Virology 261, 271–279 (1999) Article ID viro.1999.9825, available online at http://www.idealibrary.com on
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Expression of the ␣6 Integrin Confers Papillomavirus Binding upon Receptor-Negative B-Cells
Nigel A. J. McMillan,*,1 Elizabeth Payne,* Ian H. Frazer,* and Magnus Evander†
*Centre for Immunology and Cancer Research, Department of Medicine, University of Queensland, Brisbane, Australia; and †Department of Virology, Umeå University, Umeå, Sweden Received March 26, 1999; returned to author for revision April 27, 1999; accepted May 26, 1999
Papillomaviruses (PV) bind to a wide range of cell lines in a specific and saturable manner. We have recently identified a candidate receptor for papillomavirus as the ␣6 integrin (Evander et al., J. Virol. 71, 2449–2456, 1997). We have further investigated the role the ␣6 integrin plays in PV binding. Here we show that the cells expressing the ␣6 integrin, partnered with either the 4 integrin or the 1 integrin, are equally able to bind PV HPV6b L1 virus-like particles, indicating that the beta partner does not play a major role in virus binding. In order to provide definitive evidence that the ␣6 integrin is required for PV binding we undertook to genetically complement the receptor-negative B-cell line DG75 by expressing the human ␣6A gene. The transduction of the ␣6 integrin gene into DG75 cells results in the cell surface expression of the ␣6 protein and this expression confers upon DG75 cells the ability to bind laminin, a normal ligand for ␣6 integrin. Furthermore, the ␣6 protein is partnered with the 1 integrin in DG75 cells. Finally, we show that the DG75-␣6 cells were able to bind papillomavirus VLPs and this binding was inhibited by a functionally blocking anti-␣6 antibody. Together these data indicate that the ␣6 integrin is a primary cell receptor for papillomaviruses and is both necessary and sufficient for PV binding. © 1999 Academic Press
INTRODUCTION The first step in viral infection is the binding of the virus to a host cell. Previous reports have shown that Papillomaviruses (PV) are nonenveloped dsDNA tumor viruses that cause a range of proliferative lesions upon HPV binds to a wide variety of cell lines to varying infection of epithelial cells (Howley, 1996). They infect a degrees (Mu¨ller et al., 1995; Qi et al., 1996; Roden et al., wide range of species but exhibit a high degree of 1994a; Volpers et al., 1995). However, the greatest bind- species specificity (Shah and Howley, 1996). Over 70 ing was observed with epithelial and mesenchymal cells. different human subtypes of PV have been isolated and The PV particle is made of two proteins, L1 and L2. L1 is formally described (de Villiers, 1994; Halpern et al., 1996), the major coat protein, while L2 is thought to form a link each infecting either mucosal or cutaneous epithelium at between the viral DNA and the viral particle (Zhou et al., distinct sites. For example, HPV11 infects mucosal epi- 1994, 1991). PV particles bound in a saturable manner, 4 thelial cells of the genital tract but does not infect cuta- with 1–2 ϫ 10 receptors/cell, while linear L1 protein neous epithelium from other body sites (Kreider et al., binding was nonsaturable (Qi et al., 1996). Binding was 1987). PVs are the causative agent of warts (plantar, trypsin-sensitive and this, along with the wide degree of laryngopharyngeal, and genital) and are also the critical binding to cells from various species, suggests that the factor in the formation of anogenital cancer (Zur Hausen, receptor is a widely conserved protein (Qi et al., 1996; 1994). The cancer-associated subtypes (HPV16, HPV18, Volpers et al., 1995). Furthermore, the same receptor HPV33, etc.) are termed high-risk, while all others are appears to be utilized by all PVs for binding, as particles termed low-risk (Howley, 1996; Shah and Howley, 1996; from one PV species or subtype will inhibit the binding of Zur Hausen, 1994). Unfortunately, the lack of in vitro another (Mu¨ller et al., 1995; Roden et al., 1994a). For replication systems has meant that many critical aspects example, HPV6b inhibited the binding of HPV-11, -16, and of the virus life cycle remain unknown and therefore are BPV (Mu¨ller et al., 1995). not readily amenable to manipulation. However, the ad- We have recently identified the ␣6 integrin as a can- vent of virus-like particle technology has allowed some didate receptor for papillomavirus (Evander et al., 1997). aspects of PV binding and internalization to be ad- This finding was based on the observation that PV virus- dressed. like particles (VLPs) interacted with a protein consistent in size with the ␣6 integrin and were able to be coim- ␣ 1 munoprecipatated with anti- 6 antibodies, and function- To whom correspondence and reprint requests should be ad- ally blocking antibodies against ␣6 were able to reduce dressed at U.Q. Dept. of Medicine, PA Hospital, Ipswich Rd., Brisbane, ␣ QLD 4102 Australia. Fax: ϩ61 7 32 40 59 46. E-mail: nmcmillan@ VLP binding to cells. Furthermore, 6 and VLP binding medicine.pa.uq.edu.au. were colocalized upon the cell surface of the basal and
0042-6822/99 $30.00 271 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 272 MCMILLAN ET AL. suprabasal epithelial cells (the same cells to which viral RESULTS replication is restricted) and the natural ligand for ␣6, laminin, blocked VLP attachment. Finally, a binding-neg- Binding of PV to cultured cell lines correlates with ␣ ative B-cell line, DG75, did not express the ␣6 integrin. expression of 6 integrin The ␣6 integrin is termed the epithelial integrin, as it is We have previously shown that HPV is able to bind expressed most highly upon epithelial cells, the very to cells that express the ␣64 integrin and that ligands same cell type that binds PVs to the highest degree and of, and antibodies against, ␣6 are able to block virus within which primary infection is known to occur and to binding, leading to the suggestion that ␣64 is the PV which vegetative DNA replication is restricted. receptor. However, ␣64 has a limited tissue distribu- The ␣6 integrin partners with a beta integrin subunit, tion (epithelial, mesenchymal, and some neuronal either 4or1. The ␣61 integrin is expressed on a cells), while HPV binding is known to occur in a wide wide range of cells, including platelets, lymphocytes, variety of cells. The ␣6 integrin is able to heterodimer- macrophages, oocytes, and endothelial cells, whereas ize with two beta partners, 1or4. The ␣61 has a ␣64 is relatively restricted to epithelial, mesenchy- wide tissue distribution, including platelets, lympho- mal, and some neuronal cells. In stratified squamous cytes, macrophages, oocytes, and endothelial cells. epithelium the ␣64 complex is an integral part of the Thus, HPV may be able to bind to many cell types via ␣  hemidesmosome (Sonnenberg et al., 1991) and as a the 6 1 integrin. In order to test this possibility we receptor for laminins (Niessen et al., 1994) is involved analyzed two cell lines, COS-7 and HaCaT, which have been reported to express ␣61or␣64 integrin, re- in the interaction of epithelial cells with the basement spectively. We found that HaCaT cells express high membrane. The cells which have the highest degree of levels of ␣6, 1, and 4 integrin. It should be noted that PV binding match the expression profile of ␣64, al- ␣6 binds preferentially with 4 due to its higher affinity though PV does bind to cells expressing ␣61. Thus, for 4 than 1. As expected COS-7 cells express both the expression of ␣ 6 is widespread and highly con- ␣6 and 1 integrin but do not express 4 integrin (Fig. served between species, matching the criteria from 1A). We then tested these cells for their ability to bind previous in vitro results. HPV VLPs. Cells were exposed to VLPs at 4°C and We have previously proposed a model for PV infection washed, and VLPs detected using an L1-specific ␣ in which virus particles bind to the 6 integrin during monoclonal antibody. It can be seen that both HaCaT wound healing and are endocytosed during cell migra- and COS-7 bind VLPs, while the receptor-negative cell tion. The ␣64 integrin is believed to be attached to the line DG75 does not (Fig. 1B). This result indicates that microfilament network via the hemidesmosome (Gian- the beta partner does not appear to be important for cotti et al., 1992; Spinardi et al., 1993) and this may binding of VLPs but rather that it is the presence of ␣6 provide an explanation for the observation that cytocha- that is required. lasin B inhibits papillomavirus uptake in CV-1 cells (Zhou et al., 1995), suggesting that PV particles endocytosed Expression of ␣6 integrin in DG75 cells together with ␣64 integrin may be transported to the ␣ cell nucleus via a microfilament-dependent pathway. In order to prove that 6 is a critical factor required However, while our data suggest that ␣6isanHPV for PV binding we undertook to genetically comple- ␣ receptor it does not constitute definitive evidence that ment binding by transfecting the 6 gene into a cell line not able to bind PV. DG75 is a human B-cell line this is the case. Therefore, we undertook to genetically that does not express ␣6 integrin (Evander et al., complement the ability to bind HPV by expressing the ␣6 1997). The human ␣6A gene was cloned into the vector gene in the receptor-negative B-cell line DG75. First, we EBO-pLPP to allow high-level expression of the cloned show formally that PV binding occurs in cells that ex- ␣  ␣  gene and maintenance in B-cells. Following transfec- press either 6 4or 6 1. We demonstrate that the tion, cells were treated with hygromycin and resistant ␣ transduction of the 6 integrin gene into DG75 cells cells pooled. Expression of ␣6A mRNA was demon- ␣ results in the cell surface expression of 6 protein and strated by Northern blot analysis (data not shown) and that this expression confers upon these cells the ability we confirmed that ␣6 protein was expressed and pro- ␣ to bind laminin, a normal ligand for 6 integrin. Further- cessed to the cell surface using flow cytometry (Fig. 2). ␣  more, the 6 protein is partnered with the 1 integrin in It can be seen that cells transfected with the ␣6 gene DG75 cells. Finally, DG75-␣6 cells were able to bind PV (DG75-␣6) express moderate levels of ␣6 compared to VLPs and this binding was inhibited by a functionally A431-positive control cells, while cells transfected blocking anti-␣6 antibody. Together these data indicate with the control plasmid (DG75-EBO-pLPP) expressed that the ␣6 integrin is a primary cell receptor for papil- no detectable ␣6 protein. We also ascertained the lomaviruses and is both necessary and sufficient for PV expression levels of the 1 and 4 integrins. Our binding. previous work had shown that DG75 cells did not EXPRESSION OF THE ␣6 INTEGRIN CONFERS HPV BINDING 273
FIG. 1. Binding of HPV VLPs correlates with the expression of ␣6 integrin. (A) Analysis of the expression of ␣6, 1, and 4 integrins in HaCaT and Cos-7 cells by flow cytometry. Filled profiles are positive, while empty areas are the negative controls. (B) Binding of HPV6b VLPs to cells detected by immunoblotting for L1 protein. Cells were treated with (ϩ) or without (Ϫ) VLPs, incubated, washed, and L1 detected. express 4 integrin so it was of interest to see if the It can be seen that both DG75-EBO-pLPP and DG75-␣6 expression of ␣6, which must partner with 1inthe cells expressed similar levels of 1 and neither ex- absence of 4, would affect the level of 1 expression. pressed the 4 integrin. 274 MCMILLAN ET AL.
FIG. 2. Analysis of the expression of ␣6, 1, and 4 integrins in DG75 cells transfected with the ␣6A gene by flow cytometry. Filled peaks are positive, while empty peaks are the negative controls.
DG75-␣6 forms a heterodimer with 1 integrin DG75-␣6 cells two proteins of 120 and 130 kDa were precipitated by the anti-␣6 antibody. This differs from It is known that ␣6 heterodimerizes with either 1or A431 and DG75-EBO-pLPP cells and is consistent with 4 integrin but partners preferentially with 4inthe the flow cytometry data (Fig. 2) and indicates that ␣6is presence of both beta subunits (Klein et al., 1990). To partnering with the 1 integrin in the absence of 4. determine how the ␣6 integrin expressed in DG75 cells was acting we analyzed the integrin affiliation patterns by labeling cell surface proteins from A431, DG75-EBO- pLPP, and DG75-␣6 cells with 125I, preparing cell extracts, and subjecting them to immunoprecipitation using vari- ous anti-integrin antibodies (Fig. 3). The anti-␣6 antibody precipitated two major proteins of 120 and 220 kDa from A431 cells, consistent with the size of ␣6 and 4 inte- grins. The anti-1 antibody precipitated three proteins between 130 and 160 kDa, which most likely represent the 1 (130 kDa), ␣2 (165 kDa), and ␣3 (130 kDa) inte- grins. The DG75 cells transfected with the EBO-pLPP plasmid alone had no specific proteins precipitated by FIG. 3. Immunoprecipitation of 125I-labeled integrins from cells. Cell anti-␣6, while the 1 antibody appeared to precipitate surface proteins were labeled with 125I before cell lysates were pre- two bands of around 130 kDa (most likely 1 and ␣3). In pared and integrins immunoprecipitated and analyzed by SDS–PAGE. EXPRESSION OF THE ␣6 INTEGRIN CONFERS HPV BINDING 275
expression of ␣6 confers upon the DG75 cells the ability to bind PV particles, whereas control cells were not able to bind (Fig. 5A). Positive control A431 cells were able to bind PV particles, although the smaller amount of binding observed is due to the fact that five times fewer A431 cells were used. Flow cytometry analysis clearly indi- cates that A431 cells express higher levels of ␣6 than DG75-␣6 cells (Fig. 2). In order to determine if VLPs were indeed attaching via ␣6 we undertook to block binding using an anti-␣6 antibody, GoH3. We have previously shown that GoH3 is able to block binding of VLPs to HaCaT cells (Evander et al., 1997). The addition of GoH3 was able to inhibit the binding of VLPs to DG75-␣6 cells to background levels, therefore indicating that binding was occurring via the ␣6 integrin (Fig. 5B).
DISCUSSION FIG. 4. Cell adhesion assays. DG75 control and ␣6-expressing cells were plated onto laminin-coated tissue culture wells and incubated for The data presented here show that the ␣6 integrin is 60 min before being washed and stained with crystal violet, and the both necessary and sufficient for PV VLP binding and OD600 was read. that VLP–cell interaction was totally inhibited by ␣6 an- tibodies. The fact that no binding was observed in the presence of ␣6 antibodies meant studies to ascertain if ␣ DG75- 6 binds laminin this block also inhibited PV infection could not be under- We wished to determine if the ␣6 we had expressed in taken. Integrins have been shown previously to act as DG75 cells was functional and able to bind to its natural virus receptors for initial binding and/or internalization, ligand, laminin. The ␣64 integrin is normally expressed as in the case of echovirus (␣21) (Bergelson et al., in epithelial cells and functions as a receptor for laminin 5 (although it binds laminin 1 with low affinity), whereas ␣61 is expressed in a wider variety of cells types (platelets, lymphocytes, macrophages, oocytes, and en- dothelial cells) and has been reported to bind to laminins 1, 2, 4, and 5 (Niessen et al., 1994). B-cells such as DG75 express ␣21 and ␣31, which bind to collagen and fibronectin, respectively, although both these integrins are reported to bind laminin with low affinity depending on what cell type they are expressed in (Ziober et al., 1996). We undertook cell adhesion assays using DG75- EBO-pLPP and DG75-␣6 cells and tested their ability to bind to laminin-coated plates as previously described (Chao et al., 1996). It can be seen that the ␣6-expressing DG75 cells were able to bind well at all concentrations of laminin tested (Fig. 4). Control cells, however, were un- able to bind laminin to any great degree, although as the laminin concentration became higher, minor binding oc- curred. This is perhaps due to the above-mentioned ability of ␣31 to bind laminin with low affinity (Ziober et al., 1996).
Expression of ␣6 results in binding of VLPs We next wished to ascertain if the expression of ␣6 integrin in DG75 cells would result in the acquisition of the ability to bind PV particles. The cells were incubated FIG. 5. Binding of HPV6b L1 VLPs to cells detected by immunoblot- with VLPs at 4°C before being extensively washed and ting for L1 protein. (A) Cells were treated with (ϩ) or without (Ϫ) VLPs, virus attachment determined by Western blotting using incubated, and washed, and L1 was detected. (B) As for A but the cells an L1 monoclonal antibody. It can be seen that the were preincubated with an anti-␣6 antibody, GoH3 (ϩAb). 276 MCMILLAN ET AL.
1992), coxsackievirus (␣v3) (Roivainen et al., 1994), han- were able to attach to plates coated with laminin, tavirus (3) (Gavrilovskaya et al., 1998), adenovirus whereas control cells were not. (␣v3/5) (Wickham et al., 1993), and foot and mouth The molecular basis for the strict tissue and species disease virus (␣v3) (Berinstein et al., 1995). However, tropism of PV is unknown. The receptors for several binding and/or internalization by viruses through ␣v3 viruses have been shown to be the sole determinants of integrins requires a virally encoded RGD motif and vi- their cell tropism (Brown et al., 1993; Haun et al., 1993). ruses containing an RGD motif were inhibited by RGD However, in the case of PV the wide distribution of ␣6 peptides. PVs do not have an RGD motif and PV VLP integrin and PV binding are clearly not matched by any binding is not inhibited in this manner. PVs and hanatvi- productive replication in these cells. Therefore the tro- rus are the only viruses so far to bind via an RGD- pism of PVs must be at a step downstream of binding, independent mechanism. such as uptake, transport to the nucleus, uncoating, Here we show that cell lines that express either ␣61 transcription/translation, DNA replication, assembly, or or ␣64 bind VLPs. This indicates that the beta partner is release. The identification of the ␣6 integrin as a PV not important for the binding of VLP to ␣6 and agrees receptor does not exclude the fact that there may be with previous data showing that anti-␣6 antibodies block other receptors or even coreceptors, although our previ- the binding, while anti-1or-4 antibodies do not ously published data indicate that pretreatment of cells (Evander et al., 1997). Integrins are dual receptors for with laminin, the ␣6 ligand, inhibits virus binding, sug- extracellular matrix and cytoskeletal proteins and al- gesting further that the ␣6 integrin is a primary attach- though the exact role of the ␣6 subunit has not been ment receptor (Evander et al., 1997). However, many ascertained the 4 subunit has been shown to associate viruses are known to require a second receptor to facil- with the keratin filament system (Spinardi et al., 1993) itate virus uptake (e.g., chemokine receptors for HIV or and mediates signal transduction (Mainiero et al., 1997). ␣v3/5 for adenovirus). We are actively pursuing this However, studies indicate that within integrins both ␣ possibility. and  subunits contribute to ligand binding (Shih et al., There is a paucity of detail regarding the interaction of 1997). This would imply that the beta subunit does not PV and its receptor, although some conclusions can be share contact residues for PV VLPs (or contacts are not drawn from antibody studies. All neutralizing antibodies important for binding) or that the monoclonal antibodies shown to block PV infection (by xenograft, foci formation, against them do not interfere with binding. The ␣61 or early-gene PCR) recognize only conformational viral integrin is expressed on a wide range of cells, including epitopes (Christensen et al., 1994, 1996; Roden et al., platelets, lymphocytes, macrophages, oocytes, and en- 1996). Furthermore, serum raised against denatured dothelial cells, whereas ␣64 is relatively restricted to VLPs is not protective, indicating that any domain upon epithelial, mesenchymal, and some neuronal cells. The PV that inhibits infection is likely to be nonlinear. Inter- fact that VLPs bind to ␣61 may therefore explain the estingly, these neutralizing antibodies are highly type- wide range of cells that PVs are reported to be able to specific, despite the high degree of sequence conserva- bind. However, it is clear that while PVs bind to a wide tion in L1 genes, a fact that appears to be inconsistent range of cells there is no clinical infection of these cell with the data indicating that all PVs tested so far interfere types. with the ability of PVs from another species or type to It has recently been reported that VLPs from HPV type bind to cells. One could speculate therefore that the PV 11 interact with heparin and other cell surface glycos- interaction with ␣6 can occur in two ways; either a aminoglycans (Joyce et al., 1999). This report seems in diverse range of epitopes exist upon different PV sub- conflict with a previous report showing that inhibition of types which all recognize ␣6 or there is a single, highly glycosaminoglycan synthesis has no effect on VLP bind- conserved, ␣6 binding epitope upon all PVs in addition to ing (Qi et al., 1996), although this may reflect the use of an epitope(s) that interacts with a secondary receptor to different assays. However, binding of papillomavirus facilitate binding and/or uptake. The latter possibility VLPs to the ␣6 integrin does not rule out an initial appears the more likely. Indeed, it has been shown that interaction with glycosaminoglycans and it is possible neutralization occurs by more than one mechanism. that other receptors exist or that proteoglycans and in- Some antibodies to L1 can prevent virions from binding tegrins act in concert to facilitate HPV uptake. Although to the cell surface, presumably by blocking binding to a B-cells are known to express heparin it would be of receptor (Roden et al., 1994b), possibly the ␣6 integrin. extreme interest to know the status of glycosaminogly- Other neutralizing L1 antibodies do not inhibit PV bind- cans on DG-75 cells. ing, yet efficiently prevent infection (our unpublished data Our data indicate that it is possible to express ␣6 and Booy et al., 1998; Christensen et al., 1995). One integrin within a B-cell, which has not been previously suggested mechanism for the neutralizing activity of reported. The ␣6 expressed in DG75 cells functioned in these antibodies is the prevention of uncoating and in- a normal fashion in that it was expressed on the cell fection by cross-linking of capsomeres (Booy et al., surface and was partnered with 1, and transfected cells 1998). EXPRESSION OF THE ␣6 INTEGRIN CONFERS HPV BINDING 277
The elucidation of ␣6 integrin as both necessary and 60 min. The blots were washed 4 times with PBST before sufficient for PV VLP binding will allow the above hypoth- detection using enhanced chemiluminescence (Amer- esis to be tested. Furthermore, it identifies a potential sham, UK) according to the manufacturer’s instructions. therapeutic target with which to block PV infection. Fi- ␣ nally, the ␣6 integrin is known, upon ligand binding, to Cloning of 6 and transfection of DG75 cells invoke a signal transduction pathway to initiate DNA The 3.5-kbp cDNA for the ␣6A gene (a kind gift from replication in keratinocytes, a situation that would be Vito Quaranta, Scripps Institute) was excised with Not1 advantageous for viral replication. The role that the re- and cloned into the Not1 site of EBO-pLPP (Margolskee ceptor/virus ligation on the cell surface plays in prepar- et al., 1988). This vector encodes the EBV origin of rep- ing the cell for viral infection can now be addressed. lication allowing stable, episomal replication of the plas- mid in B cells, while expression of ␣6A was driven off the METHODS AND MATERIALS SV40 early promoter. The resulting clone was transfected ϫ 6 VLPs, cell lines, and antibodies into cells by electroporation. Briefly, 5 10 cells were mixed in a 0.4-cm electroporation curvette with 10–20g HPV type 6b L1 VLPs were produced in Sf9 cells by of DNA in a total volume of 250 l of complete DMEM using HPV6b L1 recombinant baculovirus and purified as and incubated for 5 min at room temperature before previously described (Qi et al., 1996). DG75 cells, an being electroporated at 230V, 960F, and Ohms limited EBV-negative, Burkitt’s lymphoma B-cell line, was pro- to 200 using a BioRad Gene Pulser. Following electropo- vided by Dr Ihor Misko, Queensland Institute of Medical ration the cells were allowed to stand for 5 min before Research, Brisbane, Australia. HaCaT human epithelial being added to 5ml of complete DMEM and incubated cells were a gift from Dr Nobert Fusenig, Deutsches for 48 h. Transfected cells were then cultured in selective Krebsforschungszentrum, Heidelberg, Germany. A431 media (complete DMEM plus 600g/ml hygromycin B). and Cos-7 cells were obtained from the American type Expression was confirmed by flow cytometry. Culture Collection. The following antibodies were used: anti-␣6 (GoH3), Serotec, England; anti-1 (P5D2), a gift Adhesion assay from Elizabeth Wagner, University of Minnesota; anti- Cell matrix proteins (human laminin or fibronectin) HPV16-L1 (Mab-8), a gift from Dr. Stephan Kelsall, De- were diluted in PBS to the indicated concentrations and partment of Microbiology, University of Western Austra- 100 l placed into 96-well plates and incubated over-  lia; and anti- 4 (3E1), Gibco-BRL, USA. night at 4°C. The wells were blocked with 100lof DMEM supplemented with 1% BSA for1hat37°C. Cells Binding assays were centrifuged, resuspended in DMEM/1% BSA and Adherent cells were detached from plates using 0.25% added in triplicate (1 ϫ 10 5 per well in a 100l volume). trypsin-0.02% EDTA, resuspended in complete DMEM Cells were incubated at 37°C for 1 h before being and held in suspension for 60 min to allow re-expression washed 3 times with DMEM/1% BSA and fixed with 100% of surface antigens. Cells were washed twice in ice cold methanol for 10 minutes. The adherent cells were binding medium (DMEM plus 0.2% BSA) and resus- stained with a solution of 0.2% crystal violet in 2% ethanol pended in 200 l of binding medium. If a blocking assay for 5 minutes and washed extensively in water before the was performed the cells were then incubated for 60 min stain was solubilized in 100 l of 1% SDS and OD600 was on ice with the appropriate antibodies (as indicated) read.
before being washed twice with binding media. VLPs 125 were added to cells as indicated and incubated on ice I labeling of cells for a further 90 min. Cells were washed extensively with Cells were surface labeled in suspension with 125I ice cold PBS, resuspended in SDS-PAGE loading buffer, (ICN, Australia) using Iodo-beads (Pierce, Australia). boiled for 10 min, and loaded on an SDS-polyacrylamide Briefly, cells (1–2 ϫ 10 6) were resuspended in PBS and gel. incubated with Iodo-beads and 125I(100Ci) for 15 min at room temperature. The beads were removed and the Immunoblotting cells washed 3 times with PBS before being solubilized ϭ Proteins separated on SDS- polyacrylamide gels were in lysis buffer (1% NP-40, 20mM Tris-HCl pH 7.5, electrically transferred (100V, 60 min) to nitrocellulose 100mM NaCl, 4mM EDTA, 1mM PMSF) for 30 min at 4°C. filters before being blocked with PBS-0.05% Tween 20 The lysates were clarified by centrifugation at 14000 xg (PBST) plus 5% skim milk powder for 60 min at room for 1 min and used in immunoprecipitation experiments. temperature. Antibodies were added as indicated and Immunoprecipitation of 125I-labeled integrins incubation continued for 60 min. Blots were washed 3 times with PBST and HRP-conjugated anti-species anti- Cell lysates were precleared by incubation with pro- body were added and incubation continued for a further tein-G sepharose for 30 min at 4°C. The lysates were 278 MCMILLAN ET AL. made up to 500l with PBS before anti-integrin antibod- Giancotti, F. G., Stepp, M. A., Suzuki, S., Engvall, E., and Ruoslahti, E. ies were added and the mixture rotated at 4°C for 60 min. (1992). Proteolytic processing of endogenous and recombinant beta Protein G–Sepharose was added and the tubes mixed 4 integrin subunit. J. Cell Biol. 118(4), 951–959. Halpern, A., Farmer, A., and Calef, C. (1996). A survey of papillomavirus for a further 45 min at 4°C. Immune complexes were variants. In “Human Papillomaviruses” (G. Myers, Ed.), pp. i43–i185. washed 3 times with PBS before the addition of SDS– Los Alamos National Laboratory, Los Alamos, NM. PAGE loading buffer, boiled for 10 min, and analyzed by Haun, G., Keppler, O. T., Bock, C. T., Herrmann, M., Zentgraf, H., and SDS–PAGE. Pawlita, M. (1993). The cell surface receptor is a major determinant restricting the host range of the B-lymphotropic papovavirus. J. Virol. 67(12), 7482–7492. Flow cytometry Howley, P. M. (1996). Papillomaviridae: The viruses and their replica- Ј Analysis of expression of cell surface molecules by tion. In “Fields Virology” (B. N. Fields, D. M. Knipe, and P. M. Howley, Eds.), 3rd ed., Vol. 2, pp. 2045–2076. Lippincott–Raven, Philadelphia. flow cytometry was performed as previously described Joyce, J. G., Tung, J. S., Przysiecki, C. T., Cook, J. C., Lehman, E. D., (Qi et al., 1996). Sands, J. A., Jansen, K. U., and Keller, P. M. (1999). The L1 major capsid protein of human papillomavirus type 11 recombinant virus- ACKNOWLEDGMENTS like particles interacts with heparin and cell-surface glycosamino- glycans on human keratinocytes. J. Biol. Chem. 274, 5810–5822. This work was supported by grants from the National Health and Klein, C. E., Steinmayer, T., Mattes, J. M., Kaufmann, R., and Weber, L. Medical Research Council of Australia, the Queensland Cancer Fund, (1990). 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