Vol. 65, No. 12JOURNAL OF VIROLOGY, Dec. 1991, p. 6528-65340022-538X/91/126528-07$02.00/0Copyright C) 1991, American Society for Microbiology

Bovine Papillomavirus with a Mutation in the E2 Serine 301Phosphorylation Site Replicates at a High Copy Number

ALISON A. McBRIDE* AND PETER M. HOWLEY

Laboratory of Tumor Virus Biology, National Cancer Institute, Bethesda, Maryland 20892

Received 16 July 1991/Accepted 22 August 1991

The E2 open reading frame of bovine papillomavirus type 1 (BPV-1) encodes at least three proteins withtranscriptional regulatory properties. The full-length E2 open reading frame encodes a transcriptionaltransactivator, and the 3' region encodes two smaller polypeptides that repress E2-mediated transactivation.The full-length gene product is also required for viral DNA replication. We have demonstrated that the BPV-1E2 polypeptides are phosphorylated primarily on two serine residues at a site adjacent to the carboxy-terminalDNA binding domain, which is common to all three E2 proteins (A. A. McBride, J. B. Bolen, and P. M.Howley, J. Virol. 63:5076-5085, 1989). These serine residues, at amino acid positions 298 and 301, were

substituted with alanine residues in the context of the entire BPV-1 genome. The mutated BPV-1 genomes wereintroduced into rodent cell lines and assayed for focus formation, viral gene expression, and extrachromosomalviral DNA replication. Viral DNAs containing the E2 serine-to-alanine substitution mutants transformed C127cells with efficiencies comparable to that of wild-type BPV-1. However, the viral genome containing theserine-to-alanine substitution at position 301 of the E2 polypeptide replicated to a copy number 20-fold higherthan that of wild-type DNA.

Bovine papillomavirus type 1 (BPV-1) is one of the best-studied papillomaviruses and provides a useful model for thestudy of eukaryotic transcriptional regulation and DNAreplication. BPV-1 is able to transform certain rodent celllines in culture; the viral genome is maintained extrachro-mosomally within these cells, and only early viral genes areexpressed (9, 15, 22). Viral gene expression and replicationare thought to be regulated, at least partially, by the productsof the E2 open reading frame (ORF).The E2 ORF of BPV-1 encodes several polypeptides with

regulatory functions. The full-length E2 gene product func-tions as a transcriptional transactivator and can activatetranscription from several viral promoters by binding tospecific DNA sequence elements located within E2-depen-dent enhancers (1, 34, 47, 48). Two smaller E2 polypeptides,encoded by the 3' portion of the ORF, inhibit E2-mediatedtransactivation. One of these repressors, E2-TR, is ex-pressed from the P3080 promoter and translated from anin-frame initiation codon at amino acid 162 of the E2 ORF.The second repressor, E8/E2, is expressed from a splicedmessage that encodes 11 amino acids from the E8 ORFspliced to E2 via the acceptor at nucleotide (nt) 3225 (6, 19,21). The full-length E2 polypeptide consists of two domainslinked by a hinge region that is nonessential for transactiva-tion. The N-terminal domain, which is present only in thefull-length transactivator, encodes the activation function(11, 14, 29). The C-terminal domain has a specific DNAbinding and dimerization function and is present in all threeE2 polypeptides (8, 29, 30). The repressors could inhibitE2-mediated transactivation by competitively binding tospecific E2 binding sites or by forming potentially inactiveheterodimers between transactivator and repressor species.Furthermore, it has recently been shown that the full-lengthE2 polypeptide and the BPV-1 El polypeptide are bothnecessary and sufficient for transient viral DNA replication(49). This finding is in agreement with previous genetic

* Corresponding author.

studies which showed that both El and E2 genes arerequired for stable plasmid replication (7, 39). However,little is known about the E2 sequences important for thisreplication function.We have previously demonstrated that the E2 polypep-

tides are phosphorylated primarily on two residues at a siteadjacent to the common C-terminal DNA binding domain(28). In other systems, phosphorylation has been shown tobe important for posttranslational regulation of factors in-volved in transcription and replication. It has been impli-cated in the regulation of DNA binding affinity and speci-ficity, polypeptide dimerization, activation of transcription,and modulation of protein-protein interaction. For example,phosphorylation by casein kinase II (CKII) inhibits DNAbinding by c-myb (23) but enhances the DNA binding activ-ity of the serum response factor (26). Protein kinase Cenhances the DNA binding activity of the cyclic AMPresponse element-binding protein, CREB, probably by mod-ulation of dimer formation (50). Protein kinase C activationalso decreases phosphorylation of several glycogen syn-thetase kinase 3 sites in c-jun. Phosphorylation of these sitesnegatively regulates the DNA binding activity of c-jun (4).The replication functions of simian virus 40 (SV40) large Tantigen are both positively and negatively regulated byphosphorylation. Phosphorylation of threonine residue 124stimulates replication activity by increasing DNA bindingspecifically to one site in the replication origin; however,phosphorylation of specific serine residues decreases repli-cation activity by reducing DNA binding, and this effectappears to be modulated by protein phosphatase 2A (re-viewed in reference 37). Transcriptional activation by CREBand yeast heat shock factor is modulated by phosphorylation(12, 46), and IKB, the inhibitor that binds to NFKB, isinactivated by phosphorylation, allowing NFKB to translo-cate to the nucleus and activate transcription (10, 44).The locations of the phosphoserine residues in the BPV-1

E2 polypeptides, at amino acids 298 and 301, are shown inFig. 1. This region has previously been designated the hingeregion because it does not appear to be essential for trans-

6528

REPLICATION OF MUTANT BPV 6529

3080- \322

2443\305

E2

162

E2-TR206

Transactivation /, DNA,,' \ Dimer

,' p p

TVPVDLASRQEEEEQSPDSTEEEPV298 301

FIG. 1. Structures of the BPV-1 E2 gene productsof the major E2 phosphorylation sites. The 410-amlength E2 polypeptide can be expressed from the P,The E2-TR repressor is expressed from the P3080initiated at codon 162 in the E2 ORF. The E8/E2encoded by a spliced message linking 11 amino acidsORF to the C-terminal 204 amino acids of E2. T}domains encode transactivation, DNA binding, andfunctions, as indicated. Molecular sizes of the pol)indicated at the right, and positions of the major ph(sites and surrounding amino acid sequence are shown

activation (29). However, this region (amino a310) is required in addition to the DNA bindingtranscriptional repression (27). Although the samino acids surrounding the phosphorylationconserved among all of the papillomaviruses, se

298 and the following two amino acids, proline a

acid, are conserved among the fibropapillomaviriBPV-2, deer papillomavirus, and European elk lrus. The region surrounding the BPV-1 phosphorhas an unusual sequence composition and constiPEST sequence. These sequences, rich in proliiand aspartic acids, serine, and threonine, are fouiproteins with a short half-life, and it has been prthey may be involved in protein turnover (41).residue at position 301 can be predicted to be;CKII site, and the serine at 298 could be a potentwhen residue 301 is phosphorylated, thus pr

necessary negative charge for CKII recogniti4Phosphorylation sites are common in PEST r

could reflect the amino acid composition of thesecould also indicate that phosphorylation playregulating the function of the PEST sequences.To examine the functional role of phosphory]

BPV-1 E2 polypeptides, we specifically mutateresidues at positions 298 and 301 to alanine re

mutated E2 polypeptides were assayed for theactivate and repress transcription through BPhancer elements. In addition, the E2 mutationstuted into the entire BPV-1 genome, and the resu

genomes were analyzed for transcription, trarand replication properties.

MATERIALS AND METHODS

Plasmid constructions. Serine-to-alanine poinwere constructed in the E2 ORF by using synthcleotides as described previously (28). The KI

fragment containing these mutations was substituted for the

410 corresponding wild-type fragment in plasmids C59 E2kZ, C59(48 Kd) E2284_410, and p142-6. C59 E2kz was constructed by replacing

4 (31Kd0 the BstEII (nt 2406)-to-BstXI (nt 3889) fragment of E2 in C59410 with the BstEII-to-BstXI fragment of pTZE2kZ. In the latter11(2f1Kd) construct, the sequences between BstEII and Sphl (nt 2622)

were replaced with synthetic oligonucleotides that providedBinding a Kozak consensus initiation codon for the E2 ORF (28). C59brization E2284-410) was constructed by replacing the BstE2-to-KpnI (nt

3460) E2 fragment of C59 with a synthetic oligonucleotidethat provided an in-frame initiation codon. p142-6 and p964have been described elsewhere (42, 47).

Transient expression assays. CV-1 cells were transfectedby the calcium phosphate technique as described previously

fTLPR (48). Briefly, 60-mm dishes of CV-1 cells were transfectedwith 4 ,ug of the chloramphenicol acetyltransferase (CAT)

and locations expression plasmid and various amounts of the expressionino-acid full- plasmid C59-E2kZ, C59-E2284-410, or p142-6. A P-galactosi-

promoter. dase expression vector, pCH110 (Pharmacia), was included)romoter and as an internal standard, and salmon sperm DNA was addedrepressor is to a total of 10 ,ug. Cells were incubated for 12 h in mediumfrom the E8 containing 3 mM sodium butyrate and harvested at 48 h

he conserved posttransfection. The assays were carried out in the lineardimerization range, and CAT activity was normalized for 3-galactosidase

ypeptides areexrsinosphorylation expression.osphorylation Focus formation assays. C127 cells were transfected by thecalcium phosphate technique. Briefly, 100-mm dishes ofcells were transfected with various amounts of p142-6 DNA

*cids 289 toor p142-6 DNA that had been cut at the BamHI site to

cids 2 to separate BPV-1 and pML2d sequences and religated at adomain for concentration of 5 jig/ml to favor intramolecular recircular-sequence of ization. Cells were stained with methylene blue 2 to 3 weekssites iS not posttransfection.rine residue Cellular DNA analysis. Total cellular DNA was preparedmnd glutamic from BPV-1-transformed cells by using standard procedures.uses BPV1, Transient replication assays were carried out as described bypapillomavi- Ustav and Stenlund (49). Briefly, C127 cells were electropo-ylation sites rated with 1 ,ug of BPV-1 DNA that had been cleaved fromtutes a good the pML2d vector and recircularized as described above.ne, glutamic Low-molecular-weight DNA was prepared from cells at 24-hnd mainly in intervals. DNA samples were digested, separated on agaroseroposed that gels, and blotted onto Nytran (Schleicher & Schuell). BPV-1

anThe senine sequences were detected by hybridization of a p142-6 probe

anCKII site generated by the nick translation or random primer tech-

oviding the niques, using kits supplied by Boehringer Mannheim.on (31, 32)..egions; thisregions but RESULTSs a role in Transcriptional properties of E2 phosphorylation site mu-

tations. To determine whether posttranslational phosphory-[ation of the lation is important for the function of the BPV-1 E2 proteins,d the serine E2 phosphorylation site mutants were assayed for theirsidues. The ability to regulate transcription. As described previously,ir ability to three serine residues at amino acid positions 290, 298, andV-1 E2 en- 301 were specifically and individually substituted with ala-were substa- nine residues. In addition, an E2 mutant was generated with.lting mutant all three serine residues replaced with alanine residues. Theisformation, serine residues at positions 298 and 301 are the major

phosphorylation sites of the E2 proteins (28). The mutationswere introduced into C59-E2kZ, a plasmid that expresses thefull-length E2 transactivator from the SV40 early promoter,and into C59-E2184410, a plasmid that expresses the C-ter-

it mutations minal 117 amino acids from the SV40 early promoter and isetic oligonu- able to repress E2-mediated transactivation. In addition,gnI-to-BstXI each of the mutations was exchanged into p142-6, a plasmid

VOL. 65, 1991

6530 McBRIDE AND HOWLEY

that contains the entire BPV-1 genome cloned into theprokaryotic vector, pML2d (42).The E2 mutations also result in amino acid substitutions in

the overlapping E3 and E4 ORFs. However, previous stud-ies have indicated that the E3 and E4 ORFs play no role intransformation, replication, or regulation of viral geneexpression in C127 cells (16, 36).Each of the C59-E2kz plasmids was assayed for its ability

to activate transcription in CV-1 cells. This was quantifiedby using the reporter plasmid p964, in which the CAT geneis expressed from the enhancer-deleted SV40 early promoterlinked to the E2-responsive element, E2RE1 (47). Expres-sion of CAT from this plasmid is dependent on the presenceof the full-length E2 transactivator protein. Various amounts(0.5, 5, 50, and 500 ng) of the C59-E2kZ plasmids containingthe serine-to-alanine substitutions were cotransfected intoCV-1 cells with 4 ,ug of p964, and E2-mediated transactiva-tion was assayed by measuring CAT activity. None of theserine-to-alanine substitutions had any effect on the ability ofthe full-length E2 protein to activate transcription in thistransient transfection assay (data not shown).The C59-E2284-410 construct containing all three serine-to-

alanine substitutions was assayed for its ability to inhibitE2-dependent transactivation from p964. Various amountsof this construct (0.1, 0.5, and 2.5 jLg) were cotransfectedwith 0.5 ,ug of C59-E2kZ and 3 ,ug of p964 into CV-1 cells. Theserine-to-alanine substitution mutant was similar to thewild-type construct (C59-E2284 410) in its ability to repressE2-mediated transactivation (data not shown).

Levels of E2-specific transactivation expressed from eachof the mutants was measured in the background of the entireBPV-1 genome. In this context, transactivation should bedependent on the relative levels and activities of the E2transactivator and repressor proteins. Increasing amounts ofthe mutated p142-6 plasmids (0.2, 1, and 5 ,ug) were cotrans-fected into CV-1 cells with the CAT reporter plasmid p964.BPV-1 genomes containing the serine-to-alanine substitutionat position 301 (p142-6 E2 A301) and alanine residues at allthree positions (p142-6 E2 AAA) expressed higher levels oftransactivation than did the wild-type genome (Fig. 2).Similar results were obtained when the prokaryotic vectorsequences were cleaved from the full-length BPV-1 genomeprior to transfection. A smaller but reproducible increase intransactivation was also observed with the E2 A301 and E2AAA full-length genomes when the experiment was carriedout in C127 cells. Since the increase in transactivation wasobserved with genomes containing either E2 A301 or E2AAA, it seemed probable that the phenotype was due to theserine-to-alanine substitution at position 301. One explana-tion for the greater transactivation observed from p142-6plasmids containing the E2 alanine substitutions at aminoacid 301 is an increase in the ratio of transactivator torepressor species. Alternatively, the function of one or moreof the E2 polypeptides may have been altered. This couldmodify viral gene expression in such a way as to result inincreased transactivation from the entire genome.

Transformation properties of the E2 phosphorylation sitemutations. Mutations in the E2 ORF of BPV-1 are pleiotro-pic, affecting transcription, transformation, and replicationfunctions of the virus (7, 39). To determine whether theserine-to-alanine mutations played a role in any of theseactivities, the E2 phosphorylation site substitution mutantswere assayed for focus formation in C127 cells. Full-lengthBPV-1 containing the serine-to-alanine 301 substitution andthe triple mutation AAA were both defective in their abilityto induce focus formation when linked to pML2d. The few

A. AAA150 r

125

iool

C.)75

501

25

AA3o1

- - -W

¢_________________ ~~~~A298

1 2 3 4 5BPV 1 DNA (ag)

FIG. 2. Levels of transactivation in BPV-1 t2 phosphorylationsite mutants. CV-1 cells were transfected with 4 ,ug of p964, variousamounts of p142-6 plasmids containing the E2 mutations, and 0.5 ,ugof pCH11O, which was included as an internal standard. Activitywas determined by normalizing the percent chloramphenicol acety-lated to the level of ,3-galactosidase expression. WT, wild type.

transformed foci that did arise after transfection with theseplasmids contained very high levels of extrachromosomalviral DNA which was rearranged. Restriction enzyme map-ping revealed that the rearrangements consisted primarily oflarge deletions in the prokaryotic vector, pML2d (data notshown). It appeared that by assaying for focus formation, wewere selecting for those viral genomes that had deleted partof the vector. This suggested that the vector sequences hadan inhibitory effect which the wild-type but not the mutantE2 alanine 301 genomes could overcome. To test this hy-pothesis, the BPV-1 sequences were cleaved from thepML2d vector and recircularized before transfection intoC127 cells. In the absence of the prokaryotic sequences, allof the E2 phosphorylation site mutants induced focus for-mation in C127 cells with wild-type efficiency (Fig. 3). Thisphenotype is reminiscent of BPV-1 mutations in the 3'enhancer region, which are also defective in their ability totransform cells when linked to the bacterial plasmid (17).

Replication properties of the E2 phosphorylation site muta-tions. To analyze the replication properties of the mutatedBPV-1 genomes, cellular DNA was prepared from pooledtransformed foci and assayed by Southern blot analysis forviral DNA sequences. In this experiment, the BPV-1 se-quences were cleaved from the prokaryotic pML2d vectorand recircularized prior to the transformation. As shown inFig. 4, each of the mutant viral DNAs was present predom-inantly as a plasmid. However, the copy number of BPV-1E2 A301 and E2 AAA was much higher than that of thewild-type viral DNA or of the genomes containing aminoacid substitutions at serine residues 290 and 298. No rear-rangement of viral DNA sequences was observed. Thehigher copy number of the BPV-1 E2 AAA and A301mutants was estimated by diluting cellular DNA from themutant containing cell lines with increasing amounts of C127cellular DNA. The hybridization analysis shown in Fig. 5

J. VIROL.

REPLICATION OF MUTANT BPV 6531

WT AAA A290 A298 A301..;''0.'''>; .s. X . Sb M.P- , v ... . t

'. ', * * , #

* 7,

.. ' '

e + f vt , _f J _ _* fl +. s t. - w w

Z -.J, . .t _

w

) m* ---. 7)

)tiSooA('0'/

\\ s -,J -,.

/

FIG. 3. Transformation of C127 cells by BPV-1 E2 phosphorylation site mutants. Various amounts of p142.6 plasmids containing the E2phosphorylation site mutations were cleaved with BamHI to separate the BPV-1 and vector sequences, recircularized, and transfected intoC127 cells. Plates were stained with methylene blue 2 weeks after transfection. WT, wild type.

demonstrated that cells containing the E2 A301 and AAAmutants contained a viral DNA copy number about 20-foldhigher than that of wild-type BPV-1, which was present atabout 50 copies per cell. Cell lines containing the mutatedgenomes were also generated by cotransfection withpSV2neo and selection for G418-resistant colonies ratherthan for focus formation. With drug selection rather thangrowth selection, the BPV-1 E2 AAA and A301 DNAs were

also present at very high copy number (data not shown).Transient replication of E2 phosphorylation site mutants.

By assaying replication in pooled transformed foci (or even

in pooled G418-resistant colonies), it is possible that there isa selection for a population of cells with a high copy numberof viral DNA molecules. To determine whether the high-copy-number phenotype was an inherent property of the E2

mutated viral genome and to analyze at what stage amplifi-cation of viral DNA occurred, replication of the BPV-1 E2AAA genome was analyzed in a transient replication assay,as described recently by Ustav and Stenlund (49). C127 cellswere electroporated with 1 ,ug of BPV-1 DNA that had beencleaved from the pML2d vector and recircularized. Low-molecular-weight DNA was extracted from the cells at 24-hintervals, digested with DpnI and HindIll, and analyzed byfilter hybridization. Replicated DNA (DpnI resistant) islinearized with HindIII and forms a band of 7,946 bp. A Bclldeletion mutant (p327-25) that removes the C-terminal 34amino acids of E2 was used as a negative control (43). Thismutation removes part of the DNA binding domain of E2,and the resulting viral genome is unable to replicate (43). As

UNCUT Xhol1 2 3 4 5 6 1 2 3 4 5 6

FIG. 4. Replication of E2 phosphorylation site mutants. CellularDNA from pooled transformed C127 cells containing the BPV-1phosphorylation site mutants was analyzed on a Southern blot. TheDNA was either uncut or cleaved with XhoI (which does not cutBPV-1) and probed with nick-translated p142-6 DNA. Forms I andII of circular episomal DNA are indicated. Lane 1 contains DNAfrom nontransformed C127 cells. The other lanes contain DNA fromC127 cells transformed by the following DNAs: lane 2, wild-typeBPV-1; lane 3, BPV-1 E2 AAA; lane 4, BPV-1 E2 A290; lane 5,BPV-1 E2 A298; lane 6, BPV-1 E2 A301.

AAAco cmj e

N- I- C1il OD " M M3.- * .. ...., .., ..

A301= CD CMr-"cso " m-)r

Amwo

--I

FIG. 5. DNA copy number of E2 phosphorylation site mutants.The increase in copy number of the BPV-1 E2 AAA and A301mutants was estimated by diluting DNA from cell lines transformedby the mutants with that of uninfected C127 cellular DNA in theratios indicated. The DNAs were cleaved with Hindlll to linearizethe viral DNA and analyzed on a Southern blot. Lane WT containsan equivalent amount of DNA (5 ,ug) from wild-type BPV-1-transformed cells which contain approximately 50 copies of viralDNA per cell. BPV-1 sequences were detected as described above.

BPVDNAng

1000

250

50

VOL. 65, 1991

II

i

6532 McBRIDE AND HOWLEY

E2 E2wt AAA

WT AAA WT+ AAA

21 2-2 2323 4 3-2 3-3 34 3-6 4-9 4-10 4-11 412

Day 1 1 2 344-]I3 341 234 4 5

OW0eSStoi a,0

4EOIiW.IU

FIG. 7. Evidence that the high copy number of the E2 phosphor-ylation site mutant is dominant. C127 cells were transfected witheither 1 pLg of wild-type BPV-1 DNA (p142.6 cleaved with BamHIand recircularized) or 1 p.g of BPV-1 E2 AAA DNA or werecotransfected with 0.5 ,ug of both viral DNAs. Individual trans-formed foci containing either wild-type BPV-1 (WT), BPV-1 E2AAA, or both viruses, as indicated, were expanded into cell lines.Cellular DNA was isolated from each line, digested with HaeII, andanalyzed on a Southern blot. BPV-1 sequences were detected byhybridization with a nick-translated probe of p142-6 DNA.

FIG. 6. Transient replication of BPV-1 DNA in C127 cells. C127cells were electroporated with 1 ,ug of BPV-1 DNA that had beencleaved from the pML2d vector and recircularized. Low-molecular-weight DNA was extracted by the Hirt method at 24-h intervals,digested with DpnI and HindIII, and analyzed on a Southern blot.BPV-1 sequences were detected by hybridization with a random-primed p142-6 DNA probe. The sets of lanes were transfected withthe following DNAs: -, none; E2 bclIA, BPV-1 DNA containing adeletion between nucleotides 3737 and 3838; E2 wt, wild-typeBPV-1; E2 AAA, BPV-1 E2 AAA.

shown in Fig. 6, the BPV-1 genome encoding E2 AAAreplicated to a higher copy number than did wild-type DNAin transient assays. Furthermore, the copy number of the E2AAA genome increased at a greater rate than did that of thewild-type genome, even although the number of cells in eachassay increased at similar rates. Similar results were ob-tained with viral DNA containing the E2 A301 mutation. Ina longer time course, the E2 A301 genome had a copynumber ranging from 2- to 5-fold greater than the wild-typecopy number in the first few days after electroporation to 10-to 20-fold greater 12 days after electroporation (data notshown). These experiments indicated that the high-copy-number phenotype was likely to be an intrinsic property ofthe mutated viral genomes and not due to selection of asubpopulation of cells with high copy number.The high-copy-number phenotype is due to a trans-acting

factor and is dominant over wild type. To determine whetherthe high-copy-number phenotype of the E2 phosphorylationsite mutant was dominant over wild type, C127 cells weretransfected with either wild-type or E2 AAA BPV-1 DNA orwith both viral DNAs. Individual transformed foci contain-ing either wild-type BPV-1, BPV-1 E2 AAA, or both DNAswere expanded into cell lines, and the levels of the respec-tive DNA sequences were determined. The wild-type and E2

AAA mutated viral DNAs could be distinguished by virtueof an additional HaeII site resulting from the serine-to-alanine substitution at residue 298. The analysis shown inFig. 7 demonstrated that the high-copy-number phenotype ofthe E2 mutant was dominant; in cells containing BPV-1 E2AAA, the wild-type genome was also amplified to the highcopy number. Furthermore, this experiment confirmed thatthe phenotype was due to a trans-acting factor and not to acis-acting mutation in the BPV-1 E2 AAA genome resultingfrom the nucleotide changes required to generate the serine-to-alanine substitutions.

DISCUSSION

The BPV-1 E2 ORF encodes a 410-amino-acid protein thatfunctions in both transcriptional activation and viral replica-tion. Two smaller E2 polypeptides act to inhibit transcrip-tional activation by the full-length E2 product and couldconceivably also have a regulatory role in viral replication.All three E2 gene products can potentially be phosphory-lated at serine residues 298 and 301 in a region adjacent tothe DNA binding domain. In the studies presented in thisreport, we have demonstrated that mutation of serine resi-due 301 results in a viral genome that replicates at a copynumber about 20 times higher than that of the wild-type viralDNA. This phenotype is due to a trans-acting factor and isdominant over wild type. A phenotype has not yet beenassociated with the other major phosphorylation site atamino acid residue 298.The increase in genome copy number could reflect a direct

role of E2 phosphorylation in viral DNA replication. It hasbeen demonstrated recently that transient replication ofBPV-1 requires only the products of the El and E2 ORFs(49) and that the El and E2 polypeptides form a complexcapable of binding to E2 DNA sites (3, 33). One could

BPVDNA

E2Bcl IA

J. VIROL.

REPLICATION OF MUTANT BPV 6533

postulate that E2 phosphorylation modulates the interactionbetween the El and E2 polypeptides, thus regulating viralDNA replication. Alternatively, phosphorylation could mod-ulate some replication-associated activity of the E1-E2 pro-tein complex. In support of the former hypothesis, Luskyand Fontane have recently demonstrated that El complexesonly with the underphosphorylated form of E2 (25).An alternative explanation for the high-copy-number phe-

notype is that the E2 mutation results in an alteration in theexpression of other viral gene products involved in replica-tion. The mutated E2 gene products could alter the levels orthe pattern of expression of the El and E2 gene products. Ithas been shown that BPV-1 mutants disrupted in the E5 genereplicate at a low copy number and are unstable (13, 39) andthat mutations in the E6 and E7 ORFs have a reduced copynumber of extrachromosomal viral DNA (2), although thislatter finding has not been observed in other laboratories (35,38). A change in the abundance of these gene products couldconceivably give rise to an increase in viral genome copynumber.The mutation in one of the major phosphorylation sites

could affect the levels of the E2 proteins by changing thestability of the polypeptides. An alteration in the ratio oftransactivator to repressor species could have far-reachingeffects on viral gene expression and DNA replication. Theincrease in E2-specific transactivation observed from theviral genome containing the E2 A301 mutation supports thehypothesis that there is some alteration in viral gene expres-sion. This result is not due to increased genome replication,since the insertion of a translational termination linker in theEl ORF has no effect on the observed increase in transac-tivation of this mutant (27). E2 DNA binding site affinity orspecificity could also be regulated by phosphorylation andplay a role in both transcription and DNA replication. OtherE2 properties such as transactivation, repression, dimeriza-tion, cooperativity, and protein-protein interaction couldpotentially be modulated. BPV-1 mutants that are unable toexpress the E2-TR repressor exhibit increased transactiva-tion and replicate at a high copy number (20, 40). However,unlike the E2 A301 mutation, these mutants also have anenhanced ability to transform cells, and therefore the phe-notype of the BPV-1 E2 A301 mutant is unlikely to be duesolely to the removal of E2-TR.These studies do not prove that the high-genome-copy-

number phenotype results from the inability of the A301polypeptides to be phosphorylated. It is conceivable that theserine-to-alanine substitution at residue 301 results in E2polypeptides with altered properties that are unrelated tophosphorylation. To establish that the phenotype does resultfrom a defect in phosphorylation and to determine whichkinase may be involved, we are currently analyzing a num-ber of additional mutations in this region of the E2 polypep-tides. Serine residue 301 is a good CKII consensus site, andpreliminary studies indicate that CKII can phosphorylatethis region of the E2 polypeptide in vitro (27). CKII is aubiquitous kinase, but it can be stimulated by treatment ofcells with serum and growth factors (5, 18, 45) and hasrecently been demonstrated to modulate the DNA bindingactivity of c-myb and the serum response factor (24, 26).We are continuing these studies to define which E2

property has been altered by the substitution at amino acid301 and to determine why this alteration results in themarked increase in viral DNA copy number. Identification ofthe kinase responsible for phosphorylation of serine residue301 may provide some insight into the cellular regulation ofpapillomavirus gene expression and DNA replication.

ACKNOWLEDGMENTSWe thank John Benson, Helen Romanczuk, and Scott Vande Pol

for helpful discussions and for critical review of the manuscript.REFERENCES

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2. Berg, L. J., K. Singh, and M. Botchan. 1986. Complementationof a bovine papilloma virus low-copy-number mutant: evidencefor a temporal requirement of the complementing gene. Mol.Cell. Biol. 6:859-869.

3. Blitz, I. L., and L. A. Laimins. 1991. The 68-kilodalton Elprotein of bovine papillomavirus is a DNA-binding phosphopro-tein which associates with the E2 transcriptional activator invitro. J. Virol. 65:649-656.

4. Boyle, W. J., T. Smeal, L. H. K. Defize, P. Angel, J. R.Woodgett, M. Karin, and T. Hunter. 1991. Activation of proteinkinase C decreases phosphorylation of c-jun at sites that nega-tively regulate its DNA binding activity. Cell 64:573-584.

5. Carroll, D., and D. R. Marshak. 1989. Serum-stimulated cellgrowth causes oscillations in casein kinase II activity. J. Biol.Chem. 246:7345-7348.

6. Choe, J., P. Vaillancourt, A. Stenlund, and M. Botchan. 1989.Bovine papillomavirus type 1 encodes two forms of a transcrip-tional repressor: structural and functional analysis of new viralcDNAs. J. Virol. 63:1743-1755.

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