Association of Bovine Papillomavirus Type 1 with Microtubules

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

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provided by Elsevier - Publisher Connector 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. Electron microscopy showed BPV-1 virions in association with microtubules in the cytoplasm, a finding confirmed by co-immunoprecipitation 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

INTRODUCTION been widely used as a model system for studying PV infection. The infectious entry pathway of BPV-1 starts Before viruses can infect and replicate in a host cell, with the binding of the virus to specific cell receptors, they must cross the plasma membrane and then target including ␣6-integrin protein (Evander et al., 1997) and their genome and accessory proteins to the correct or- heparin-like molecules (Joyce et al., 1999). BPV-1 crosses ganelle, where gene transcription, nucleic acid replica- the plasma membrane by an unknown mechanism and tion, and viral maturation can take place. After a virus moves through the cytosol to the nuclear membrane penetrates the cell membrane, the cytoplasm imposes a (Muller et al., 1995), which it must penetrate to replicate diffusion barrier. Within the eukaryotic cytoplasm, or- in the nucleus. Papillomaviruses are not expected to ganelles, solutes, and a complex latticelike mesh of move by free diffusion through the cytoplasm because of microtubule, actin, and intermediate filament networks their size, but little is known about their mechanism of restrict free diffusion of macromolecular complexes intracytoplasmic transport. We therefore analyzed the larger than 500 kDa (Luby-Phelps, 2000; Seksek et al., transport of BPV-1 virions, purified from cattle warts, to 1997). Movement of large complexes, including mem- the nucleus of a cultured cell line, and this appears to be brane organelles and ribonucleoprotein particles, gener- an energy-dependent process involving intact microtu- ally involves either actin or the microtubule (MT) network bules. (Grunert and St Johnston, 1996; Hirokawa et al., 1998; Holleran et al., 1998; Mermall et al., 1998). The role of MT RESULTS in the intracellular transport of particles and organelles has been documented (Toomre et al., 1999). Physiological temperature is needed for nuclear Some nonenveloped viruses use similar transport targeting of BPV-1 mechanisms to macromolecules within the cell. Adeno- BPV-1 particles are targeted to the cell nucleus after viruses, for example, are carried by microtubules to the uptake into the cytoplasm (Zhou et al., 1995). Diffusion cell nucleus where they are decapsidated near nuclear of artificial particles larger than 50 nm in diameter is pores to allow DNA to enter the nucleus (Suomalainen et restricted by the structural organization of the cyto- al., 1999), while herpes simplex and rabies viruses are plasm (Luby-Phelps, 2000). BPV-1 particles are ap- transported retrogradely in neuronal axons to infect the proximately 50 nm and thus would be expected to cell body (Kristensson et al., 1986; Miranda-Saksena et have minimal diffusion mobility under physiological al., 2000). Papillomaviruses (PV) are epitheliotropic dou- conditions. To test whether BPV-1 is actively trans- ble-stranded DNA viruses. The virions are nonenveloped ported in the cytoplasm by an energy-dependent pro- and have a 55-nm icosahedral structure (DeLap et al., cess, we bound BPV-1 virions to cells for1hat4°C, 1977; Galloway and McDougall, 1989). Bovine PV has washed off unbound virus, and increased the temperature to 37°C for 20 min. During this time, virions were taken up into the cytoplasm where they appeared as 1 To whom reprint requests should be addressed. Fax: ϩ61-7-3240 small, intensely labeled spots (data not shown). Cells 5946. E-mail: [email protected]. were then cooled to 4°C and held for up to 60 min:

FIG. 1. Bovine papillomavirus transport to the nucleus requires physiological temperature. CV-1 cells were infected by BPV-1 virions for 60 min at 4°C (A, B, D, E, F, H) or were similarly infected in taxol-containing medium after a 30-min preincubation of cells at 37°C in taxol (20 ␮M) (C, G). All cells were then held at 37°C for 20 min to allow virus internalization. Cells were then held at 37°C for 60 min (A, E) or held for the same period at 4°C (other groups). Some cells (D, H) were then held at 37°C for a further 60 min. Cells were stained for BPV-1 L1 protein (A–D; green) or ␤-tubulin (E–H; red). FIG. 2. Colocalization of incoming BPV-1 virions with microtubules. CV-1 cells were infected by BPV-1 virions at an m.o.i. of 1000. 30 min and 60 min after infection, cells were fixed with 80% ethanol and double labeled with anti-BPV-1 L2 (green) and anti-␤-tubulin (red). At 30 min postinfection (PI), capsids are scattered in the cytoplasm and colocalize with microtubules (A). At 60 min PI, most of the capsids are localized on the nuclear membrane, and the other capsids are colocalized with microtubules (B). In some cells, BPV-1 viral antigen is located in microtubule organization like centers (C). control cells were kept at 37°C. Since exposure of was released by incubating cells at 37°C for another cells to reduced temperatures causes depolymeriza- 60 min, most of the BPV-1 L1 was located around the tion of MTs (Fig. 1F), targeting was analyzed in cells nucleus (Fig. 1D). These results confirmed that incom- that have been pretreated with the MT stabilizing drug ing BPV-1 virions do not move through the cytoplasm taxol (Fig. 1G). In cells held at 37°C, most of the BPV-1 by diffusion and suggested that transport is actively antigen could be detected in the perinuclear zone at associated with microtubules. 60 min (Fig. 1A), in agreement with previous results (Zhou et al., 1995). In contrast, in cells held at 4°C with Binding of BPV-1 virions to microtubules or without taxol treatment, virus antigen was predom- inantly localized in numerous small vesicle-like struc- To investigate the role of microtubule-mediated tures throughout the cytoplasm at 60 min (Figs. 1B and transport in the perinuclear location of BPV, CV-1 cells 1C) and at 90 min (data not shown), indicating a failure were incubated for2hat37°C with 20 ␮M of taxol, of the viral antigen to reach the nuclear membrane. which prevents the disassembly of microtubules, and Staining for tubulin of cells held at 4°C showed exten- then were infected with BPV-1 virions. Colocation of sive disruption of microtubules compared with cells ␤-tubulin and BPV-1 was examined by double immu- held at 37°C, or cells held at 4°C after taxol treatment nofluorescence. At 30 min postinfection, most of the (Figs. 1E, 1F, and 1G). When the temperature block BPV-1 particles appeared to be restricted to small BOVINE PAPILLOMAVIRUS TYPE 1 MICROTUBULES 239

nucleus depended on intact MTs, CV-1 cells were incubated with nocodazole, a specific inhibitor of tubulin polymerization into microtubules (De Brabander et al., 1977), for 2 h. Virus was subsequently bound to the cell surface for 60 min at 4°C. Cells were then held at 37°C in nocodazole-containing medium for 60 min. Under these conditions, MTs were completely depolymerized, as shown by anti-␤-tubulin immunostaining, and localization of BPV-1 virus to the nuclear envelope was inhibited (Figs. 4C and 4D). Similar results were obtained if incubation at 37°C was extended to 135 min. In contrast, untreated cells and cells treated with taxol accumulated BPV-1 virions on the nuclear membrane (Figs. 4A and 4B). Nocodazole blocked nuclear trafficking of BPV-1 when added up to 30 min after exposure of the cells to virus, 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, whereby the MT had repolymerized, and previously dispersed capsids were now concentrated on the nuclear rim, similar to cells never treated with nocodazole (Fig. 4F). FIG. 3. Colocalization of incoming BPV-1 capsids with microtubules Taken together, these results suggested that intact MTs on conventional EM. CV-1 cells were infected at an m.o.i. of 1000. are required for targeting of incoming virus particles to Infected CV-1 cells were treated by CSK buffer and fixed by glutaraldehyde. At 30 min, numerous incoming viral capsids were found local- the nucleus. ized to microtubules (MT) (A–C). At 60 min, virions were seen within the nucleus where disrupted forms were seen, suggesting that the virus had started to uncoat and release DNA (arrow). Bar, 100 nm. cytoplasmic vacuole-like structures (Fig. 2A). These fluorescent vacuoles were scattered around the cytoplasm and bound with the tubule network. At 60 min, the majority of BPV-1 antigen accumulated in the perinuclear zone, while some was still found in the cytoplasm and bound with tubule networks (Fig. 2B). In some cells, BPV-1 antigen was observed to accumulate in structures resembling the microtubule organization-like center (MTOC; Fig. 2C). Electron microscopy confirmed the close association of BPV-1 capsids with MT. At 30 min after infection, 55-nm size BPV-1 particles could be seen in association with cytoplasmic filaments with MT-like morphology and the expected 24-nm width. The MT-associated capsids had an electron-dense DNA core (Figs. 3A–3C). At later time points (60 min), hundreds of intact capsids with electron-dense cores were seen bound to the nuclear membrane. Disrupted virus particles were also observed (Fig. 3D), suggesting that BPV-1 virion disassembly and release of viral DNA was occurring by this time. FIG. 4. CV-1 cells exposed to nocodazole or taxol were infected with BPV-1 virions and then held at 37°C for 60 min. Cells were then fixed and permeabilized and labeled with either anti-BPV-1 L1 (A, B, D, F) or Reduced capsid transport to the nucleus without anti-␤-tubulin (C, E) antibody. Under control conditions (A, E) or in taxol microtubules treated cells (B), BPV-1capsids accumulate around the cell nucleus. In the cells pretreated with nocodazole, capsids are scattered throughout As shown above, BPV-1virions were observed in close the entire cytoplasm and microtubules are disrupted (C, D). Nocoda- association with MTs immediately after infection of the zole-treated BPV-1 infected cells, washed and held without nocodazole cell. To determine whether transport of BPV-1 to the for 60 min, accumulate virus at the cell nucleus (F). 240 LIU ET AL.

FIG. 5. Depolymerization of the microtubule network does not affect internalization of BPV-1. BPV-1 virions were bound to CV-1 cells at 4°C. Cells were then brought to 37°C in the presence (lanes 7–12) or absence (lanes 1–6) of nocodazole and held for the indicated times. Cells were then held at 4°C for 1 h with or without (lanes 1, 7) added trypsin to remove bound external virus, washed, and subjected to SDS–PAGE and immunoblotting with anti-L1 Ab for detection of internalized virus.

Depolymerization of the microtubule network does min. Entry at 20-min postwarming was close to 75% with not down-regulate BPV-1 binding and internalization or without nocodazole. Thus, BPV-1 virus binding to the cell surface and internalization does not require an intact To establish whether cell-surface expression of BPV-1 microtubule network, in keeping with the observation receptors was down-regulated or virus endocytosis was that nocodazole has no effect on the formation of endo- impaired following nocodazole treatment of cells, we cytic vesicles (Gruenberg et al., 1989). measured binding and internalization of BPV-1 into drug- treated cells using an assay that measures virus uptake Interaction of BPV-1 with microtubules in vivo by detecting a trypsin-resistant major capsid protein L1. Nocodazole-treated and untreated CV-1 cells bound sim- Double-labeling immunofluorescence microscopy and ilar amounts of BPV-1 virions after 1 h exposure to virus electron microscopy demonstrates an association of at 4°C (Fig. 5). Approximately 50% of the virus particles BPV-1 with microtubules after entry into the cell. To were endocytosed by 10 min after nocodazole-treated demonstrate whether the BPV-1 virion L1 capsid protein cells were warmed to 37°C, whereas for untreated cells interacts with tubulin protein, a reciprocal immunopre- the half-time of internalization was slightly less than 10 cipitation experiment was performed (Fig. 6). Lysate from

FIG. 6. Coimmunoprecipitation of L1 protein or tubulin from extracts of CV-1 cells infected with BPV-1 (V), or not so infected (NV), using monoclonal antibody against tubulin or using rabbit polyclonal antibody against BPV-1 virions. BPV-1 virion infected or uninfected CV-1 cell extracts were precipitated by antitubulin antibody (anti-tub V/anti-tub NV) in A; meanwhile the same extracts were precipitated by anti-BPV-1 virions (anti-L1 V/anti-L1 NV) in B. Precipitated proteins and control precipitations with antibody to BPV-1 virions (BPV-1cont) and to tubulin (tub cont) were separated in 10% SDS–polyacrylamide gels and transferred to nitrocellulose membranes which were labeled with rabbit anti-BPV-1 L1 antibody (A) or with monoclonal antitubulin antibody (B) followed by second antibody incubation. Arrows mark L1 and tubulin proteins. (C) Specific co-immunoprecipitation of BPV-1 virion from BPV-1 infected/uninfected CV-1 cell extracts suing antitubulin (anti-tub V/lane anti-tub NV) or anti-pan-cytokeratin (lane anti-p-ker). L1 protein is indicated by arrow. BOVINE PAPILLOMAVIRUS TYPE 1 MICROTUBULES 241

CV-1 cells infected or not infected with BPV-1 virions was toskeleton, and poliovirus and SV40 virus interact with immunoprecipitated with either a monoclonal antibody microfilaments (Miles et al., 1980; Lycke and Tsiang, against ␤-tubulin (Fig. 6A) or a rabbit antiserum against 1987; Ben Ze’ev, 1984; Sharpe et al., 1982; Nedellec et al., BPV-1 virions (Fig. 6B). Precipitates were probed with the 1998). rabbit anti-L1 serum (Fig. 6A) or the monoclonal antibody Nocodazole, which almost completely inhibited BPV-1 against ␤-tubulin (Fig. 6B). L1 protein was coprecipitated transport in the present study, disrupts the microtubule with ␤-tubulin antibody (Fig. 6A, lane 2) and conversely, network by inhibiting polymerization of tubulin mono- tubulin protein was coprecipitated by anti-BPV-1 virion mers (Cleveland et al., 1981; Lau et al., 1986) and has antibody (Fig. 6). To confirm the specificity of the inter- been widely used to study the association of viruses with action of BPV-1 with tubulin, antibodies against cytoker- microtubules (Storrie et al., 1998; Candurra et al., 1999; atins were used in co-immunoprecipitation, and L1 pro- Hirschberg et al., 1998; Bayer et al., 1998). Glu-tubulin, a tein was only detected after co-precipitation with anti-␤- nocodazole-resistant minor variant of ␣-tubulin (Khawaja tubulin antibody (Fig. 6C). These observations suggest et al., 1988; Thyberg and Moskalewski, 1989), might ex- that complexes composed of L1 and tubulin exist in plain any nocodazole-resistant BPV-1 transport. Nocoda- extracts from BPV-1 virions infected CV-1 cells. zole does not inhibit cell-surface protein expression (Kizhatil and Albritton, 1997), and in the present study did DISCUSSION not alter BPV-1 binding and uptake. As no significant inhibition of virus transport was observed when nocoda- In this study, we have analyzed the mechanism by zole was added 1 h postinfection, the microtubule-de- which BPV-1 virions are transported to the nucleus after pendent early step of PV entry was complete within this internalization within an infected cell and have demon- time, in keeping with previous data about the timing of strated that this process requires energy and an intact arrival of BPV-1 virions at the nucleus (Muller et al., 1995). microtubule network. Further, we have shown that BPV-1 The MT network of an interphase cell is a dynamic L1 capsid protein is associated within the cell in a polarized structure. Relatively stable minus ends are complex with ␤-tubulin. Finally, we have confirmed the localized to the MT organizing center, which is typically finding of others that after exposure of a cell to BPV-1, located at perinuclear position in culture cells (Man- intact virions appear in close relation to the nuclear delkow and Mandelkow, 1995). The dynamic, fast grow- membrane and shown that this process is also energy ing, and fast-shrinking plus ends extend toward the cell and microtubule dependent. Thus, it seems probable periphery (Desai and Mitchison, 1997). Directional move- that the process of infection with BPV-1 requires the ment along MTs is mediated by motor proteins, which transport of intact virions by microtubules to the nuclear hydrolyze ATP to induce conformational changes in their membrane. Formal confirmation of this would require structure. Motors are classified according to the se- demonstration that transformation of C127 cells by BPV-1, quences of their motor domains and the directionality of the only currently available quantitative in vitro assay for their motion (Vale and Fletterick, 1997; Hirokawa, 1998). BPV-1 replication (Law et al., 1981), is inhibited by micro- Kinesin superfamily motors typically move toward the MT tubule depolymerization. Attempts to carry out this ex- plus ends, whereas dynein motors mediate minus end- periment have not been successful, probably because directed movement. Since BPV-1 virions move from cell prolonged exposure to nocodazole arrests the cell cycle surface to the nucleus, we hypothesized that dynein (Zieve et al., 1980) and focus formation requires cycling might facilitate the movement of virions. However, we cells, while short-term exposure to nocodazole disrupts failed to detect any dynein protein in the immunoprecipi- microtubules only transiently (De Brabander et al., 1976; tates, possibly because of the specificity and affinity of Hamilton and Snyder, 1982), allowing the cell cycle and the available antibody. PV infection to progress. Viral entry into cells can occur through incorporation The cytoskeleton, which consists of microtubules, mi- into endosomes, by uptake into clathrin-coated pits, or, crofilaments, and intermediate filaments, is believed to for membrane-coated viruses, by membrane fusion. After play a role in cell shape, movement, and division as well exposure of cells to BPV-1, virus can be seen in lipid layer as in endocytosis, and various components of the cy- surrounded vesicles (Zhou et al., 1995), suggesting that toskeleton are used by viruses for intracellular transport. at least some viral uptake is endosome mediated. How- An integral microtubule network is an essential factor in ever, in this and previous studies, free virus is also the replication of adenovirus types 2 and 5 and reovirus observed by electron microscopy in the cytoplasm and, type 3 (Dales and Chardonnet, 1973; Quesada and Gut- as large quantities of virus have been used in such terman, 1986). Herpes simplex virus type 1 also uses the studies to allow visualization of intracellular virions, it microtubule network for axonal transport (Kristensson et has been uncertain whether endocytosis represents a al., 1986; Sodeik et al., 1997). The polyoma virus, adeno- significant route of uptake of virus for infection. Transport virus type 2 and 5, reovirus and Theiler’s murine enceph- from early to late endosomes is microtubule dependent alomyelitis virus all interact with 10-nm filaments of cy- (Aniento et al., 1993; Clague et al., 1994; Gruenberg and 242 LIU ET AL.

Howell, 1989), and the observation of small cytoplasmic Indirect immunofluorescence vacuole-like structures in BPV-1 virus infected CV-1 cells Cells grown on glass coverslips were infected with suggested that there is a microtubule-dependent trans- BPV-1 virions at multiplicity of infection (m.o.i.) of 1000 fer step within the cytoplasm. These results, taken to- virions per cell. This high dose of input BPV-1 virion was gether with previous work by ourselves and others dem- required to visualize the signal from incoming viral pro- onstrating that cytochalasin B, which can destroy micro- teins by immunofluorescence (IF). Virus was bound to filaments, affects receptor-mediated endocytosis of cells in the presence of 10% FBS at 4°C for 1 h with BPV-1 virions, allows the hypothesis that virus uptake gentle shaking. Cell monolayers were washed with ice- consists of an initial microfilament-dependent endocy- cold DMEM to remove unbound virions. To allow inter- totic process. Release of virions from endocytotic vesi- nalization of virions, monolayers were shifted to 37°C cles is apparently by an acid-independent step (Zhou et and analyzed by IF. For the drug treatment, as indicated, al., 1995) in contrast to the acid-dependent release of cells were incubated for2hinmedium containing 20 ␮M adenovirus (Greber et al., 1993) and subsequent trans- nocodazole, or 20 ␮M taxol (Sigma) at 37°C, before port of naked virions to the cell nucleus is in association infection with BPV-1. After the BPV-1 virions were bound with microtubules. This mechanism is similar to that to the cells at 4°C for 1 h, the monolayers were washed employed by the other papovaviridiae, including poly- with DMEM medium to remove free BPV-1 virions. Me- omavirus and SV40 (Clever et al., 1991; Mackay and dium with or without chemicals was added again and the Consigli, 1976; Barbanti-Brodano et al., 1970). cells were incubated at 37°C for different times. The cells were then washed with phosphate-buffered saline (PBS), lysed in cytoskeleton buffer (CSK; 100 mM NaCl, MATERIALS AND METHODS 300 mM sucrose, 10 mM PIPES, pH 6.8, 3 mM MgCl2,1 Cells and antibodies, chemicals mM EGTA, 1 mM phenylmethyl sulfonyl fluoride (PMSF), and 0.5% Triton X-100, 1 ␮M taxol) for 30 s, fixed with 80% CV-1 cells were cultured in DMEM (Gibco) with 10% ethanol for 10 min, and processed for indirect IF. Mono- fetal bovine serum (FBS), nonessential amino acids, 100 clonal antibody MC15 was used at a 1:1000 dilution for U/ml penicillin, 100 ␮g/streptomycin, and 2 mM glu- the detection of BPV-1L1, and the monoclonal Mab C1 tamine. Cell lines were maintained as adherent cultures against BPV-1L2 surface epitope at a 1:1000 dilution was ina5%CO2 humid incubator at 37°C and passaged applied to identify the L2 protein. After washing to re- twice weekly. Rabbit anti-BPV-1 antiserum (Dako), anti- move unbound antibodies, the secondary antibody FITC- tubulin CY3-conjugated antibody, and FITC-conjugated conjugated anti-mouse IgG (Sigma) was used to detect goat anti-mouse antibody (Sigma) were purchased com- L1ϩL2 (green) proteins of BPV-1 virions. For double mercially. Mouse monoclonal antibody (Mab) MC15 (Kul- staining identifying both tubulin protein and BPV-1 cap- ski et al., 1998) against BPV-1L1 and a mouse Mab C1 sids, the primarily mouse Mab C1 anti-BPV-1L2 was (Liu et al., 1997) against an epitope of BPV-1L2 exposed applied to cells infected by BPV-1virions, followed by on the BPV-1 viral surface have been previously de- secondary antibodies anti-mouse IgG (FITC-conjugated) scribed. Drugs were dissolved as 1000ϫ stocks in to detect BPV-1L2 protein. Cy3-conjugated antitubulin DMSO and used as indicated. Control treatments in- antibody was then added for identified of the tubulin cluded appropriately diluted DMSO. protein.

Immunoprecipitation and immunoblotting Purification of BPV-1 virions from cattle warts Coprecipitation experiments were performed using a Purification of BPV-1 virions has been previously de- modified method of Maxwell and Arlinghaus (1981). CV-1 scribed (Liu et al., 1997). Briefly, cattle warts, typed as cells (2 ϫ 10 6) infected with BPV-1 virions, or not infected, BPV-1 by PCR, were minced and homogenized in extrac- were harvested by scraping and lysed in 1 ml of 50 mM tion buffer (20 mM HEPES, pH 7.4, 10 mM MgCl2,50mM TN buffer (Tris–HCl, pH 8.0, 1% Nonidet-P40) for 30 min at CaCl2, 150 mM NaCl, 0.01% Triton X-100, and 1 mM 4°C. Nuclei were removed from lysate by centrifugation PMSF). The minced tissue lysate was cleared by centrif- at 10,000 rpm for 2 min at 4°C. The cytoplasmic fractions ugation in a Beckman SW-28 rotor for 20 min at 6000 rpm, were preadsorbed with normal rabbit/mouse serum for and virions were concentrated by pelleting them in a 30 min at 4°C. Specific antiserum (1–2 ␮l) was added to Beckman SW-28 rotor for2hat27,000 rpm. The pellets the mixed extract and rocked for4hat4°C. Immune were resuspended in extraction buffer, and virions en- complexes were harvested by absorption onto Protein A riched by centrifugation through a 40% sucrose cushion. beads, washed vigorously 3 times in TN buffer, and then Virions were further purified by CsCl density gradient. prepared for electrophoresis by disruption in 40 ␮lof Purified BPV-1 virions were examined on a 10% SDS gel SDS sample buffer (2% SDS, 1.5% dithiothreitol, 10% glyc- for purity. erol, 160 mM Tris, pH 6.8, 12 g/ml bromphenol blue). After BOVINE PAPILLOMAVIRUS TYPE 1 MICROTUBULES 243 the samples were separated on 10% polyacrylamide– Cleveland, D. W., Lopata, M. A., Sherline, P., and Kirschner, M. W. (1981). SDS gels, samples were transferred onto nitrocellulose Unpolymerized tubulin modulates the level of tubulin mRNAs. Cell membranes by electroblotting in transfer buffer (25 mM 25, 537–546. Clever, J., Yamada, M., and Kasamatsu, H. (1991). Import of simian virus Tris, 190 mM glycine, 20% methanol). Membranes were 40 virions through nuclear pore complexes. Proc. Natl. Acad. Sci. blocked for1hat37°C with 5% nonfat milk in PBS USA 88, 7333–7337. containing 0.05% Tween 20. After three washes, the blots Dales, S., and Chardonnet, Y. (1973). Early events in the interaction of were incubated for1hat37°C with rabbit anti-BPV-1 adenoviruses with HeLa cells. IV. Association with microtubules and antiserum (Dako) at 1:2000 dilution or mouse antitubulin the nuclear pore complex during vectorial movement of the inocu- lum. Virology 56, 465–483. monoclonal antibody (Sigma). After further three washes, De Brabander, M., De May, J., Joniau, M., and Geuens, G. (1977). the membranes were incubated with horseradish perox- Ultrastructural immunocytochemical distribution of tubulin in cul- idase (HRP)-conjugated sheep anti-mouse IgG or with tured cells treated with microtubule inhibitors. Cell Biol. Int. Rep. 1, HRP-conjugated anti-rabbit IgG antibodies at 1:10,000 177–183. dilutions. Specific protein bands were visualized using De Brabander, M., Van, D., V, Aerts, F., Geuens, S., and Hoebeke, J. (1976). A new culture model facilitating rapid quantitative testing of the enhanced chemiluminescence detection system mitotic spindle inhibition in mammalian cells. J. Natl. Cancer Inst. 56, (Amersham). 357–363. DeLap, R. J., Yanagi, K., and Rush, M. G. (1977). Analysis of the structure Electron microscopy of human papilloma virus DNA. Arch. Virol. 54, 263–269. Desai, A., and Mitchison, T. J. (1997). Microtubule polymerization dy- To visualize BPV-1 virions attached to microtubules in namics. Annu. Rev. Cell Dev. Biol. 13, 83–117. cells, CV-1 cells grown on culture dishes for 1 day to 80% Evander, M., Frazer, I. H., Payne, E., Qi, Y. M., Hengst, K., and McMillan, confluency were washed with PBS, and approximately N. A. (1997). Identification of the alpha6 integrin as a candidate receptor for papillomaviruses. J. Virol. 71, 2449–2456. 1000 PFU BPV-1 virions/cell was added to the cell mono- Galloway, D. A., and McDougall, J. K. (1989). Human papillomaviruses layers. After incubation at 4°C for 1 h, cells were washed and carcinomas. Adv. Virus Res. 37, 125–171. with PBS, removing unbound BPV-1 virions, and cells Greber, U. F., Willetts, M., Webster, P., and Helenius, A. (1993). Stepwise were extracted with 0.5% Triton X-100 in MT CSK for 1 dismantling of adenovirus 2 during entry into cells. Cell 75, 477–486. min at room temperature and fixed for 30 min with 1% Gruenberg, J., Griffiths, G., and Howell, K. E. (1989). Characterization of the early endosome and putative endocytic carrier vesicles in vivo glutaraldehyde (GA). After fixation, the cells were con- and with an assay of vesicle fusion in vitro. J. Cell Biol. 108, 1301– trasted using 2% OsO4 for 10 min and 2% uranyl acetate 1316. in 50 mM maleate buffer, pH 5.2, for 1 h. They were Gruenberg, J., and Howell, K. E. (1989). Membrane traffic in endocyto- embedded in Epon and ultrathin sections parallel to the sis: Insights from cell-free assays. Annu. Rev. Cell Biol. 5, 453–481. substrate were prepared. All sections were further con- Grunert, S., and St Johnston, D. (1996). RNA localization and the devel- opment of asymmetry during Drosophila oogenesis. Curr. Opin. trasted using lead citrate and uranyl acetate. Genet. Dev. 6, 395–402. Hamilton, B. T., and Snyder, J. A. (1982). Rapid completion of mitosis and ACKNOWLEDGMENTS cytokinesis in PtK cells following release from nocodazole arrest. Eur. J. Cell Biol. 28, 190–194. This work was supported by grants from the University of Queens- Hirokawa, N. (1998). Kinesin and dynein superfamily proteins and the land, the Queensland Cancer Fund, and the Princess Alexandra Hos- mechanism of organelle transport. Science 279, 519–526. pital Foundation. Hirokawa, N., Noda, Y., and Okada, Y. (1998). Kinesin and dynein superfamily proteins in organelle transport and cell division. Curr. REFERENCES Opin. Cell Biol. 10, 60–73. Hirschberg, K., Miller, C. M., Ellenberg, J., Presley, J. F., Siggia, E. D., Aniento, F., Emans, N., Griffiths, G., and Gruenberg, J. (1993). Cytoplas- Phair, R. D., and Lippincott-Schwartz, J. (1998). Kinetic analysis of mic dynein-dependent vesicular transport from early to late endo- secretory protein traffic and characterization of golgi to plasma somes [published erratum appears in J. Cell Biol. 124(3), 397 (1994)]. membrane transport intermediates in living cells. J. Cell Biol. 143, J. Cell Biol. 123, 1373–1387. 1485–1503. Barbanti-Brodano, G., Swetly, P., and Koprowski, H. (1970). Early events Holleran, E. A., Karki, S., and Holzbaur, E. L. (1998). The role of the in the infection of permissive cells with simian virus 40: Adsorption, dynactin complex in intracellular motility. Int. Rev. Cytol. 182, 69–109. penetration, and uncoating. J. Virol. 6, 78–86. Joyce, J. G., Tung, J. S., Przysiecki, C. T., Cook, J. C., Lehman, E. D., Bayer, N., Schober, D., Prchla, E., Murphy, R. F., Blaas, D., and Fuchs, R. Sands, J. A., Jansens, K. U., and Keller, P. M. (1999). The L1 major (1998). Effect of bafilomycin A1 and nocodazole on endocytic trans- capsid protein of human papillomavirus type 11 recombinant virus- port in HeLa cells: Implications for viral uncoating and infection. like particles interacts with heparin and cell-surface glycosamino- J. Virol. 72, 9645–9655. glycans on human keratinocytes. J. Biol. Chem. 274, 5810–5822. Ben Ze’ev, A. (1984). Inhibition of vimentin synthesis and disruption of Khawaja, S., Gundersen, G. G., and Bulinski, J. C. (1988). Enhanced intermediate filaments in simian virus 40-infected monkey kidney stability of microtubules enriched in detyrosinated tubulin is not a cells. Mol. Cell. Biol. 4, 1880–1889. direct function of detyrosination level. J. Cell Biol. 106, 141–149. Candurra, N. A., Lago, M. J., Maskin, L., and Damonte, E. B. (1999). Kizhatil, K., and Albritton, L. M. (1997). Requirements for different com- Involvement of the cytoskeleton in Junin virus multiplication. J. Gen. ponents of the host cell cytoskeleton distinguish ecotropic murine Virol. 80(Pt. 1), 147–156. leukemia virus entry via endocytosis from entry via surface fusion. Clague, M. J., Urbe, S., Aniento, F., and Gruenberg, J. (1994). Vacuolar J. Virol. 71, 7145–7156. ATPase activity is required for endosomal carrier vesicle formation. Kristensson, K., Lycke, E., Roytta, M., Svennerholm, B., and Vahlne, A. J Biol. Chem. 269, 21–24. (1986). Neuritic transport of herpes simplex virus in rat sensory 244 LIU ET AL.

neurons in vitro. Effects of substances interacting with microtubular A. B., Alonso, A., Zentgraf, H., and Zhou, J. (1995). Papillomavirus function and axonal flow [nocodazole, taxol and erythro-9–3-(2-hy- capsid binding and uptake by cells from different tissues and spe- droxynonyl)adenine]. J. Gen. Virol. 67(Pt. 9), 2023–2028. cies. J. Virol. 69, 948–954. Kulski, J. K., Sadleir, J. W., Kelsall, S. R., Cicchini, M. S., Shellam, G., Nedellec, P., Vicart, P., Laurent-Winter, C., Martinat, C., Prevost, M. C., Peng, S. W., Qi, Y. M., Galloway, D. A., Zhou, J., and Frazer, I. H. (1998). and Brahic, M. (1998). Interaction of Theiler’s virus with intermediate Type specific and genotype cross reactive B epitopes of the L1 filaments of infected cells. J. Virol. 72, 9553–9560. protein of HPV16 defined by a panel of monoclonal antibodies. Quesada, J. R., and Gutterman, J. U. (1986). Psoriasis and alpha-inter- Virology 243, 275–282. feron. Lancet 1(8496), 1466–1468. Lau, J. T., Pittenger, M. F., Havercroft, J. C., and Cleveland, D. W. (1986). Seksek, O., Biwersi, J., and Verkman, A. S. (1997). Translational diffusion Reconstruction of tubulin gene regulation in cultured mammalian of macromolecule-sized solutes in cytoplasm and nucleus. J. Cell cells. Ann. N.Y. Acad. Sci. 466, 75–88. Biol. 138, 131–142. Law, M. F., Lowy, D. R., Dvoretzky, I., and Howley, P. M. (1981). Mouse Sharpe, A. H., Chen, L. B., and Fields, B. N. (1982). The interaction of cells transformed by bovine papillomavirus contain only extrachro- mammalian reoviruses with the cytoskeleton of monkey kidney CV-1 mosomal viral DNA sequences. Proc. Natl. Acad. Sci. USA 78, 2727– cells. Virology 120, 399–411. 2731. Sodeik, B., Ebersold, M. W., and Helenius, A. (1997). Microtubule- Liu, W. J., Gissmann, L., Sun, X. Y., Kanjanahaluethai, A., Mu¨ller, M., mediated transport of incoming herpes simplex virus 1 capsids to the Doorbar, J., and Zhou, J. (1997). Sequence close to the N-terminus of nucleus. J. Cell Biol. 136, 1007–1021. L2 protein is displayed on the surface of bovine papillomavirus type Storrie, B., White, J., Rottger, S., Stelzer, E. H., Suganuma, T., and 1 virions. Virology 227, 474–483. Nilsson, T. (1998). Recycling of golgi-resident glycosyltransferases Luby-Phelps, K. (2000). Cytoarchitecture and physical properties of through the ER reveals a novel pathway and provides an explanation cytoplasm: Volume, viscosity, diffusion, intracellular surface area. Int. for nocodazole-induced Golgi scattering. J. Cell Biol. 143, 1505–1521. Rev. Cytol. 192, 189–221. Suomalainen, M., Nakano, M. Y., Keller, S., Boucke, K., Stidwill, R. P., and Lycke, E., and Tsiang, H. (1987). Rabies virus infection of cultured rat Greber, U. F. (1999). Microtubule-dependent plus and minus end- sensory neurons. J. Virol. 61, 2733–2741. directed motilities are competing processes for nuclear targeting of Mackay, R. L., and Consigli, R. A. (1976). Early events in polyoma virus adenovirus. J. Cell Biol. 144, 657–672. infection: Attachment, penetration, and nuclear entry. J. Virol. 19, 620–636. Thyberg, J., and Moskalewski, S. (1989). Subpopulations of microtu- Mandelkow, E., and Mandelkow, E. M. (1995). Microtubules and micro- bules with differential sensitivity to nocodazole: Role in the structural tubule-associated proteins. Curr. Opin. Cell Biol. 7, 72–81. organization of the Golgi complex and the lysosomal system. J. Maxwell, S., and Arlinghaus, R. B. (1981). In vitro proteolytic cleavage of Submicrosc. Cytol. Pathol. 21, 259–274. Gazdar murine sarcoma virus p65gag. J. Virol. 39, 963–967. Toomre, D., Keller, P., White, J., Olivo, J. C., and Simons, K. (1999). Mermall, V., Post, P. L., and Mooseker, M. S. (1998). Unconventional Dual-color visualization of trans-Golgi network to plasma membrane myosins in cell movement, membrane traffic, and signal transduc- traffic along microtubules in living cells. J. Cell Sci. 112(Pt. 1), 21–33. tion. Science 279, 527–533. Vale, R. D., and Fletterick, R. J. (1997). The design plan of kinesin Miles, B. D., Luftig, R. B., Weatherbee, J. A., Weihing, R. R., and Weber, motors. Annu. Rev. Cell Dev. Biol. 13, 745–777. J. (1980). Quantitation of the interaction between adenovirus types 2 Zhou, J., Gissmann, L., Zentgraf, H., Mu¨ller, H., Picken, M., and Mu¨ller, and 5 and microtubules inside infected cells. Virology 105, 265–269. M. (1995). Early phase in the infection of cultured cells with papillo- Miranda-Saksena, M., Armati, P., Boadle, R. A., Holland, D. J., and mavirus virions. Virology 214, 167–176. Cunningham, A. L. (2000). Anterograde transport of herpes simplex Zieve, G. W., Turnbull, D., Mullins, J. M., and McIntosh, J. R. (1980). virus type 1 in cultured, dissociated human and rat dorsal root Production of large numbers of mitotic mammalian cells by use of the ganglion neurons. J. Virol. 74, 1827–1839. reversible microtubule inhibitor nocodazole. Nocodazole accumu- Muller, M., Gissmann, L., Cristiano, R. J., Sun, X. Y., Frazer, I. H., Jenson, lated mitotic cells. Exp. Cell Res. 126, 397–405.