Functional analysis of a carboxyl-terminal phosphorylation mutant of the bovine papillomavirus E1 protein

doi:10.1006/jmbi.2001.5375 available online at http://www.idealibrary.com on J. Mol. Biol. (2002) 316, 599±609

Functional Analysis of a Carboxyl-terminalPhosphorylation Mutant of the Bovine PapillomavirusE1 Protein

Michael Lentz1*, Thomas Zanardi2, Robyn Filzen2, Jena Carter1

and Maria Hella1

1Department of BiologyUniversity of North FloridaJacksonville, FL 32224, USA2Department of Biochemistryand Biophysics, Texas A&MUniversity, College StationTX 77843, USA

E-mail address of the [email protected]

Present address: T. Zanardi, SierrNV, USA.

Abbreviations used: BPV, bovine1; HPV, human papillomavirus.

0022-2836/02/030599±11 $35.00/0

The papillomavirus E1 protein is essential for viral DNA replication, andphosphorylation of E1 appears to regulate protein function and DNAreplication. Serine 584 of bovine papillomavirus E1 is in a conservedmotif resembling a CK2 consensus site, and is phosphorylated by CK2in vitro. Mutation of serine 584 to alanine eliminates replication of theviral genome in transient replication assays. Wild-type and mutant E1proteins were expressed from recombinant baculoviruses and used toassess biochemical functions of the amino acid 584 substitution. Helicaseenzyme activity, E1 binding to the viral E2 protein and to cellular DNApolymerase alpha-primase were all unaffected in the mutant protein.Binding of E1 to viral replication origin DNA sequences was reducedin the mutant, but not eliminated. The carboxyl-terminal region of theprotein appears to play a role in regulating E1 function, and adds to acomplex picture emerging for papillomavirus DNA replication control.

# 2002 Elsevier Science Ltd.

Keywords: DNA replication; phosphorylation; helicase; papillomavirus;E1 protein*Corresponding author

Introduction

Papillomaviruses infect epithelial cells of a widevariety of vertebrate hosts. Their unique infectiouscycle provides an attractive model for investigatingregulation of DNA replication in eukaryotic cells.A latent infection is established in the basal cells ofthe epithelium, where the viral DNA is maintainedas an extrachromosomal episome.1 During latency,viral DNA synthesis is regulated to maintain a con-stant DNA copy number per cell, and viral geneexpression is limited to those genes required formaintenance of the viral DNA in the undifferen-tiated cell.2 ± 5 Progeny of dividing basal cellsmigrate towards the epithelial surface, initiating adifferentiation pathway. Differentiation of theinfected cell triggers changes in the pattern of viralgene expression, leading to ampli®cation of viral

ing author:

a Biomedical, Reno,

papillomavirus type-

DNA, synthesis of virus structural proteins, andassembly of progeny virions.6

This complicated viral life cycle requires severalmodes of DNA replication. Transient ampli®cationis required to establish the latent infection, fol-lowed by regulated replication to maintain lowcopy viral DNA in infected basal cells. In differen-tiated cells, higher replication rates are required toamplify copy number for assembly of viral pro-geny. Since the viral genome is not known toencode any regulatory functions, the host cell mustimpose mechanisms for regulation of viral DNAreplication in order to maintain control of DNAcopy number. Analysis of papillomavirus DNAreplication may therefore provide valuable insightsinto the host cell DNA replication regulatorymachinery.

Maintenance of low copy number during latentinfection is mimicked by monolayer culture ofinfected cells. Fifty to 200 copies of viral DNA percell are maintained by synchronous replication ofviral DNA in S phase, averaging once per cell cycleper viral genome.4,5,7,8 Three viral components arerequired for maintenance of viral DNA: a cis-actingreplication origin, and two viral proteins, E1 andE2.9 ± 12 The replication origin sequence contains

# 2002 Elsevier Science Ltd.

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Figure 1. Transient DNA replication assay.(a) Southern blot of low molecular weight DNAharvested from C127 cells on days (d.) 2-6 followingelectroporation. DNA was digested with EcoRI andDpnI prior to electrophoresis. The blot was probed withrandom-primed BPV DNA. The position of the viralgenome is indicated in the marker (M) lane. (b) The blotin (a) was stripped and reprobed with random-primedDNA from the rat cytochrome oxidase 2 gene tomeasure mitochondrial DNA synthesis as a control forrecovery of low molecular weight DNA (see Materialsand Methods).

600 C-terminal E1 Phosphorylation Mutant

binding sites for both E1 and E2, and a model hasemerged in which interaction between the E1 andE2 proteins, and between each protein and itsrespective DNA binding site, facilitates ef®cientassembly of E1 at the origin.10 In vitro or underarti®cially high levels of E1 protein, E2 is notrequired for viral DNA replication, observationswhich support an auxiliary role for E2 in the repli-cation process.13

The E1 protein of bovine papillomavirus type-1(BPV) is a 605 amino acid residue nuclear phos-phoprotein required for initiation of viral DNAsynthesis.14 E1 origin DNA binding induces distor-tion of the helix, followed by unwinding of DNAat the origin and at the replication fork throughATP-dependent helicase activity.11,15,16 DNA syn-thesis is carried out by host cell replicationenzymes, attracted to the viral origin through inter-action with E1.17,18

Recent work by several laboratories has pro-vided strong evidence that E1 activity in initiatingviral DNA replication is regulated at least in partby phosphorylation of the protein. Several pos-itions in the amino-terminal third of E1 are phos-phorylated by protein kinases. Enzymes implicatedin these modi®cations include Cdks, PKC, andCK2.19 ± 23 We have recently identi®ed serine 584near the carboxyl terminus of BPV E1 to be phos-phorylated in vivo and in vitro.24 Serine 584 is in amotif resembling a CK2 consensus site, and CK2ef®ciently phosphorylates E1 in vitro at thisposition.24 This CK2 motif is highly conserved inall human and animal papillomavirus E1sequences examined. Here, we demonstrate thatmutation of serine 584 severely disrupts viral DNAreplication, while only causing moderate defects inbiochemical function of puri®ed protein.

Results

DNA replication

We have recently shown that serine 584 of BPVE1 is phosphorylated both in vivo and in vitro.24 Totest the relevance of this modi®cation on E1 func-tion, a mutant viral genome was constructed,encoding alanine at position 584 (E1-S584A) inplace of serine. The alanine side-chain is similar insize and shape to serine, but lacks the phospho-acceptor hydroxyl group, eliminating the potentialfor phosphorylation in the mutant. Full-lengthwild-type and S584A mutant viral genomes wereelectroporated into mouse C127 ®broblast cells,and assayed for viral DNA replication in a transi-ent assay. Cells were harvested at 24 hour intervalson days 2 through 6 following electroporation.Low molecular weight DNA was isolated fromcells, and cleaved with restriction enzymes EcoRIand DpnI. EcoRI cleavage facilitates electrophoresisand linearizes the viral DNA. DpnI cleavage ismethylation-sensitive, and will not cleave DNAthat has been replicated in eukaryotic cells. There-fore, DpnI resistance is diagnostic for replicated

DNA. Digested DNA was separated by electro-phoresis and transferred to a nylon membrane.The blot was probed with radiolabeled BPV-speci®c DNA, and analyzed by autoradiography.As shown in Figure 1(a), replicated wild-typeDNA accumulated during the six days of cellgrowth. This signal represents establishment andmaintenance of viral DNA copy number as celldensity increases over the course of the exper-iment. In contrast, there was no detectable replica-tion of the S584A E1-containing mutant viral DNA,suggesting a role for serine 584 in establishingand/or maintaining viral DNA in the host cell.

The blot in Figure 1(a) was stripped andreprobed with labeled DNA from the mitochon-drial cytochrome oxidase II gene.25 MitochondrialDNA is ef®ciently recovered along with viral gen-omes in the DNA puri®cation procedure, and thecytochrome oxidase gene serves as a recovery con-trol. As shown in Figure 1(b), low molecularweight DNA was ef®ciently recovered from wild-type and mutant samples, eliminating recoveryproblems as an explanation for the replicationresult.

In an effort to determine whether establishmentor maintenance was affected by the mutation,stable replication assays were performed. Pools offoci from both wild-type and E1-S584A mutant

C-terminal E1 Phosphorylation Mutant 601

transformed cells were analyzed for the state ofviral DNA. Foci from mutant transformed cellswere smaller and slower to form than those fromwild-type. The pooled E1-S584A transformed cellswere unstable, and reverted over several passagesto poor-growing, ``revertant'' lines that appearedmore like untransformed C127 cells than theirwild-type transformed counterparts. Total cellularDNA was isolated from early passage cell poolsand analyzed for presence and stability of viralDNA. As shown in Figure 2, pools of wild-typetransformed cells maintained episomal viral DNAand exhibited high molecular weight replicativeintermediate DNA forms, expected for mainten-ance of replication. In the E1 mutant cell lines, areduced viral genome copy number was observed,and no replicative intermediate forms were seen.The viral DNA present in the E1-S584A mutanttransformed cells remained episomal in all poolsobserved.

In a separate transient replication experiment,glutamic acid was substituted for serine at position584 in the full-length viral genome E1(E1-S584E).Glutamic acid restores some of the negative charge

Figure 2. Stable replication assay. Total cellular DNAwas isolated from early passage pooled foci from eitherwild-type BPV transformed cells, or cells transformedby E1 S584A mutant genomes. DNA was subject to elec-trophoresis following digestion with XhoI, then trans-ferred to a nylon membrane and probed with random-primed BPV DNA. Results from three independentpools of wild-type and mutant genomes are shown. FI,monomeric (supercoiled) form I viral DNA; FII, nickedmonomeric viral DNA; RI, multimeric replicative inter-mediate viral DNA.

character associated with phosphorylation. Whenanalyzed in a transient replication assay asdescribed above, the S584E mutant replicated todetectable levels of about 10 to 30 % of wild-type(see Figure 3). While full replication was notrestored by the glutamic acid substitution, thedifferent replication phenotypes for the alanineand glutamic acid mutants suggests that phos-phorylation of the wild-type serine may play a rolein E1 function in transient replication. Puri®edwild-type and mutant proteins expressed fromrecombinant baculoviruses show similar stabilityduring puri®cation and storage; however, due tothe low levels of expression from the viral genome,it is not possible to assess potential differences inprotein stability during the replication assay.

It is possible that the replication defect seen inthe alanine substitution mutant could be an indir-ect effect resulting from altered E2 proteinexpression from the viral genome. It has beendemonstrated that E1 can modulate E2-dependenttranscription of viral genes.26,27 The S584Amutation may alter E1 transcriptional repressionfunction without a direct effect on replication func-tion. Furthermore, the position of the serine 584codon mutated (nucleotides 2598-2600) is near theP2443 promoter that can direct expression of theviral E2 protein.28,29 Disruption of a cis elementcould alter E2 expression and DNA replicationwithout directly altering E1 replication activities.

To address these potential indirect replicationeffects, a trans-expression replication assay wasperformed. Expression plasmids carrying the wild-type or mutant E1 genes, along with an E2-expres-sing plasmid, were tested for their ability to sup-port replication of BPV origin-containing DNA. Asshown in Figure 3, results from this replicationassay correlate well with the whole genome repli-cation assay. Wild-type E1 supports levels of repli-cation readily detectable by day three, while thealanine mutant replicates to only minimally detect-able levels by the end of the assay (day ®ve). Theglutamic acid mutant has an intermediate replica-tion phenotype. These results support the con-clusion that the phenotypes observed for the E1

Figure 3. Trans-expression replication assay. E1 andE2 expressing plasmids along with a BPV-origin con-taining plasmid (pORI) were electroporated into C127cells. Low molecular mass DNA was harvested one,three, and ®ve days post-electroporation, treated withEcoRI and DpnI, and subject to electrophoresis andSouthern blotting. The blot was probed with random-primed pORI DNA.

Table 1. C127 cell transformation assay

Wild-type E1 S584A E1 S584E

Expt 1 345 39 167Expt 2 228 22 190Expt 3 210 9 106

Cells were electroporated with either 5 mg (expt 1) or 2 mg (expt 2 and 3) viral DNA. Plates were ®xed and stained after 14 days,and transformed foci were counted.

Figure 4. E1-E2 interaction: 100 or 300 ng of wild-typeor S584A mutant E1 was incubated with [35S]methion-ine-labeled E2. Complexes were recovered by immuno-precipitation with antibodies against E1, and visualizedby electrophoresis and ¯uorography. The position of E2protein is indicated to the left of the gel. Complex for-mation with BPV L1 was used as a negative control(Neg.). I.P. represents input in vitro translated E2 pro-tein.


mutants are a result of some change in the replica-tion function of the E1 protein, and not indirecteffects on viral gene transcription.

A cell transformation assay was also performedto assess maintenance mode DNA replication.30

Culture plates from the viral genome replicationassay were allowed to grow to con¯uency andwere maintained for 14 days following electropora-tion. Cells were ®xed and stained, and transformedfoci were counted. Results from three independentexperiments are presented in Table 1, and closelymatch the results from both types of replicationassay. Wild-type E1-containing genomes ef®cientlytransformed mouse C127 cells, whereas the E1S584A mutant was severely impaired for this func-tion. The E1 S584E mutant was intermediatebetween the wild-type and alanine substitutionmutant. As described above, E1 S584A mutant fociwere slow to form and smaller than those fromwild-type or E1 S584E mutant transformed cells.

Protein binding assays

Several key functions of E1 in viral DNA replica-tion depend on the ability of E1 to recognize andinteract with other proteins. These protein-proteininteractions can be reconstituted in vitro using puri-®ed components. Wild-type and mutant E1 pro-teins were expressed and puri®ed from insect cellsinfected with recombinant baculoviruses.9,23 TheE1 proteins were ``tagged'' with eight amino acidresidues at the amino terminus corresponding tothe epitope of the FLAG antibody system (Sigma),and referred to as fE123. All protein preparationsused appeared as a single 70 kDa band onCoomassie-stained polyacrylamide gels.

Assembly of E1 on the replication origin in vivorequires interaction with the viral E2 protein.10 E1forms a stable complex with E2, and this inter-action has been shown to facilitate recognition andbinding to origin DNA. We tested the ability ofpuri®ed wild-type or S584A mutant fE1 to interactwith BPV E2 protein. E2 was synthesized andlabeled by a coupled in vitro transcription/trans-lation system from the E2 gene under control of aT7 RNA polymerase promoter, and labeled with[35S]methionine during translation. Puri®ed fE1and labeled E2 were mixed under conditionsdescribed in Materials and Methods. Puri®edFLAG-tagged viral L1 protein was used as a nega-tive control in these E2 binding assays. Followingincubation, complexes were immunoprecipitated

with M2 monoclonal antibodies directed againstthe FLAG peptide on fE1. Complexes were dis-rupted by boiling in gel loading buffer followed bypolyacrylamide gel electrophoresis and ¯uorogra-phy. As shown in Figure 4, there was no signi®-cant difference in the ability of the mutant E1protein to form a stable complex with E2. Disrup-tion of this interaction therefore cannot account forthe observed replication defect.

E1 is required to interact with the cellular repli-cation apparatus to recruit replication enzymes tothe viral replication fork. This interaction isachieved through binding to cellular proteins,including DNA polymerase alpha-primase (DNApola).18 To assess E1 interaction with DNA pola, arecombinant baculovirus expressing the p180 sub-unit of human DNA pola (a gift from E. Fanning,Vanderbilt University) was used to infect culturedSf9 cells, followed by labeling with [35S]methionine48 hours post-infection (p.i.). Nuclear extracts con-taining labeled recombinant p180 were prepared asdescribed. Puri®ed wild-type or S584A mutant fE1proteins were mixed and incubated with labeledextracts, and analyzed as described for E2 inter-action above. Figure 5 demonstrates that wild-typeand mutant fE1 proteins were equally able to formcomplexes with the p180 subunit of DNA pola inthis assay. While this experiment does not precludeloss of interaction with other cellular components,it demonstrates that the serine 584 mutant is

Figure 6. E1-origin DNA binding. Increasing amountsof puri®ed wild-type or S584A mutant fE1 were incu-bated with mixed ori� and oriÿ end-labeled DNA frag-ments. E1-DNA complexes were recovered byimmunoprecipitation, followed by puri®cation of boundDNA and analysis by electrophoresis and autoradiog-raphy. The positions of input ori� and oriÿ DNA frag-ments are indicated.

Figure 5. E1-DNA pola (p180) interaction. Nuclearextracts of recombinant baculovirus-infected cells con-taining overexpressed p180 subunit of DNA pola wereincubated with puri®ed wild-type or S584A mutant fE1.Complexes were recovered by immunoprecipitationwith antibodies against the FLAG peptide, and visual-ized by electrophoresis and ¯uorography. Negative(Neg.) and positive (Pos.) controls included FLAG-immunoprecipitated nuclear extract with no E1 proteinadded, and nuclear extract precipitated with anti-p180antibody, respectively. The position of the pola p180subunit is indicated to the left of the gel.


capable of interacting with the cellular polymeraseimplicated in initiating DNA synthesis.

Origin DNA binding

E1 recognizes and binds to an 18 bp invertedrepeat within the replication origin.31 ± 34 This E1binding site is highly conserved among papilloma-virus origins. Binding to the origin site initiateschanges in DNA structure that lead to the for-mation of the replication fork. To assess DNAbinding activity of mutant and wild-type E1 pro-teins, puri®ed fE1 was incubated under establishedconditions with 32P-end-labeled DNA fragments.Binding reactions contained a 200 bp fragmentwith the intact origin sequence (ori�) and an 80 bpfragment containing an 8 bp insertion within the18 bp inverted repeat E1 binding site (oriÿ). Thetwo DNA fragments were incubated with puri®edwild-type or mutant fE1 proteins, followed byimmunoprecipitation of E1-DNA complexes by theM2 monoclonal antibody directed against theFLAG peptide. Immune complexes were washed,followed by phenol/chloroform-extraction toremove proteins and purify the bound DNAfragments. Bound DNA was then analyzed by

polyacrylamide gel electrophoresis and autoradiog-raphy. A representative autoradiograph is shownin Figure 6. The S584A mutant is capable of recog-nizing and binding to the origin, and maintainsselective binding to origin-containing DNA. Noorigin-minus DNA was bound by either protein inthis experiment. Over several trials, there was areproducible reduction in the amount of DNAbound by the mutant protein, with the mutantbinding from 30-80 % origin DNA compared towild-type. Differences were most pronounced athigher concentrations of fE1, relative to DNA frag-ments. This assay is used to assess sequence-speci®c recognition and binding by E1. It does notaddress possible changes in speci®c nucleotide con-tacts or subsequent distortion of the DNA helixleading to unwinding of the origin.

DNA helicase activity

E1 functions as a helicase, unwinding DNA atthe origin and at the replication fork.11,15,16 It istherefore an essential component of the replicationmachinery for the viral genome. To analyze thehelicase function of wild-type and mutant E1 pro-teins, puri®ed fE1 was incubated with a helicaseassay substrate, consisting of a 24 nucleotide 32P-end-labeled DNA fragment hybridized to a single-stranded, circular plasmid DNA molecule. Helicaseactivity is necessary under experimental conditionsto displace the labeled oligonucleotide from the lar-ger molecule, resulting in a low molecular weightradiolabeled DNA fragment as a helicase reactionproduct.16 Reaction products were separated bypolyacrylamide gel electrophoresis and quanti®edby phosphorimage analysis. At each time point,the percentage of oligonucleotide displaced fromthe substrate molecule was calculated for wild-type and mutant fE1, and a negative control (the

Figure 7. Helicase assay. The substrate for E1 helicasereactions is a short, single-stranded, radiolabeled DNAfragment bound to circular single-stranded DNA. Heli-case activity displaces the oligonucleotide from the cir-cular DNA. The percentage of radiolabeledoligonucleotide displaced over the course of the reactionis displayed, with the standard error of the mean (errorbars). BPV fL1 protein was used as a negative control.(^) fL1 (negative control); (*) wild-type fE1; (~)S584A fE1.


viral L1 protein). The results are graphed inFigure 7. Both wild-type and mutant fE1 proteinsretained ef®cient helicase function in this assay,with the mutant consistently showing slightlyhigher initial reaction rates, but similar overallfunction compared to wild-type. Altered helicaseactivity therefore does not appear to account forthe transient replication phenotype.

E1 has recently been shown to form a hexamer,a structural form associated with helicasefunction.15 Since the mutant and wild-type E1 pro-teins behaved similarly in the helicase assay, it isassumed that E1-E1 interactions necessary for hex-amer formation are not signi®cantly disrupted bythe S584A substitution.

Discussion

Here, we present results that add to a complexpicture emerging about regulation of BPV E1 pro-tein function in viral DNA replication. In replica-tion assays, serine 584 is shown to be essential forproper protein function in DNA replication. Notransient replication is observed from mutant viralgenomes carrying an alanine substitution, andlong-term maintenance of viral DNA does notoccur in mutant cell lines. An intermediate level ofreplication is recovered from genomes carrying E1genes encoding a glutamic acid substitution inplace of the wild-type serine. This observation sup-ports our hypothesis that phosphorylation plays arole in serine 584 function. Since the glutamic acidside-chain is negatively charged at neutral pH, itmay mimic the negative charge associated withphosphorylation of serine in the wild-type protein,

permitting recovery of some replication functioncompared to the alanine mutant.

The wild-type and alanine mutant proteins wereexpressed in cultured insect cells from recombinantbaculoviruses, and puri®ed by immunoaf®nitychromatography. The mutant protein wasexpressed at comparable levels to wild-type, andpuri®ed and stored with similar stability. It there-fore is unlikely that the observed replicationdefects can be blamed on global structural changesin the mutant that affect protein folding or stab-ility, although stability could not be assessedwithin cells harboring the viral genome.

Several diverse biochemical activities of E1 wereassayed using puri®ed wild-type and mutant pro-teins. No single activity was as severely impairedas the transient replication phenotype of themutant; however, there was reproducibly reducedaf®nity for the replication origin DNA sequence bythe alanine mutant. Over several independentexperiments, the mutant E1 protein bound consist-ently less origin DNA than wild-type. The differ-ences were most pronounced at higher E1concentrations (see Figure 6). It should be notedthat during viral latency in infected cells, E1 andother viral proteins are expressed at very lowlevels. Even the lowest concentrations of E1 usedin our in vitro biochemical assays are likely to farexceed the concentrations seen in the course of thenatural infection. If the ratio of E1 protein to viralDNA is ®ne-tuned by the virus and critical forproper regulation and maintenance of viral gen-omes during latency, then small perturbations ofDNA binding function could potentially accountfor the severe replication defect observed for themutant protein. This possibility is also supportedby a comparison of replication ef®ciency in thewhole genome assay and the three-plasmid replica-tion assay. Both mutants and the wild-type E1replicated to higher levels in the latter assay, whilemaintaining the same overall relative replicationef®ciencies. Higher levels of E1 and E2 expressionfrom heterologous promoters could explain theobserved replication differences.

A complex picture is emerging as biochemicaland genetic analyses of BPV and human papillo-mavirus (HPV) E1 mutants are conducted. TheCdk class of kinases is well known to regulate pas-sage through the cell cycle, at both the G1/S andG2/M boundaries. We have previously shown thatthreonine 102 of BPV E1 is a site for phosphoryl-ation by Cdk2, however mutation of this site hadno detectable effect on viral DNA replication.20 Itshould be noted that Cdk2 is associated with theG2/M cell cycle transition. Several more recent stu-dies have strongly implicated Cdk phosphorylationin the regulation of papillomavirus E1 function.Mutation of putative Cdk sites in HPV 11 E1showed dramatic defects in DNA replication.21 Inthe BPV replication experiments, a single phos-phorylation site (threonine 102) was altered, whilein the HPV experiments, all four putative Cdk tar-get sites were eliminated at once. It is possible that


threonine 102 is not the relevant Cdk target forregulation of DNA replication. Alternatively, therole of phosphorylation on E1 protein regulationcould be different between BPV and HPV. Itwould be interesting to independently mutate eachof the four putative Cdk phosphorylation sites inthe HPV protein, as well as the other sites in BPV.Using a Xenopus egg extract replication system,Cueille et al.19 recently identi®ed Cdk phosphoryl-ation as essential to BPV replication; however, therelevant amino acid targets on the E1 protein werenot identi®ed. Both threonine 126 and serine 283are in the context for Cdk phosphorylation in BPVE1, in addition to threonine 102. It is also notablethat the BPV E1 protein contains the conservedRXL motif responsible for binding to cyclin pro-teins, and shown to interact with several cyclins inthe HPV 11 E1.21

We have previously shown that mutation of ser-ine 109 of BPV E1 to alanine increases replicationin transient assays.23 This activity correlates wellwith an increase in origin DNA binding of the pur-i®ed mutant protein compared to wild-type E1.This position is ef®ciently phosphorylated by pro-tein kinase A and protein kinase C in vitro, andappears to be phosphorylated in vivo as well,although the in vivo kinase was not identi®ed. Allof these combined results suggest that the BPV E1protein is regulated both positively and negativelyby phosphorylation. These regulatory features areconserved among other members of the papova-virus family, most notably SV40. SV40 alsoencodes a replication initiator protein, the largetumor antigen (T antigen), which has been well-characterized. T antigen carries out many functionsdirectly analogous to the papillomavirus E1 pro-tein, including origin binding, DNA helicaseactivity, and interaction with cellular replicationproteins.35 These activities of T antigen are bothpositively and negatively regulated by phosphoryl-ation. Kinases implicated in regulation of T antigenreplication function include Cdks and CK1.

Several functional domains of BPV E1 have beenmapped by deletions and point mutations. Interest-ingly, the DNA binding domain maps to aminoacid residues 142 to 308,36 ± 38 quite distant fromserine 584, even though DNA binding is the onlybiochemical phenotype associated with thismutant. The crystal structure of the DNA bindingdomain was recently determined to 1.9 AÊ

resolution.37 This fragment retains all of the func-tions required for initial interaction with the origin,including cooperative interaction with the viral E2protein and E1 dimerization. Since the full-lengthprotein structure is not known, it is not clearwhether the carboxyl terminus of the protein andserine 584 could be in a position to interact withthis domain. The capacity of E1 to bind to the E2protein has mapped to different domains underdifferent conditions. There appear to be amino andcarboxyl-terminal regions of E1 that interact withE2.39,40 From the results presented here, serine 584

does not appear to be directly involved in thisinteraction.

Serine 584, analyzed in the experimentsdescribed here, is in a consensus motif for CK2,and is ef®ciently phosphorylated by CK2 in vitro.24

CK2 is a highly conserved, ubiquitous kinase withmany substrates; however, its role in cellular regu-lation is largely unexplored. Recent experiments ina variety of systems implicate CK2 as a player inregulation of cell cycle progression, both at theG1/S and G2/M transitions.41 ± 46 It therefore isreasonable to consider CK2 motifs in E1 as poten-tial regulators of E1 function in cell cycle-regulatedviral DNA replication. A conserved serine in aCK2 motif at position 48 of BPV E1 was recentlyshown to be required for proper origin-dependentreplication in a transient system;47 however, nobiochemical defect was identi®ed to account forthe severe replication phenotype.

A clear understanding of papillomavirus DNAreplication control will require further mappingand analysis of regulatory modi®cations. No singleposition is likely to account for the observed regu-lation; rather a network of small-scale modi®-cations of function may ®ne-tune the behavior ofthe E1 protein in concert with cellular conditions.

Materials and Methods

Cells and viruses

Mouse C127 cells were grown and maintained in Dul-becco modi®ed Eagle Medium (Gibco BRL) sup-plemented with 10 % (v/v) fetal bovine serum (JRHBiosciences), penicillin, and streptomycin. Culture ofSpodoptera frugiperda Sf9 cells and their infection byrecombinant baculoviruses was as previouslydescribed.9,48 Generation of recombinant baculovirusexpressing either wild-type or mutant BPV fE1 protein,or BPV fL1 protein, utilized the BaculoGold1 (Pharmin-gen) viral DNA in a lipofection protocol (Lipofectin,Gibco BRL) as described.23 Sf9 cells (3 � 106) were platedonto 60 mm dishes in Graces medium with no sup-plements and allowed to adhere for one hour. A mixtureof 0.2 mg of BaculoGold DNA, 2-5 mg of recombinantDNA, and 40 ml of medium was added to 20 ml of Lipo-fectin in 30 ml of medium and incubated 15 minutes atroom temperature. Medium was added to 1 ml, thenadded to cells followed by incubation for four hours at28 �C, rocking the plates occasionally. The DNA-contain-ing medium on the cells was replaced by 3 ml of com-plete medium, and cells were incubated for four days.Recombinant viruses were plaque-puri®ed.48

DNA manipulations

Synthesis of a full-length, FLAG-tagged clone of theBPV E1 gene (gGEMfE1) has been described.23 pGEMfE1was used as a template for generating serine 584 mutantsusing the protocol of Deng and Nickoloff,24,49 for sub-cloning into baculovirus transfer vector pVL1393 (Phar-Mingen). Alternatively, the 1342 bp EcoRI to Acc65Ifragment of the BPV genome that overlaps the 30 end ofthe E1 gene (pMLBPV50) was subcloned into vectorpGEM (Promega) as a template for creating the same ser-ine 584 substitutions. This alternate vector was required


for subcloning the mutant E1 fragment back into theviral genome (pMLBPV) for transient replication assays.Subcloning was carried out using established protocols.51

E1 expression and purification

E1 genes were placed downstream of the strong bacu-lovirus polyhedrin promoter in the vector pVL1393(PharMingen). These vectors were used to constructrecombinant baculoviruses by lipofection (LipoFectin,Gibco BRL) with BaculoGold1 DNA (PharMingen) asdescribed.23 Suspension cultures of Sf9 cells (100-200 ml)were infected as described.9 At 48 hours p.i., cells wererecovered by centrifugation at 1000 g for ®ve minutes,rinsed with phosphate-buffered saline, and recentri-fuged. Cell pellets were used directly or stored atÿ70 �C. fE1 was puri®ed from salt-washed nuclei asdescribed.9 Immunoaf®nity columns were prepared from1-2 ml of FLAG M2 monoclonal antibody complexed toagarose (Sigma) and used according to the manufac-turer's instructions. Bound E1 was eluted with syntheticFLAG peptide (Sigma). Fractions of eluted fE1 were ana-lyzed by polyacrylamide gel electrophoresis. fE1-contain-ing fractions were pooled, concentrated by dialysisagainst solid sucrose, dialyzed into storage buffer(50 mM Tris-HCl (pH 7.8), 1 mM EDTA, 1 mM dithio-threitol (DTT), 12.5 mM MgCl2, 100 mM KCl, 0.3 MNaCl, 10 % (v/v) glycerol), and stored at ÿ70 �C in20-100 ml aliquots. A full-length clone with amino-terminal FLAG tag of the BPV L1 protein (fL1) wasexpressed as described above for fE1, and used as a con-trol protein for some experimental assays. All proteinsused in assays appeared as a single band on Coomassie-stained polyacrylamide gels.

DNA replication

Analysis of transient replication of BPV DNA inmouse C127 cells was as described.20,23 Brie¯y, full-length wild-type or mutant BPV genomes were removedfrom the pML plasmid background by digestion withBamHI: 1-5 mg of linear DNA was introduced into cellsby electroporation. Replicated BPV DNA was isolated bymodi®ed HIRT extraction on days 2-6 following electro-poration.52 Isopropanol-precipitated DNA was collectedby centrifugation, washed with 70 % ethanol, and airdried. DNA was dissolved in 15 ml of water containing20 mg/ml of RNase A and incubated at 65 �C for 20 min-utes. After cooling, 15 ml of enzyme mix containing man-ufacturer-supplied buffer and DpnI and EcoRI restrictionenzymes, was added to the samples and incubated at37 �C for 12-16 hours. Reaction products were separatedon 0.8 % agarose gels and covalently bound to chargednylon membrane by capillary transfer and UV treatment.Blots were probed with a radiolabeled BPV-speci®cprobe, generated by random primed synthesis using the``Prime-a-Gene'' Labeling System (Promega) as describedby the manufacturer. Linear BPV genome was used asa template. Blots were washed, dried and exposed toX-ray ®lm. Following analysis of BPV replication, blotswere stripped by washing in boiling 0.5 % (w/v) sodiumdodecyl sulfate, then reprobed with a random-primedfragment of pIL7 containing the rat cytochrome oxidaseII gene as a recovery control.25

For stable replication assays, total cellular DNA wasisolated by detergent lysis and proteinase K digestion,followed by extensive dialysis against TE (10 mM Tris(pH 7.8), 1 mM EDTA):51 10 mg of each DNA was

digested with XhoI, which does not cut within the BPVgenome, but will facilitate electrophoresis and transfer ofcellular DNA. Digests were electrophoresed, transferredto charged nylon membranes, and probed as describedabove.

A three-plasmid trans-expression system was alsoused to assess replication function.12,20 C127 cells wereelectroporated as described above with 5 mg of vectorpSG5 expressing wild-type or mutant fE1 proteins, 5 mgof pMTE2 expressing the viral E2 protein, and 2 mg ofpORI. pORI consists of the cloning vector pGEM (Prome-ga Corp.) carrying a 204 bp PCR fragment spanning theviral origin from nucleotides 7829 to 87. Expression offunctional E1 and E2 from the CMV or adenovirus majorlate promoter, respectively, will support replication ofthe origin-containing DNA. pORI replication wasassayed as described above, except that blots wereprobed with random-primed pORI DNA.

Protein binding

Co-immunoprecipitation of protein complexes wasused to assess E1 interaction with either the viral E2 pro-tein or the p180 subunit of DNA pola-primase. E2 pro-tein was prepared in a coupled transcription/translationreaction using a cloned E2 gene under control of the bac-teriophage T7 promoter (pGEM, Promega). The PromegaTnT system was used as described by the manufacturer.Reactions were carried out with [35S]methionine to createlabeled E2 extracts. 35S-labeled p180 was prepared byinfecting cultured Sf9 cells on 60 mm dishes asdescribed.20 p180-expressing recombinant baculoviruswas a generous gift from E. Fanning. At 48 hours p.i.cells were washed twice in methionine-free Graces med-ium, followed by growth in the same medium sup-plemented with 100 mCi of [35S]methionine (Amersham)for four hours. Nuclear extracts were prepared asdescribed9, and used directly.

For E1/E2 interactions, 100 or 300 ng of wild-type ormutant fE1 protein was incubated in a 40 ml binding mixcontaining 5 ml of [35S]methionine-labeled E2, 30 mMHepes (pH 7.4), 7 mM MgCl2, and 20 mM potassiumglutamate. Following a 30-60 minute room temperatureincubation, 160 ml of TBS and 0.5-1 mg of anti-FLAG M2monoclonal antibody were added, and a second incu-bation of 60 minutes was carried out on ice. Immunecomplexes were recovered by precipitation with proteinA-Sepharose beads as described.53 fE1 immune com-plexes were washed twice with TBS, disrupted by boil-ing in gel loading buffer (1 % sodium dodecyl sulfate,2.5 % b-mercaptoethanol, bromophenol blue), and ana-lyzed by electrophoresis and ¯uorography. As a negativecontrol, reactions were carried out with FLAG-taggedBPV L1 protein in place of E1.

For E1/DNA pola interaction, 100 ml of [35S]methion-ine p180 was incubated with 200-500 ng of puri®edwild-type or mutant fE1, 20 mM Tris-HCl (pH 7.8),1 mM EDTA, 1 mM dithiothreitol (DTT), 12.5 mMMgCl2, 100 mM KCl, 0.3 M NaCl in 500 ml for fourhours at 4 �C. Then 1 mg of anti-FLAG M2 monoclonalantibody was added, and a second incubation of 60 min-utes was carried out on ice. Immune complexes wererecovered by precipitation with protein A-Sepharosebeads as described.53 E1 immune complexes werewashed with 0.5 M LiCl and TBS, disrupted by boilingin gel loading buffer, and analyzed by electrophoresisand ¯uorography. For the negative control, fE1 was leftout of the reaction, and as positive control, a reaction


without E1 was precipitated with monoclonal antibody2CT25, directed against p180 (a gift from E. Fanning).

DNA binding

An immunoprecipitation procedure was used to ana-lyze origin DNA binding of puri®ed E1.39,40 Radiolabeled200 bp origin-containing (ori�) or 80 bp origin-lacking(oriÿ) DNA probes were generated by PCR, using pri-mers labeled at their 50 ends by reaction with T4 polynu-cleotide kinase (Promega) and [g-32P]ATP:51 40, 120, or360 ng of puri®ed fE1 protein was precipitated from sto-rage buffer by binding to FLAG M2 monoclonal anti-body followed by binding to protein A-Sepharose beads(Sigma). Immune complexes were washed with 20 mMTris-HCl (pH 8.0), 5 mM KCl, 1 mM MgCl2, 1 mM DTT,0.3 M NaCl, then resuspended in 20 ml of binding buffercontaining 30 mM Hepes (pH 7.4), 7 mM MgCl2, 2 mg ofpoly[d(I-C)], 20 mM potassium glutamate and 1000 cpmeach of ori� and oriÿ DNA fragments followed by incu-bation at 30 �C, for 30 minutes. Unbound probe waswashed from the immune complexes, which were thentreated with 70 ml of dissociation buffer (1 % SDS,50 mM EDTA, pH 8.0), for 15 minutes at 65 �C. BoundDNA was puri®ed by phenol/chloroform-extraction andethanol-precipitation, followed by separation on 5 %polyacrylamide gels in TBE buffer.51 The gels were driedand exposed to X-ray ®lm, then quanti®ed on a phos-phorimager.

Helicase assay

32P-end-labeled, 24 nucleotide, M13/pUC primer(New England Biolabs) (5 mg) was annealed to 2.5 mgM13mp18 single-stranded circular DNA by boiling for®ve minutes, then slow cooling to room temperature.Annealed substrate was puri®ed by ®ve successive spinsthrough a Microcon-100 microconcentrator (Millipore)and the ®nal product was adjusted to 50 ml with 10 mMTris-HCl (pH 7.6), 1 mM EDTA. Helicase reactions werecarried out in a 20 ml volume containing 200 ng of wild-type or mutant fE1 protein, 2 ml of annealed substrate,20 mM Tris-HCl (pH 7.6), 7.5 mM MgCl2, 1 mM DTT,2 mM ATP, 20 mg of phosphocreatine, and 2 mg of cre-atine phosphokinase. Reactions were incubated for 60minutes at 37 �C. At 0, 15, 30, and 60 minutes, 5 ml ofreaction was removed and added to 5 ml of stop buffer(50 % glycerol, 3 % SDS, 150 mM EDTA), followed by10 ml of water, and stored in an ice bath. Reaction pro-ducts were analyzed by electrophoresis through 8 %polyacrylamide gels in 1 � TBE buffer.

Gels were dried and reactions were quanti®ed on aphosphorimager.

Acknowledgments

We are grateful to Dr Ellen Fanning (Vanderbilt Uni-versity) for recombinant baculoviruses expressing DNApola subunits, and for monoclonal antibodies againstthose proteins. Dr Van Wilson (Texas A&M University)provided valuable insights and critique of the manu-script. Dr FracËoise Cuzin, UniversiteÂ de Nice, kindly pro-vided plasmid pIL7. M.L. was supported by PublicHealth Service Award CA59426 from the National Insti-tutes of Health.

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Edited by K. Yamamoto

(Received 25 May 2001; received in revised form 15 October 2001; accepted 19 December 2001)

Documents

Functional analysis of a carboxyl-terminal phosphorylation mutant of the bovine papillomavirus E1 protein