saccharomyces cerevisiae is permissive for replication of...

JOURNAL OF VIROLOGY, Dec. 2002, p. 12265–12273 Vol. 76, No. 230022-538X/02/$04.00�0 DOI: 10.1128/JVI.76.23.12265–12273.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Saccharomyces cerevisiae Is Permissive for Replication of BovinePapillomavirus Type 1

Kong-Nan Zhao* and Ian H. FrazerCentre for Immunology and Cancer Research, The University of Queensland, Princess Alexandra Hospital,

Brisbane, Queensland 4102, Australia

Received 10 June 2002/Accepted 19 July 2002

We recently demonstrated that Saccharomyces cerevisiae protoplasts can take up bovine papillomavirus type1 (BPV1) virions and that viral episomal DNA is replicated after uptake. Here we demonstrate that BPVvirus-like particles are assembled in infected S. cerevisiae cultures from newly synthesized capsid proteins andalso package newly synthesized DNA, including full-length and truncated viral DNA and S. cerevisiae-derivedDNA. Virus particles prepared in S. cerevisiae are able to convey packaged DNA to Cos1 cells and to transformC127 cells. Infectivity was blocked by antisera to BPV1 L1 but not antisera to BPV1 E4. We conclude that S.cerevisiae is permissive for the replication of BPV1 virus.

Papillomaviruses (PVs) are exclusively epitheliotropic vi-ruses and have evolved a unique replication strategy that de-pends upon the differentiation program of keratinocytes (15).Though transient viral episome replication can occur in a num-ber of in vitro cells (37), only keratinocytes, or cells with thepotential for squamous maturation, can be productively in-fected, since viral capsid proteins are synthesized and virionsare assembled only in terminally differentiated keratinocytes.PV capsid proteins, expressed in mammalian cells (61), insectcells (22), and E. coli (20, 27), can be used to study virionassembly and DNA encapsidation (43, 44, 52, 57–60). How-ever, there remain large gaps in the understanding of PV lifecycle.

Kreider et al. (24) first reported the use of athymic mousexenograft culture to produce infectious human PV type 11(HPV11) in vivo. In vitro raft culture systems have alloweddifferentiation-specific viral amplification, late gene expres-sion, and virion morphogenesis for HPV31 (9, 46) and otherPV types (2, 34). Recently, infectious particles have been pro-duced (2, 8, 31, 35, 40), although the viral yield is generallysmall compared to input virions. However, only a small num-ber of HPV types can be successfully grown in athymic and scidmouse xenograft systems or raft culture systems (55), andpropagation of large numbers of viral particles in vitro is yet tobe achieved (2).

Lambert et al. (26) first used the S. cerevisiae system to studythe expression and function of the bovine PV type 1 (BPV1)E2 gene. Dostatni et al. (5) used S. cerevisiae to express full-length BPV1 E2 protein and assayed in vitro its capacity tomodulate transcription. Prakash et al. (41) reported that BPV1E2 protein regulates viral transcription by binding as a dimerto the DNA sequence ACCGN4CGGT. According to previousstudies of viral DNA replication in yeast (21, 42), the basicrequirements for viral cis and trans elements for episome rep-lication are similar between S. cerevisiae and mammalian cells.

We have recently observed that S. cerevisiae protoplasts, whichhave extensive endocytotic activity (10), can take up BPV1virions, and the BPV1 episome can replicate (56). In thepresent study, we have studied whether S. cerevisiae exposed toPV virions can support production of infectious virions.

MATERIALS AND METHODS

S. cerevisiae protoplast culture and virus infection. BPV1 virions were pre-pared from bovine papillomas as described previously (28). S. cerevisiae proto-plast culture and virus infection were carried out as described previously (56). Inbrief, S. cerevisiae cells were cultured to 108 cells/ml in liquid medium andharvested by centrifugation. The harvested S. cerevisiae cells were incubated in anenzyme buffer at 30°C for 3 h. The enzyme-cell mixture was checked microscop-ically to determine when the enzyme digestion was sufficient to produce S.cerevisiae protoplasts. S. cerevisiae protoplasts were washed with STC buffer (1 Msorbitol, 10 mM CaCl2, 10 mM Tris-HCl; pH 7.5) twice and resuspended in S.cerevisiae medium containing 0.8 M sorbitol and 0.2 M glucose, and the densitywas adjusted to 5 � 107 cells/ml for virus infection. Virion suspensions weredialyzed against 0.15 M phosphate-buffered saline (pH 7.4) (PBS) for 30 min.The dialyzed virus was then used to infect S. cerevisiae protoplasts. Infected oruninfected S. cerevisiae cultures were placed on a shaker with gentle agitation at28°C in the dark. Fresh medium without sorbitol was added to the S. cerevisiaecell cultures once a day to reduce the osmoticum at the beginning of culture andsubsequently based on experimental requirements.

Immunofluorescence examination of BPV L1 protein in S. cerevisiae. S. cer-evisiae protoplast culture(10 ml) was fixed by the addition of 1 ml of 37%formaldehyde in PEM buffer (100 mM Na-PIPES [piperazine-N,N�-bis(2-eth-anesulfonic acid], pH 6.9; 1 mM EGTA; 1 mM MgSO4), with gentle agitation for1 min, and of 88 �l of 25% glutaraldehyde (Sigma, St. Louis, Mo.; electronmicroscopy grade at a final concentration of 0.2% [vol/vol]). Fixed S. cerevisiaecells were agitated for 90 min in a water bath, pelleted at 1,000 � g for 5 min, andwashed with 2 ml of PEM buffer three times. Washed S. cerevisiae cells wereresuspended at a density of 5 � 107 cells/ml in PEMS (PEM, 1 M sorbitol) buffercontaining 20,000 U of lyticase (Sigma)/ml to digest the cell walls at 37°C for ca.2 to 3 h. Digested cells were resuspended after three washes with 2 ml of PEMbuffer in 2 ml of PEM containing 1% of Triton X-100 and held for 1 min. TritonX-100-treated cells were washed with 2 ml of PEM buffer three times and treatedwith 2 ml of fresh sodium borohydride (1 mg/ml in PEM) twice for 5 min. Cellswere resuspended in 0.5 ml of PEMBAL (PEM, 0.1 M lysine, 1% globulin-freebovine serum albumin, and 0.1% sodium azide) and incubated for 1 h withcontinuous inversion. Cells were then pelleted and incubated in 100 �l of mono-clonal antibody (MAb; 1:1,000) against BPV L1 capsid protein (MC15 [58]) ona rotary inverter overnight. MAb-labeled cells were washed with 1 ml of PEM-BAL three times and incubated with 100 �l of FITC conjugated anti-mouseimmunoglobulin secondary antibody, diluted 1/50 in PEMBAL, on a rotaryinverter overnight. Antibody-labeled cells were washed with 0.5 ml of PEMBALthree times and mounted.

* Corresponding author. Mailing address: Centre for Immunologyand Cancer Research, The University of Queensland, Princess Alex-andra Hospital, Brisbane, QLD 4102, Australia. Phone: 61-7-3240-5912. Fax: 61-7-3240-5946. E-mail: [email protected].

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VLP preparation. Virus-like particles (VLPs) were prepared from S. cerevisiaecultures similarly to the method used for Cos1 cells (57, 59). Briefly, cells werecollected by centrifugation at 3,000 rpm for 10 min, and washed with PBScontaining 2-mM phenylmethylsulfonyl fluoride (PMSF). Pellets were resus-pended in 20 ml of SCE buffer (1 M sorbitol, 0.1 M sodium citrate, 10 mMEDTA; pH 6.8) containing 20 mM �-mercaptoethanol and digested with 200 �lof lyticase at 50,000 U/ml for 2 to 3 h. The digested cells were pelleted andresuspended in 5 ml of PBS containing 2 mM PMSF and homogenized in aDounce homogenizer with a tight-fitting pestle for 10 min. Released nuclei werecollected by centrifugation at 3,000 rpm at 4°C for 15 min, resuspended in 10 mlof PBS with PMSF, and sonicated for 40 s. Lysate was layered over 20% sucroseand pelleted by centrifugation at 26,000 rpm for 2 h with a Beckman SW26 rotor.Pellets were resuspended in 11.5 ml of PBS containing 5.5 g of CsCl andcentrifuged in a Beckman SW41 rotor at 40,000 rpm at 21°C for 20 h. From theresulting gradient, 22 0.5-ml fractions were collected.

Immunoblotting of L1 and L2 protein. Samples (50 �l) from CsCl gradientswere dialysed in PBS, precipitated with 3 volumes of acetone at �70°C for 4 h,pelleted, resuspended in 20 �l of 1� Laemmli buffer (25), and boiled for 8 min.Samples were separated on 10% (wt/vol) sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) gels and electrotransferred onto nitrocellulosemembranes (Bio-Rad). Blots were washed with PBS for 10 min, blocked in PBScontaining 5% nonfat milk for 1 h, and probed with L1 or L2 specific MAbs (58)at 4°C overnight. Blots were then incubated with anti-mouse secondary antibodyconjugated with horseradish peroxidase (Silenus Australia) and developed byenhanced chemiluminescence (Amersham Australia).

Radiolabeling of VLPs. S. cerevisiae protoplast cultures (40 ml) were incubatedin Cys/Met-free medium for 5 h prior to addition of 150 �Ci of [35S]Cys�Met(ICN). Cultures were incubated at 28°C for 4 days, with the addition of 20-mlfresh Cys/Met-free medium every day. S. cerevisiae cells were lysed, and fractionsof density corresponding to VLPs prepared as described above. Fractions (300�l) were incubated with 0.3 �l of L1- and L2-specific MAb for 2 h and then withprotein G-Sepharose (Sigma) beads at 4°C overnight. Beads were washed withradioimmunoprecipitation assay buffer (100 mM Tris, pH 7.5; 150 mM NaCl; 5mM CaCl2; 0.1% Triton X-100) five times and with sterile water once and thenboiled in Laemmli buffer; next, supernatant was applied to an SDS–10% poly-acrylamide gel. Gels were dried and exposed to film at �70°C for 48 h.

[3H]thymidine labeling. Fifty milliliters of S. cerevisiae protoplasts (108 cells/ml) was infected with 10 �g of BPV1 virus as described above, 4 h prior to theaddition of 200 �Ci of [3H]thymidine. At 24 h, 50 ml of fresh S. cerevisiaemedium was added. After 4 days, fractions of VLP density were prepared asdescribed above, dialyzed against PBS, and incubated with 10 U of DNase I in10� DNase buffer at 37°C for 30 min. Fractions were mixed with 50 �l of 3 NNaOH and boiled for 5 min, and the amount of incorporated 3H was determinedby liquid scintillation counting (Beckman LS 7000).

DNA packaging analysis. Extraction of packaged DNA from individual frac-tions was as described previously (57, 59). Briefly, 300 �l of fraction was dialyzedagainst PBS, 50 �l of 10� DNase buffer containing10 U of DNase I was added,and suspensions were held at 37°C for 30 min. After the addition of 100 �l of10% SDS, and 500-�l phenol fractions were held at 65°C for 1 h. The aqueousphase was extracted twice with equal volumes of phenol and chloroform, andDNA precipitated with 50 �l of 3 M sodium acetate and 2 volumes of 100%ethanol at �70°C for 2 h. DNA was pelleted, resuspended in 20 �l of TE buffer,and electrophoresed on a 1% agarose gel.

Characteristic analysis of packaged DNA. DNA samples with or without priordigestion were electrophoresed on 1% agarose gel, blotted onto nylon mem-brane, and probed with 32P-labeled BPV1 DNA or BPV1 L1 DNA.

Analysis of L1 protein and viral DNA delivery in VLP-infected mammaliancells. BPV1 VLP suspension (50 �l) was dialyzed against PBS, digested withDNase, and added to Cos1 cell cultures grown in Dulbecco modified Eaglemedium supplemented with 10% fetal bovine serum. For some experiments,antibody specific for BPV1 L1 (58) or E4 (Kindly provided by John Doobar) wasadded to particles for 1 h prior to infection of Cos1 cell. The particle-infectedCos1 cells were harvested for Hirt DNA and protein preparations after 48 h. Forprotein preparation, cells were resuspended in 1� Laemmli buffer and sonicatedfor 40 s. Protein sample (10 �g) was boiled for 8 min and applied to a SDS–10%polyacrylamide gel. Immunoblotting assay for BPV L1 was as described above. Inaddition, cells were resuspended in lysate buffer (10 mM Tris-HCl, pH 7.5; 10mM EDTA; 0.2% Triton X-100). Episomal DNA was prepared by the Hirtmethod (12) with some modifications (57, 59), digested with BamHI, and elec-trophoresed on 1% agarose gel. Southern blots were hybridized with 32P-labeledBPV DNA. Extracted DNA was also used for PCR amplification by usingoligonucleotides specific for the six early and two late genes of BPV1.

Focus formation assay. Different volume of BPV1 VLP suspension was dia-lyzed against PBS, digested with DNase, and added to confluent C127 cellcultures grown in 6- or 12-well plates with Dulbecco modified Eagle mediumsupplemented with 2% fetal bovine serum. The medium was changed every 3days. After 4 to 5 weeks, cells were washed with PBS twice, fixed with coldmethanol for 3 min, and stained with 0.5% methylene blue plus 0.25% carbolfuschin for 15 min. Foci were counted in four separate experiments.

RESULTS

BPV virus particles are present in BPV1-infected S. cerevi-siae cells. Recent studies by ourselves (56) and others (1) havedemonstrated that BPV can replicate its episome after intro-duction of natural virions or episomal PV DNA into S. cerevi-siae cells. To investigate the extent to which BPV1 can repli-cate in S. cerevisiae, we first examined the extent and locationof the expression of BPV1 L1 capsid protein at 20 h afterexposure of S. cerevisiae cells to BPV1. Immunofluorescencemicroscopy of BPV1-infected S. cerevisiae cells demonstratedBPV1 L1 protein within S. cerevisiae nuclei (Fig. 1A) but notwithin S. cerevisiae not exposed to BPV1 (Fig. 1B). A total of30 to 40% of S. cerevisiae cells showed significant L1 staining,which was confirmed by immunoblotting of individual colonies.Infected cells were plated and individual colonies were assayedfor L1 by immunoblotting. In six experiments, 34.3% � 13.2%of colonies were positive for L1 protein (data not shown). Inaddition, studies of BPV1-infected S. cerevisiae cells with cy-cloheximide treatment by using immunoblotting assay indi-cated that, at 20 h, L1 protein was at least partly newly syn-thesized (Fig. 1D).

Newly synthesized L1 and L2 proteins assemble into VLPsin S. cerevisiae. To determine whether the capsid proteins ofPV observed in BPV1-infected S. cerevisiae were newly synthe-sized, we first established that virus particles could be purifiedfrom infected cells by density gradient separation (Fig. 1C).We examined the migration of L1 and L2 proteins across acesium chloride gradient to determine whether these proteinswere associated with material of density typical of empty or fullBPV1 virions. The majority of the L1 protein was found in the1.31- to 1.29-g/ml (fractions 16 to 20) and 1.34- to 1.36-g/ml(fractions 4 to 9) gradient fractions (Fig. 2A), which corre-spond to the expected densities of empty and full PV virions,and VLPs could be observed in this material by electron mi-croscopy (Fig. 1C). L2 protein was also found across the gra-dient, although rather more was present in the L1 containinghigher-density fractions (Fig. 2A). The results confirm thatBPV L1/L2 proteins in BPV1-infected S. cerevisiae culturesprefer to adopt either a heavy 1.34- to 1.36-g/ml configurationor a light 1.29- to 1.31-g/ml configuration and are thereforelikely to be configured as VLPs, in keeping with prior obser-vations that the expression of HPV L1 in S. cerevisiae results inthe assembly of VLPs (16)

To show that L1 and L2 proteins incorporated into VLPs inBPV1-infected S. cerevisiae cells were newly synthesized, welabeled BPV1-infected cell cultures with 35S-labeled methio-nine and cysteine and precipitated L1 and L2 from fractions ofthe CsCl gradient with specific MAbs (Fig. 2B). Proteins of 55and 77 kDa, corresponding to the known molecular masses ofthe BPV1 L1 and L2 proteins, were detected in material pre-cipitated by L1 and L2 specific antibodies from fractions of aCsCl gradient prepared from BPV1-infected cultures at 4 days

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after infection (Fig. 2B) and also at 2 days postinfection (datanot shown). Other labeled proteins were also precipitated, inlesser abundance. However, the bands corresponding in size toL1 and L2 proteins that were precipitated from infected la-beled cells were not precipitated from cultures not exposed toBPV1 virus (data not shown). Incorporation of 35S-labeledmethionine and cysteine into proteins of appropriate molecu-lar mass and within material of the density of PV VLPs andreactive with L1 and L2 antibodies confirms that BPV1-in-fected S. cerevisiae produced newly synthesized BPV1 VLPs.

DNA encapsidated by VLPs is heterogeneous. To investigatewhether the BPV1 VLPs in BPV1-infected S. cerevisiae cellscould incorporate DNA, we purified DNA from dense frac-tions (fractions 7 and 8) and light fractions (fractions 18 and19) from a CsCl gradient, prepared from S. cerevisiae infected4 days previously with BPV1 virions, and also from naturalBPV1 virions, in each case after DNase I treatment of thestarting material to remove any DNA not packaged withinparticles. DNA was recovered from the fractions of the CsClgradient corresponding to dense and light VLPs, indicatingthat BPV1 VLPs assembled in BPV1-infected S. cerevisiaepackage DNA internally (Fig. 3A). However, the electrophore-sis pattern of DNA prepared from natural virions was differentfrom that of the DNA encapsidated by BPV1 particles pro-duced in S. cerevisiae, since the DNA encapsidated by VLPsincluded, in addition to the species of about the same size asPV genome found in natural virions, variable amounts of anadditional species of ca. 4 kb (Fig. 3A). Hybridization of DNApurified from S. cerevisiae-produced BPV1 VLPs with a BPV1genomic DNA probe showed that this DNA was of the samemobility as DNA from BPV1 natural virions (Fig. 3A), indi-cating that S. cerevisiae-produced BPV1 VLPs encapsidate full-length BPV1 genomic DNA. The additional 4-kb DNA bandpurified from S. cerevisiae produced VLPs did not hybridize toBPV1 DNA, suggesting that BPV1 VLPs produced in S. cer-evisiae may also encapsidate DNA of S. cerevisiae origin.

To determine whether the BPV1 DNA encapsidated by theVLPs produced in BPV1-infected S. cerevisiae represents acomplete BPV1 episome, the restriction pattern of the DNAfrom both dense and light VLPs was compared with that fromnatural BPV1 virions after digestion with three enzymes(BamHI, EcoRI, and HindIII) with unique site in the BPV1genome (Fig. 3B). A single band representing an intact BPV1episome at 7.95 kb hybridized with a BPV L1 gene probe inboth BPV1 virion and dense VLP DNA samples (Fig. 3B).

FIG. 1. Detection of BPV1 L1 protein in S. cerevisiae cells infectedwith BPV1 virions. S. cerevisiae cells exposed to (A) or not exposed to(B) BPV1 were probed with an L1 specific MAb 20 h after infection.(C) Electron micrograph of BPV1 virus particles purified by densitygradient centrifugation from BPV1-infected S. cerevisiae cultures 4days after infection. The size bar represents 50 nm. (D) Time courseanalysis of BPV L1 protein in BPV1-yeast cultures without or withcycloheximide treatment (5 �g/ml) by immunoblotting assay. A set of1 ml of yeast culture (5 � 107cells/ml) was infected with 60 ng of BPV1virus. At different time points (as shown in Fig. 1D), 100 �l of yeastculture was collected for protein preparation. The collected yeast cul-ture, after pelleted and washed with PBS twice, was resuspended in 50�l of 1� Laemmli buffer and sonicated for 40 s. Then, 25 �l of proteinsample was boiled for 8 min and applied subjected to SDS-PAGE. Atotal of 100 ng of BPV1 virus was used as positive control (V). Immu-noblotting assay for BPV L1 was as described in Materials and Meth-ods section.

FIG. 2. BPV1 capsid proteins in BPV1-infected S. cerevisiae cul-tures. (A) BPV1-infected S. cerevisiae cells were homogenized andfractionated on a CsCl density gradient. Fractions were separated bySDS-PAGE and probed for L1 and L2 proteins with specific MAbs.Band of 55 and 77 kDa, corresponding to L1 and L2, respectively, areindicated. (B) PV-infected S. cerevisiae cells were labeled with 35S-labeled methionine and cysteine and fractionated as in panel A. VLPswere immunoprecipitated, and proteins were separated by SDS-PAGE. Arrows indicate L1 and L2 proteins at 55 and 77 kDa, respec-tively. The numbers represent fractions of CsCl gradients from thebottom (1.45 g/ml) to the top (1.26 g/ml).

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However, DNA purified from light VLPs, after digestion withthe three enzymes described above, produced a more complexpattern with the BPV1 genome probe, with BamHI having anextra 2.6-kb band, three EcoRI bands (at 2.4, 3.5, and 4.0 kb)and HindIII two bands (2.5 and 4.0 kb) (data not shown). Theresults suggest that the DNA encapsidated by light particlesmight be different from that found in dense VLPs. Therefore,we used eight restriction enzyme treatments to restrict furtherthe DNA purified from light VLPs and L1 gene probe to doSouthern hybridization (Fig. 3C). The predicted hybridization

pattern for BPV1 with an L1 gene probe (Fig. D and E) isshown for comparison with the pattern observed from lightVLPs. Uncut DNA prepared from light VLPs shows BPV1 L1specific hybridization to three bands, suggesting that episomalforms are present. Although the predicated L1 hybridizationswere generally present in digested DNA, extra bands wereobserved for individual enzyme restriction, particularly for thecombination of BamHI and EcoRI (Fig. 3C), indicating thatheterogeneous DNA species incorporating at least a portion ofthe BPV1 L1 gene was encapsidated by light VLPs.

FIG. 3. Characterization of DNA extracted from high (1.35-g/ml)- and low (1.30-g/ml)-density virus-like particles prepared from BPV1-infectedS. cerevisiae. (A) Undigested DNase of BPV1 virions (V), dense (D, 1.35 g/ml), and light (L, 1.30 g/ml) VLPs from BPV1-infected S. cerevisiae wereelectrophoresed on 1% agarose (I) and subjected to Southern blot hybridization with a 32P-labeled BPV1 genomic probe (II). (B) DNAs of naturalvirions and dense (1.35 g/ml) VLPs were digested with three enzymes as shown and hybridized with 32P-labeled BPV1 L1 gene. (C) DNA of light(1.30 g/ml) VLPs was digested with eight restriction enzymes as shown and hybridized with 32P-labeled BPV1 L1 gene. (D) Predicted hybridizationpattern of BPV1 DNA with 32P-labeled BPV1 L1. (E) Schema showing the restriction sites of eight enzymes for the BPV1 genome and the locationof the L1 gene probe.



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Packaged DNA is newly replicated. To determine whetherthe DNA packaged by BPV1 VLPs produced in S. cerevisiaecells was newly synthesized, all cultures were labeled with[3H]thymidine 4 h after BPV1 infection. Fractions of VLPdensity were prepared by CsCl gradient separation. Incorpo-ration of [3H]thymidine into the extracted DNA was then ex-amined (Fig. 4A). Cell cultures not exposed to BPV1 had nosignificant incorporation of 3H into DNA in the fractions ofVLP density, whereas fractions from BPV1-infected culturesincorporated significant 3H. Moreover, the maximal 3H activitywas seen in the 1.30- and 1.35-g/ml fractions, showing thatDNase-resistant DNA packaged within VLPs included newlysynthesized viral and/or cellular DNA. In separate experi-ments, aliquots of each fraction of the CsCl gradient weredialyzed and treated with DNase I and DNA extracted (Fig.4B). Inclusion of PV-associated DNA within these fractionswas confirmed by hybridization with a BPV1 DNA probe (Fig.4C).

VLPs produced in BPV1-infected S. cerevisiae are infectious.To determine whether the VLPs produced in BPV1-infected S.cerevisiae cells were infectious, VLPs after dialysis and DNasedigestion were added to Cos1 cells in culture. After 48 h, cellswere collected for protein and Hirt DNA preparations. L1protein was detected in protein samples prepared from Cos1cells infected with VLPs or with E4 antibody-neutralized VLPs

(Fig. 5A) but not from Cos1 infected with L1 antibody-neu-tralized VLPs (Fig. 5A), confirming that VLP rather thanDNA was taken up by mammalian cells. DNA Southern blothybridization revealed that episomal BPV1 DNA was detectedin samples prepared from Cos1 cells exposed to dense or lightVLPs (Fig. 5B), suggesting that the VLPs produced in BPV1-infected S. cerevisiae cell cultures can effectively convey pack-aged viral DNA to mammalian cells. PCR analysis with prim-ers specific for different BPV1 genes revealed further that allORFs of BPV1 could be detected within Hirt DNA samplesfrom infected Cos1 cells (Fig. 5C).

To further confirm the infectivity of BPV1 VLPs producedin S. cerevisiae, we carried out a focus formation assay withC127 cells (43). VLPs from the dense or light fractions of aCsCl gradient were added to C127 cells. After 28 days, trans-formation of C127 monolayers exposed to dense or light VLPswas observed (Fig. 6). Data from four focus assay experimentshas shown that the number of foci produced by BPV1 virus was(3.9 � 1.4) � 103/�g of L1 protein, the number of foci pro-duced by the dense VLPs was (3.1 � 1.2) � 103/�g of L1protein, and the number of foci produced by the light VLPs(1.2 � 0.4) � 103/�g of L1 protein. The infectivity of the denseVLPs was 2.5 times higher than the light VLPs in C127 cells.Taken together, these experiments indicate that BPV1 virionscan reproduce its life cycle in S. cerevisiae cells.

To estimate the efficiency of BPV1 reproduction in S. cer-

FIG. 4. Characterization of DNA extracted from 22 fractions of aCsCl gradient prepared from S. cerevisiae cells infected with BPV1virions labeled with (A) or without (B and C) [3H]thymidine. (A) In-corporated radioactivity was determined by scintillation counting. Thedata presented are the average of three independent experiments, andthe standard deviations are indicated by error bars. (B) DNA withinfractions is characterized by gel electrophoresis (1% agarose).(C) DNA within fractions (see panel B) was blotted and characterizedby hybridization with a 32P-labeled BPV1 genomic probe. The numbersrepresent CsCl gradient fractions from 1 (1.45 g/ml) to 22 (1.26 g/ml).

FIG. 5. Delivery of viral DNA by VLPs prepared from BPV1-in-fected S. cerevisiae protoplast cultures. Cos1 cells were exposed todense (1.35 g/ml) or light (1.30 g/ml) VLPs. (A) Assay of BPV1 L1protein in Cos1 cells exposed to dense (1.35 g/ml) VLPs. Cells wereexposed as follows: lane 1, VLPs; lane 2, VLPs pretreated with poly-clonal L1 antibody (1:1,000); lane 3, VLPs pretreated with monoclonalL1 antibody (1:1,000); lane 4, VLPs pretreated with E4 antibody (1:1,000). (B) Southern hybridization of Hirt DNA from Cos1 cells ex-posed to VLPs as indicated and probed with BPV1 genomic DNA.Lanes: 1, � DNA; 2, 1.35 g of VLPs/ml; 3, 1.35 g of VLPs/ml, pre-treated with polyclonal L1 antibody (1:1,000); 4, 1.35 g of VLPs/ml,pretreated with monoclonal L1 antibody (1:1,000); 5, 1.35 g of VLPs/ml, pretreated with E4 antibody (1:1,000); 6, 1.30 g of VLPs/ml; 7,1.30 g of VLPs/ml, pretreated with polyclonal L1 antibody (1:1,000); 8,1.30 g of VLPs/ml, pretreated with monoclonal L1 antibody (1:1,000);9, 1.30 g of VLPs/ml, pretreated with E4 antibody (1:1,000). (C) PCRanalysis for BPV1 early and late genes of Hirt DNA prepared fromCos1 cells infected with dense VLPs (1.35 g/ml).



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evisiae, we calculated the yields of VLP production afterBPV1-infection. L1 protein in the dense (1.35-g/ml) and light(1.30-g/ml) VLP fractions was quantified by immunoblottingand densitometric analysis, with defined quantities of purified

L1 protein of natural BPV1 virions serving as a standard (Fig.7). Based on six independent experiments, 8.9 � 4.5 �g ofVLPs were recovered in a 0.6-ml fraction at a density of 1.35g/ml and 13.1 � 1.9 �g of VLPs were recovered in a 0.6-mlfraction at a density of 1.30 g/ml at 4 days postinfection. Inthese experiments, cells were exposed to 8.6 � 1.8 �g of nat-ural BPV1 virions. The data, taken together with the distribu-tion of L1 over the entire CsCl gradient (Fig. 2A), allow aminimal estimate of (i) the output of VLPs (measured as L1)at least five times higher than that of the input L1 protein and(ii) the output of infectious particles that was at least similar tothe input of infectious virus.

DISCUSSION

Using an S. cerevisiae cell culture system, we demonstratedthe DNA replication, RNA transcription, and L1 protein trans-lation of BPV1 in S. cerevisiae cells infected with natural BPV1virions (56). The capacity of a range of types of PV genomes,including BPV1, HPV6b, HPV11, HPV16, HPV18, andHPV31, to replicate in S. cerevisiae has been demonstrated byothers (1). In the present study, by using immunofluorescencemicroscopy, we have determined that the major capsid protein(L1) of the BPV1 is localized in the nuclei of BPV1-infected S.cerevisiae cells and that VLPs can be isolated from a lysate ofinfected S. cerevisiae cells. VLPs of densities typical of emptyand full PV virions were present in infected cells, and thesewere assembled from newly synthesized L1 protein in S. cer-evisiae cultures. VLPs encapsidated both viral and cellularDNA and were able to deliver encapsidated BPV1 DNA andto transform susceptible cells. S. cerevisiae exposed to naturalBPV1 virions can thus produce infectious BPV1 particles, andthis represents a promising model for propagation of PV viri-ons in vitro.

The host range of the PVs is narrow. In contrast, PV capsidsare able to bind to a wide variety of cells derived from a diversenumber of species, indicating that a specific cellular receptor isnot responsible for the narrow host range (36). Multiple re-ceptor and uptake mechanisms have been demonstrated forPVs. PVs are believed to infect basal epithelial cells via the6�4 integrin receptor (7, 32). Other receptors, including hep-arin and glycosaminoglycans have also been reported (19). Thecarboxyl-terminal portion of HPV11 interacts with heparin,and that this region appears to be crucial for interaction withthe cell surface (19). Glycosaminoglycans, on the cell surface,are one group of molecules able to serve as a putative receptor(62). Heidenreich and Dierich (11) reported an integrin-likeprotein in C. albicans. In an independent study, Edwards et al.(6) found that the MAb Mo-1 raised against human comple-ment receptor type 3 bound specifically to C. albicans. Severalintegrin-like proteins identified on the cell surface have beenreported in different yeast species (14). Recently, an integrin-like protein identified at 37 kDa was immunoprecipitated withantibodies to the 5�1 and v�3 integrins and showed 75%homology at the nucleotide sequence level to alcohol dehydro-genase of S. cerevisiae (23). It is possible that a receptor ofrelatively low specificity permits uptake of BPV1 by S. cerevi-siae cells after cell wall digestion. However, a recent studyobserved that 6 integrin is not the obligatory cell receptor forBPV4 (47). As an alternative, therefore, virus may be internal-

FIG. 6. Focus formation assay. C127 cells were cultured in 12-wellplate and exposed to different volumes (vol/vol) of BPV1 virions(V) and light (1.30 g/ml, L) and dense (1.35 g/ml, D) VLPs. A controlculture was left without infection (Mock). Cells were stained with 0.5%methylene blue plus 0.25% carbol fuschin for 15 min at 4 weeks afterinfection.

FIG. 7. Estimation of VLP production from BPV1-infected S. cer-evisiae cells with BPV1 virions as a standard. (A) Coomassie bluestaining of SDS-PAGE gel loaded with three concentrations of BPV1virions (V) and dense (D, 1.35-g/ml) and light (L, 1.30-g/ml) fractionspurified from BPV1-infected S. cerevisiae by using a CsCl gradient.(B) Immunoblot of duplicate of panel A with an MAb (MC15) to L1protein. Densitometry scanning was used to estimate the L1 signal.The signal for 1 �g of BPV1 virions was 7,306 � 1,846 arbitrary units.Over six experiments, the signal for 1.35 g of VLPs/ml was 5,585 �4,130, a value equivalent to 0.72 �g of L1/50 �l, and the signal for1.30 g of VLPs/ml was 7,651 � 2,556, a value equivalent to 1.09 �g ofL1/50 �l.



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ized by a endocytotic route since S. cerevisiae protoplasts areendocytotic. Exposure of S. cerevisiae protoplasts to BPV1virions is followed by ongoing autonomous episome replication(1, 56), transcription of the viral RNA (K.-N. Zhao et al.,unpublished data) and translation of L1 and L2 capsid. Theexpression of L1 and L2 capsid proteins lead to the assembly ofvirus particles and DNA encapsidation observed in the presentstudy.

Since the early 1980s, various yeasts have been used as anexpression system for the production of VLPs. Hepatitis B(53), poliovirus (18), and a range of PVs VLPs, includingHPV6 (13, 38, 45, 55), cottontail rabbit PV (16), HPV16 (39,44, 45), HPV18 (13), and HPV11 (3, 19, 30), have been pro-duced in yeasts. Yeasts have also been used to study the as-sembly of PV VLPs and the efficiency of VLP production. Forexample, the L2 protein of HPV6 and HPV16 is not incorpo-rated into the VLPs synthesized in S. pombe (45). Joyce et al.(19) reported that HPV11 L1 forms particulate structures re-sembling native virus with an average particle diameter of 50 to60 nm. VLPs assembled in S. cerevisiae can interact with hep-arin and with cell surface glycosaminoglycans resembling hep-arin on keratinocytes and CHO cells (19). The efficiency ofVLP production depends on the expression of the PV L1 gene.Neeper et al. (38) observed that few HPV11 VLPs were pro-duced in S. cerevisiae because of a truncation of the HPV L1mRNA in their experiments. Yeager et al. (55) generatedHPV11 pseudovirions in S. cerevisiae in which VLPs are cou-pled to the �-lactase gene and used them to define neutralizingantibodies. Thus, PV virions produced in S. cerevisiae are heldto be useful for studies of natural PV infection. However, all ofthe studies were carried out after transformation of S. cerevi-siae with PV genes. In recent studies we introduced the au-thentic viral genome of BPV1 into yeast protoplasts to studythe episomal replication of the BPV1 genome in S. cerevisiae(53). In the present study, we have extended the utility of S.cerevisiae production of virions to study the life cycle of BPV1.Generally, epithelial differentiation is critical for efficient PVreplication (15) since cellular differentiation is also necessaryfor optimal replication in other virus systems, including cyto-megalovirus (54), Friend virus, (17), and human immunodefi-ciency virus (4). However, the complete life cycle of HPV16has recently been displayed in cultured placental trophoto-blasts (29). The present study suggests also that epithelial dif-ferentiation is not an absolute requirement for BPV1 virionproduction. Thus, studies of the replication of PV in S. cerevi-siae, in placental trophotoblasts, and in epithelial cells shouldcontribute to our understanding of the epithelial cell specificfactors assisting viral production.

Different PV protein expression systems, including vacciniavirus and baculovirus, have allowed DNA encapsidation intoexpressed PV VLPs (43, 57–60). Recently, Rossi et al. (44)reported that S. cerevisiae as a PV expression system alsoallows DNA encapsidation into the expressed VLPs of HPV16.In the present study, two types of VLPs are identified in BPV1-infected S. cerevisiae cells. Dense VLPs encapsidate an intact8-kb BPV1 genome, and cellular DNA was also packaged.Less-dense VLPs incorporate a mixture of virus and cell-de-rived DNA, and not all virus DNA is intact episome. The datasupport previous studies that VLPs assembled with PV L1 andL2 capsid proteins can package whole-length BPV1 genome

(43, 60). Previously, we observed that the DNA packaged in1.30 g of BPV VLPs/ml is preferentially 5 kb in both thevaccinia virus and baculovirus expression systems (58, 59). Inthe present study, the 1.30-g BPV1 VLPs packaged 8-kb BPV1genomic DNA and 4-kb cellular DNA. The DNA packaged byVLPs in the present study was DNase I resistant, in contrast toa recent report that DNA associated with light VLPs in S.cerevisiae was not DNase I resistant (44). Mechanisms of DNApackaging by BPV1 VLPs produced in virus-infected S. cerevi-siae cells may be different from those for VLPs produced in aS. cerevisiae transformation system. Dense VLPs (1.35 g) as-sembled in BPV1-infected S. cerevisiae cells preferentially en-capsidate BPV1 genome, suggesting that mechanisms for as-sembly of the virus particles and encapsidation of the viralgenome in BPV1-infected S. cerevisiae cultures may be analo-gous to those in epithelial and basal cells of the papillomas.

After Kreider et al. (24) succeeded in using raft culturetechnique to produce HPV11 infectious particles, different raftculture systems and several keratinocyte cell lines have beendeveloped to establish the PV life cycle and produce infectiousvirions of different PV types, including HPV11 (24, 49),HPV16 (8, 48), HPV31b (34), HPV18 (35), and BPV1 (31).More recently, Meyers et al. (33) reported that HPV18/16infectious chimeric particles could also be produced in raftculture. However, no study on the output and input of PVvirions in raft cultures has been reported. Low yields of VLPswere observed for several PV types in different expressionsystems (38, 50), which is also an issue for raft cultures becausethere are no models for propagation of free PV viral particles(2). Production of PV VLPs presumably reflects in part levelsof L1 protein expression (51, 58). As in the baculovirus expres-sion system (58), S. cerevisiae cultures produce significantlymore BPV1 L1 protein than they receive as infectious virus,and at least half of the packaged DNA is BPV1 derived in thecurrent culture system. Further, we have estimated the outputof infectious virions achieved in the S. cerevisiae system byusing a focus assay, and this was of the same magnitude after4 days in culture as the input virus. However, viral replicationcontinues to occur over much longer period periods of yeastculture, and we are currently assessing the efficiency of prop-agation of PV episomes through multiple yeast division and ofthe production of PV virions in longer-term yeast cultures.

In conclusion, BPV1 VLPs produced in the S. cerevisiaesystem are infectious, and the output yield of both L1 proteinand infectious virus particles was similar to the amount ofinput virus in short-term (4 days) cultures. Thus, this systemmay be a promising model for the propagation of free virusparticles of BPV1 in vitro and will allow further genetic anal-ysis of the life cycle of BPV1.

ACKNOWLEDGMENTS

The late Jian Zhou strongly supported the initiation of this work.We thank Quan Mei Tu for helping with immunofluorescence micros-copy and Nigel McMillan and Wen Jun Liu for helpful discussions.

This work was funded in part by the Queensland Cancer Fund, theNational Health and Medical Research Council of Australia, and thePrincess Alexandra Hospital Foundation.

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