells. At reduced temperature(4°C), BPV-1 binding to CV-1 cells was unaffected but there was no transport of virions across the cytosol. Electronmicroscopy showed BPV-1 virions in association with microtubules in the cytoplasm, a finding confirmed by co-immunopre-cipitation of L1 protein and tubulin. Internalization of virus was unimpaired in cells treated with the microtubule-depolymer-izing drug nocodazole but virions were retained in cytoplasmic vesicles and not transported to the nucleus. We conclude that

a microtubule transport mechanism in CV-1 cells moves intact BPV-1 virions from the cell surface to the nuclear membrane.© 2001 Academic Press

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INTRODUCTION

Before viruses can infect and replicate in a host cell,they must cross the plasma membrane and then targettheir genome and accessory proteins to the correct or-ganelle, where gene transcription, nucleic acid replica-tion, and viral maturation can take place. After a viruspenetrates the cell membrane, the cytoplasm imposes adiffusion barrier. Within the eukaryotic cytoplasm, or-ganelles, solutes, and a complex latticelike mesh ofmicrotubule, actin, and intermediate filament networksrestrict free diffusion of macromolecular complexeslarger than 500 kDa (Luby-Phelps, 2000; Seksek et al.,1997). Movement of large complexes, including mem-brane organelles and ribonucleoprotein particles, gener-ally involves either actin or the microtubule (MT) network(Grunert and St Johnston, 1996; Hirokawa et al., 1998;Holleran et al., 1998; Mermall et al., 1998). The role of MTn the intracellular transport of particles and organellesas been documented (Toomre et al., 1999).

Some nonenveloped viruses use similar transportechanisms to macromolecules within the cell. Adeno-

iruses, for example, are carried by microtubules to theell nucleus where they are decapsidated near nuclearores to allow DNA to enter the nucleus (Suomalainen etl., 1999), while herpes simplex and rabies viruses are

ransported retrogradely in neuronal axons to infect theell body (Kristensson et al., 1986; Miranda-Saksena etl., 2000). Papillomaviruses (PV) are epitheliotropic dou-le-stranded DNA viruses. The virions are nonenvelopednd have a 55-nm icosahedral structure (DeLap et al.,977; Galloway and McDougall, 1989). Bovine PV has

1 To whom reprint requests should be addressed. Fax: 161-7-3240946. E-mail: ifrazer@medicine.pa.uq.edu.au.

237

een widely used as a model system for studying PVnfection. The infectious entry pathway of BPV-1 starts

ith the binding of the virus to specific cell receptors,ncluding a6-integrin protein (Evander et al., 1997) andeparin-like molecules (Joyce et al., 1999). BPV-1 crosses

he plasma membrane by an unknown mechanism andoves through the cytosol to the nuclear membrane

Muller et al., 1995), which it must penetrate to replicaten the nucleus. Papillomaviruses are not expected to

ove by free diffusion through the cytoplasm because ofheir size, but little is known about their mechanism ofntracytoplasmic transport. We therefore analyzed theransport of BPV-1 virions, purified from cattle warts, tohe nucleus of a cultured cell line, and this appears to ben energy-dependent process involving intact microtu-ules.

RESULTS

hysiological temperature is needed for nuclearargeting of BPV-1

BPV-1 particles are targeted to the cell nucleus afterptake into the cytoplasm (Zhou et al., 1995). Diffusion

of artificial particles larger than 50 nm in diameter isrestricted by the structural organization of the cyto-plasm (Luby-Phelps, 2000). BPV-1 particles are ap-proximately 50 nm and thus would be expected tohave minimal diffusion mobility under physiologicalconditions. To test whether BPV-1 is actively trans-ported in the cytoplasm by an energy-dependent pro-cess, we bound BPV-1 virions to cells for 1 h at 4°C,washed off unbound virus, and increased the temper-ature to 37°C for 20 min. During this time, virions weretaken up into the cytoplasm where they appeared as

small, intensely labeled spots (data not shown). Cellswere then cooled to 4°C and held for up to 60 min:

0042-6822/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

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238 LIU ET AL.

control cells were kept at 37°C. Since exposure ofcells to reduced temperatures causes depolymeriza-tion of MTs (Fig. 1F), targeting was analyzed in cellsthat have been pretreated with the MT stabilizing drugtaxol (Fig. 1G). In cells held at 37°C, most of the BPV-1antigen could be detected in the perinuclear zone at60 min (Fig. 1A), in agreement with previous results(Zhou et al., 1995). In contrast, in cells held at 4°C withor without taxol treatment, virus antigen was predom-inantly localized in numerous small vesicle-like struc-tures throughout the cytoplasm at 60 min (Figs. 1B and1C) and at 90 min (data not shown), indicating a failureof the viral antigen to reach the nuclear membrane.Staining for tubulin of cells held at 4°C showed exten-sive disruption of microtubules compared with cells

FIG. 1. Bovine papillomavirus transport to the nucleus requires phys4°C (A, B, D, E, F, H) or were similarly infected in taxol-containing medcells were then held at 37°C for 20 min to allow virus internalization. C4°C (other groups). Some cells (D, H) were then held at 37°C for a furt(E–H; red).

FIG. 2. Colocalization of incoming BPV-1 virions with microtubules.min after infection, cells were fixed with 80% ethanol and double label(PI), capsids are scattered in the cytoplasm and colocalize with micromembrane, and the other capsids are colocalized with microtubules (Bcenters (C).

held at 37°C, or cells held at 4°C after taxol treatment(Figs. 1E, 1F, and 1G). When the temperature block

was released by incubating cells at 37°C for another60 min, most of the BPV-1 L1 was located around thenucleus (Fig. 1D). These results confirmed that incom-ing BPV-1 virions do not move through the cytoplasmby diffusion and suggested that transport is activelyassociated with microtubules.

Binding of BPV-1 virions to microtubules

To investigate the role of microtubule-mediatedtransport in the perinuclear location of BPV, CV-1 cellswere incubated for 2 h at 37°C with 20 mM of taxol,which prevents the disassembly of microtubules, andthen were infected with BPV-1 virions. Colocation ofb-tubulin and BPV-1 was examined by double immu-

l temperature. CV-1 cells were infected by BPV-1 virions for 60 min atter a 30-min preincubation of cells at 37°C in taxol (20 mM) (C, G). Allere then held at 37°C for 60 min (A, E) or held for the same period atmin. Cells were stained for BPV-1 L1 protein (A–D; green) or b-tubulin

lls were infected by BPV-1 virions at an m.o.i. of 1000. 30 min and 60anti-BPV-1 L2 (green) and anti-b-tubulin (red). At 30 min postinfection

s (A). At 60 min PI, most of the capsids are localized on the nucleare cells, BPV-1 viral antigen is located in microtubule organization like

iologicaium afells w

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CV-1 ceed with

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nofluorescence. At 30 min postinfection, most of theBPV-1 particles appeared to be restricted to small

239BOVINE PAPILLOMAVIRUS TYPE 1 MICROTUBULES

cytoplasmic vacuole-like structures (Fig. 2A). Thesefluorescent vacuoles were scattered around the cyto-plasm and bound with the tubule network. At 60 min,the majority of BPV-1 antigen accumulated in the pe-rinuclear zone, while some was still found in the cy-toplasm and bound with tubule networks (Fig. 2B). Insome cells, BPV-1 antigen was observed to accumu-late in structures resembling the microtubule organi-zation-like center (MTOC; Fig. 2C). Electron micros-copy confirmed the close association of BPV-1 capsidswith MT. At 30 min after infection, 55-nm size BPV-1particles could be seen in association with cytoplas-mic filaments with MT-like morphology and the ex-pected 24-nm width. The MT-associated capsids hadan electron-dense DNA core (Figs. 3A–3C). At latertime points (60 min), hundreds of intact capsids withelectron-dense cores were seen bound to the nuclearmembrane. Disrupted virus particles were also ob-served (Fig. 3D), suggesting that BPV-1 virion disas-sembly and release of viral DNA was occurring by thistime.

Reduced capsid transport to the nucleus withoutmicrotubules

As shown above, BPV-1virions were observed in close

FIG. 3. Colocalization of incoming BPV-1 capsids with microtubuleson conventional EM. CV-1 cells were infected at an m.o.i. of 1000.Infected CV-1 cells were treated by CSK buffer and fixed by glutaral-dehyde. At 30 min, numerous incoming viral capsids were found local-ized to microtubules (MT) (A–C). At 60 min, virions were seen within thenucleus where disrupted forms were seen, suggesting that the virushad started to uncoat and release DNA (arrow). Bar, 100 nm.

association with MTs immediately after infection of thecell. To determine whether transport of BPV-1 to the

nucleus depended on intact MTs, CV-1 cells were incu-bated with nocodazole, a specific inhibitor of tubulinpolymerization into microtubules (De Brabander et al.,1977), for 2 h. Virus was subsequently bound to the cellsurface for 60 min at 4°C. Cells were then held at 37°Cin nocodazole-containing medium for 60 min. Underthese conditions, MTs were completely depolymerized,as shown by anti-b-tubulin immunostaining, and local-ization of BPV-1 virus to the nuclear envelope was inhib-ited (Figs. 4C and 4D). Similar results were obtained ifincubation at 37°C was extended to 135 min. In contrast,untreated cells and cells treated with taxol accumulatedBPV-1 virions on the nuclear membrane (Figs. 4A and4B). Nocodazole blocked nuclear trafficking of BPV-1when added up to 30 min after exposure of the cells tovirus, but was ineffective if added at 1 h (data not shown).To test whether the effect of nocodazole was reversible,the cells were analyzed 1 h after drug removal, wherebythe MT had repolymerized, and previously dispersedcapsids were now concentrated on the nuclear rim, sim-ilar to cells never treated with nocodazole (Fig. 4F).Taken together, these results suggested that intact MTsare required for targeting of incoming virus particles tothe nucleus.

FIG. 4. CV-1 cells exposed to nocodazole or taxol were infected withBPV-1 virions and then held at 37°C for 60 min. Cells were then fixedand permeabilized and labeled with either anti-BPV-1 L1 (A, B, D, F) oranti-b-tubulin (C, E) antibody. Under control conditions (A, E) or in taxoltreated cells (B), BPV-1capsids accumulate around the cell nucleus. Inthe cells pretreated with nocodazole, capsids are scattered throughoutthe entire cytoplasm and microtubules are disrupted (C, D). Nocoda-

zole-treated BPV-1 infected cells, washed and held without nocodazolefor 60 min, accumulate virus at the cell nucleus (F).

nd exte

240 LIU ET AL.

Depolymerization of the microtubule network doesnot down-regulate BPV-1 binding and internalization

To establish whether cell-surface expression of BPV-1receptors was down-regulated or virus endocytosis wasimpaired following nocodazole treatment of cells, wemeasured binding and internalization of BPV-1 into drug-treated cells using an assay that measures virus uptakeby detecting a trypsin-resistant major capsid protein L1.Nocodazole-treated and untreated CV-1 cells bound sim-ilar amounts of BPV-1 virions after 1 h exposure to virusat 4°C (Fig. 5). Approximately 50% of the virus particleswere endocytosed by 10 min after nocodazole-treatedcells were warmed to 37°C, whereas for untreated cellsthe half-time of internalization was slightly less than 10

FIG. 5. Depolymerization of the microtubule network does not affectwere then brought to 37°C in the presence (lanes 7–12) or absence (laat 4°C for 1 h with or without (lanes 1, 7) added trypsin to remove bouwith anti-L1 Ab for detection of internalized virus.

FIG. 6. Coimmunoprecipitation of L1 protein or tubulin from extracts oantibody against tubulin or using rabbit polyclonal antibody againstprecipitated by antitubulin antibody (anti-tub V/anti-tub NV) in A; meV/anti-L1 NV) in B. Precipitated proteins and control precipitations within 10% SDS–polyacrylamide gels and transferred to nitrocellulose memonoclonal antitubulin antibody (B) followed by second antibody incuba

of BPV-1 virion from BPV-1 infected/uninfected CV-1 cell extracts suing antitubuL1 protein is indicated by arrow.

min. Entry at 20-min postwarming was close to 75% withor without nocodazole. Thus, BPV-1 virus binding to thecell surface and internalization does not require an intactmicrotubule network, in keeping with the observationthat nocodazole has no effect on the formation of endo-cytic vesicles (Gruenberg et al., 1989).

Interaction of BPV-1 with microtubules in vivo

Double-labeling immunofluorescence microscopy andelectron microscopy demonstrates an association ofBPV-1 with microtubules after entry into the cell. Todemonstrate whether the BPV-1 virion L1 capsid proteininteracts with tubulin protein, a reciprocal immunopre-cipitation experiment was performed (Fig. 6). Lysate from

lization of BPV-1. BPV-1 virions were bound to CV-1 cells at 4°C. Cells) of nocodazole and held for the indicated times. Cells were then heldrnal virus, washed, and subjected to SDS–PAGE and immunoblotting

cells infected with BPV-1 (V), or not so infected (NV), using monoclonalvirions. BPV-1 virion infected or uninfected CV-1 cell extracts were

the same extracts were precipitated by anti-BPV-1 virions (anti-L1y to BPV-1 virions (BPV-1cont) and to tubulin (tub cont) were separateds which were labeled with rabbit anti-BPV-1 L1 antibody (A) or with

rows mark L1 and tubulin proteins. (C) Specific co-immunoprecipitation

internanes 1–6

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lin (anti-tub V/lane anti-tub NV) or anti-pan-cytokeratin (lane anti-p-ker).

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241BOVINE PAPILLOMAVIRUS TYPE 1 MICROTUBULES

CV-1 cells infected or not infected with BPV-1 virions wasimmunoprecipitated with either a monoclonal antibodyagainst b-tubulin (Fig. 6A) or a rabbit antiserum against

PV-1 virions (Fig. 6B). Precipitates were probed with theabbit anti-L1 serum (Fig. 6A) or the monoclonal antibodygainst b-tubulin (Fig. 6B). L1 protein was coprecipitated

with b-tubulin antibody (Fig. 6A, lane 2) and conversely,ubulin protein was coprecipitated by anti-BPV-1 virionntibody (Fig. 6). To confirm the specificity of the inter-ction of BPV-1 with tubulin, antibodies against cytoker-tins were used in co-immunoprecipitation, and L1 pro-

ein was only detected after co-precipitation with anti-b-tubulin antibody (Fig. 6C). These observations suggestthat complexes composed of L1 and tubulin exist inextracts from BPV-1 virions infected CV-1 cells.

DISCUSSION

In this study, we have analyzed the mechanism bywhich BPV-1 virions are transported to the nucleus afterinternalization within an infected cell and have demon-strated that this process requires energy and an intactmicrotubule network. Further, we have shown that BPV-1L1 capsid protein is associated within the cell in acomplex with b-tubulin. Finally, we have confirmed thefinding of others that after exposure of a cell to BPV-1,intact virions appear in close relation to the nuclearmembrane and shown that this process is also energyand microtubule dependent. Thus, it seems probablethat the process of infection with BPV-1 requires thetransport of intact virions by microtubules to the nuclearmembrane. Formal confirmation of this would requiredemonstration that transformation of C127 cells by BPV-1,the only currently available quantitative in vitro assay for

PV-1 replication (Law et al., 1981), is inhibited by micro-ubule depolymerization. Attempts to carry out this ex-eriment have not been successful, probably becauserolonged exposure to nocodazole arrests the cell cycle

Zieve et al., 1980) and focus formation requires cyclingells, while short-term exposure to nocodazole disruptsicrotubules only transiently (De Brabander et al., 1976;amilton and Snyder, 1982), allowing the cell cycle andV infection to progress.

The cytoskeleton, which consists of microtubules, mi-rofilaments, and intermediate filaments, is believed tolay a role in cell shape, movement, and division as wells in endocytosis, and various components of the cy-

oskeleton are used by viruses for intracellular transport.n integral microtubule network is an essential factor in

he replication of adenovirus types 2 and 5 and reovirusype 3 (Dales and Chardonnet, 1973; Quesada and Gut-erman, 1986). Herpes simplex virus type 1 also uses the

icrotubule network for axonal transport (Kristensson etl., 1986; Sodeik et al., 1997). The polyoma virus, adeno-

irus type 2 and 5, reovirus and Theiler’s murine enceph-lomyelitis virus all interact with 10-nm filaments of cy-

f(

toskeleton, and poliovirus and SV40 virus interact withmicrofilaments (Miles et al., 1980; Lycke and Tsiang,1987; Ben Ze’ev, 1984; Sharpe et al., 1982; Nedellec et al.,1998).

Nocodazole, which almost completely inhibited BPV-1transport in the present study, disrupts the microtubulenetwork by inhibiting polymerization of tubulin mono-mers (Cleveland et al., 1981; Lau et al., 1986) and hasbeen widely used to study the association of viruses withmicrotubules (Storrie et al., 1998; Candurra et al., 1999;Hirschberg et al., 1998; Bayer et al., 1998). Glu-tubulin, a

ocodazole-resistant minor variant of a-tubulin (Khawajaet al., 1988; Thyberg and Moskalewski, 1989), might ex-plain any nocodazole-resistant BPV-1 transport. Nocoda-zole does not inhibit cell-surface protein expression(Kizhatil and Albritton, 1997), and in the present study didnot alter BPV-1 binding and uptake. As no significantinhibition of virus transport was observed when nocoda-zole was added 1 h postinfection, the microtubule-de-pendent early step of PV entry was complete within thistime, in keeping with previous data about the timing ofarrival of BPV-1 virions at the nucleus (Muller et al., 1995).The MT network of an interphase cell is a dynamicpolarized structure. Relatively stable minus ends arelocalized to the MT organizing center, which is typicallylocated at perinuclear position in culture cells (Man-delkow and Mandelkow, 1995). The dynamic, fast grow-ing, and fast-shrinking plus ends extend toward the cellperiphery (Desai and Mitchison, 1997). Directional move-ment along MTs is mediated by motor proteins, whichhydrolyze ATP to induce conformational changes in theirstructure. Motors are classified according to the se-quences of their motor domains and the directionality oftheir motion (Vale and Fletterick, 1997; Hirokawa, 1998).Kinesin superfamily motors typically move toward the MTplus ends, whereas dynein motors mediate minus end-directed movement. Since BPV-1 virions move from cellsurface to the nucleus, we hypothesized that dyneinmight facilitate the movement of virions. However, wefailed to detect any dynein protein in the immunoprecipi-tates, possibly because of the specificity and affinity ofthe available antibody.

Viral entry into cells can occur through incorporationinto endosomes, by uptake into clathrin-coated pits, or,for membrane-coated viruses, by membrane fusion. Afterexposure of cells to BPV-1, virus can be seen in lipid layersurrounded vesicles (Zhou et al., 1995), suggesting that

t least some viral uptake is endosome mediated. How-ver, in this and previous studies, free virus is alsobserved by electron microscopy in the cytoplasm and,s large quantities of virus have been used in suchtudies to allow visualization of intracellular virions, itas been uncertain whether endocytosis represents aignificant route of uptake of virus for infection. Transport

rom early to late endosomes is microtubule dependentAniento et al., 1993; Clague et al., 1994; Gruenberg and

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242 LIU ET AL.

Howell, 1989), and the observation of small cytoplasmicvacuole-like structures in BPV-1 virus infected CV-1 cellssuggested that there is a microtubule-dependent trans-fer step within the cytoplasm. These results, taken to-gether with previous work by ourselves and others dem-onstrating that cytochalasin B, which can destroy micro-filaments, affects receptor-mediated endocytosis ofBPV-1 virions, allows the hypothesis that virus uptakeconsists of an initial microfilament-dependent endocy-totic process. Release of virions from endocytotic vesi-cles is apparently by an acid-independent step (Zhou etal., 1995) in contrast to the acid-dependent release ofadenovirus (Greber et al., 1993) and subsequent trans-port of naked virions to the cell nucleus is in associationwith microtubules. This mechanism is similar to thatemployed by the other papovaviridiae, including poly-omavirus and SV40 (Clever et al., 1991; Mackay andConsigli, 1976; Barbanti-Brodano et al., 1970).

MATERIALS AND METHODS

Cells and antibodies, chemicals

CV-1 cells were cultured in DMEM (Gibco) with 10%fetal bovine serum (FBS), nonessential amino acids, 100U/ml penicillin, 100 mg/streptomycin, and 2 mM glu-tamine. Cell lines were maintained as adherent culturesin a 5% CO2 humid incubator at 37°C and passagedtwice weekly. Rabbit anti-BPV-1 antiserum (Dako), anti-tubulin CY3-conjugated antibody, and FITC-conjugatedgoat anti-mouse antibody (Sigma) were purchased com-mercially. Mouse monoclonal antibody (Mab) MC15 (Kul-ski et al., 1998) against BPV-1L1 and a mouse Mab C1(Liu et al., 1997) against an epitope of BPV-1L2 exposedon the BPV-1 viral surface have been previously de-scribed. Drugs were dissolved as 10003 stocks inDMSO and used as indicated. Control treatments in-cluded appropriately diluted DMSO.

Purification of BPV-1 virions from cattle warts

Purification of BPV-1 virions has been previously de-scribed (Liu et al., 1997). Briefly, cattle warts, typed asBPV-1 by PCR, were minced and homogenized in extrac-tion buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2, 50 mM

aCl2, 150 mM NaCl, 0.01% Triton X-100, and 1 mMMSF). The minced tissue lysate was cleared by centrif-gation in a Beckman SW-28 rotor for 20 min at 6000 rpm,nd virions were concentrated by pelleting them in aeckman SW-28 rotor for 2 h at 27,000 rpm. The pellets

were resuspended in extraction buffer, and virions en-riched by centrifugation through a 40% sucrose cushion.Virions were further purified by CsCl density gradient.

Purified BPV-1 virions were examined on a 10% SDS gelfor purity.

Indirect immunofluorescence

Cells grown on glass coverslips were infected withBPV-1 virions at multiplicity of infection (m.o.i.) of 1000virions per cell. This high dose of input BPV-1 virion wasrequired to visualize the signal from incoming viral pro-teins by immunofluorescence (IF). Virus was bound tocells in the presence of 10% FBS at 4°C for 1 h withgentle shaking. Cell monolayers were washed with ice-cold DMEM to remove unbound virions. To allow inter-nalization of virions, monolayers were shifted to 37°Cand analyzed by IF. For the drug treatment, as indicated,cells were incubated for 2 h in medium containing 20 mM

ocodazole, or 20 mM taxol (Sigma) at 37°C, beforeinfection with BPV-1. After the BPV-1 virions were boundto the cells at 4°C for 1 h, the monolayers were washedwith DMEM medium to remove free BPV-1 virions. Me-dium with or without chemicals was added again and thecells were incubated at 37°C for different times. Thecells were then washed with phosphate-buffered saline(PBS), lysed in cytoskeleton buffer (CSK; 100 mM NaCl,300 mM sucrose, 10 mM PIPES, pH 6.8, 3 mM MgCl2, 1mM EGTA, 1 mM phenylmethyl sulfonyl fluoride (PMSF),and 0.5% Triton X-100, 1 mM taxol) for 30 s, fixed with 80%ethanol for 10 min, and processed for indirect IF. Mono-clonal antibody MC15 was used at a 1:1000 dilution forthe detection of BPV-1L1, and the monoclonal Mab C1against BPV-1L2 surface epitope at a 1:1000 dilution wasapplied to identify the L2 protein. After washing to re-move unbound antibodies, the secondary antibody FITC-conjugated anti-mouse IgG (Sigma) was used to detectL11L2 (green) proteins of BPV-1 virions. For doublestaining identifying both tubulin protein and BPV-1 cap-sids, the primarily mouse Mab C1 anti-BPV-1L2 wasapplied to cells infected by BPV-1virions, followed bysecondary antibodies anti-mouse IgG (FITC-conjugated)to detect BPV-1L2 protein. Cy3-conjugated antitubulinantibody was then added for identified of the tubulinprotein.

Immunoprecipitation and immunoblotting

Coprecipitation experiments were performed using amodified method of Maxwell and Arlinghaus (1981). CV-1cells (2 3 106) infected with BPV-1 virions, or not infected,were harvested by scraping and lysed in 1 ml of 50 mMTN buffer (Tris–HCl, pH 8.0, 1% Nonidet-P40) for 30 min at4°C. Nuclei were removed from lysate by centrifugationat 10,000 rpm for 2 min at 4°C. The cytoplasmic fractionswere preadsorbed with normal rabbit/mouse serum for30 min at 4°C. Specific antiserum (1–2 ml) was added tothe mixed extract and rocked for 4 h at 4°C. Immunecomplexes were harvested by absorption onto Protein Abeads, washed vigorously 3 times in TN buffer, and thenprepared for electrophoresis by disruption in 40 ml of

SDS sample buffer (2% SDS, 1.5% dithiothreitol, 10% glyc-erol, 160 mM Tris, pH 6.8, 12 g/ml bromphenol blue). After

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243BOVINE PAPILLOMAVIRUS TYPE 1 MICROTUBULES

the samples were separated on 10% polyacrylamide–SDS gels, samples were transferred onto nitrocellulosemembranes by electroblotting in transfer buffer (25 mMTris, 190 mM glycine, 20% methanol). Membranes wereblocked for 1 h at 37°C with 5% nonfat milk in PBScontaining 0.05% Tween 20. After three washes, the blotswere incubated for 1 h at 37°C with rabbit anti-BPV-1antiserum (Dako) at 1:2000 dilution or mouse antitubulinmonoclonal antibody (Sigma). After further three washes,the membranes were incubated with horseradish perox-idase (HRP)-conjugated sheep anti-mouse IgG or withHRP-conjugated anti-rabbit IgG antibodies at 1:10,000dilutions. Specific protein bands were visualized usingthe enhanced chemiluminescence detection system(Amersham).

Electron microscopy

To visualize BPV-1 virions attached to microtubules incells, CV-1 cells grown on culture dishes for 1 day to 80%confluency were washed with PBS, and approximately1000 PFU BPV-1 virions/cell was added to the cell mono-layers. After incubation at 4°C for 1 h, cells were washedwith PBS, removing unbound BPV-1 virions, and cellswere extracted with 0.5% Triton X-100 in MT CSK for 1min at room temperature and fixed for 30 min with 1%glutaraldehyde (GA). After fixation, the cells were con-trasted using 2% OsO4 for 10 min and 2% uranyl acetatein 50 mM maleate buffer, pH 5.2, for 1 h. They wereembedded in Epon and ultrathin sections parallel to thesubstrate were prepared. All sections were further con-trasted using lead citrate and uranyl acetate.

ACKNOWLEDGMENTS

This work was supported by grants from the University of Queens-land, the Queensland Cancer Fund, and the Princess Alexandra Hos-pital Foundation.

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