Bovine papillomavirus type 1 E2 protein heterodimer is functional in papillomavirus DNA replication in vivo

Bovine papillomavirus type 1 E2 protein heterodimer is functional in papillomavirus DNA replication in vivo
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  Bovine papillomavirus type 1 E2 protein heterodimer is functional in papillomavirusDNA replication in vivo Reet Kurg a, ⁎ , Piia Uusen a , Toomas Sepp c , Mari Sepp c , Aare Abroi b , Mart Ustav a,b a Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia b Estonian Biocentre, Tartu, Estonia c Institute of Molecular and Cellular Biology, University of Tartu, Estonia a b s t r a c ta r t i c l e i n f o  Article history: Received 13 October 2008Returned to author for revision26 December 2008Accepted 16 January 2009Available online 20 February 2009 Keywords: PapillomavirusReplicationE2 proteinHeterodimer Papillomaviruses are small DNA viruses that induce epithelial lesions in their host. The viral life cycle isregulated by the family of proteins encoded by the E2 open reading frame. In addition to the full-length E2protein, the BPV-1 genome encodes two truncated E2 proteins, E2C and E8/E2, which maintain the DNA-binding-dimerization domains, but lack the activation domain. Heterodimers formed between the full-lengthE2 and truncated E2 proteins serve as activators of E2-dependent transcription and papillomavirus DNAreplication. We show that the single activation domain of E2 is suf  fi cient for interactionwith viral helicase E1and for initiation of DNA replication from different papillomavirus srcins. Single-chain E2 heterodimer isable to activate papillomavirus DNA replication in the context of entire BPV genome in the absence of otherE2 proteins. These data suggest that E2 heterodimers with single activation domain are functional ininitiation of papillomavirus replication in vivo.© 2009 Elsevier Inc. All rights reserved. Introduction Papillomaviruses are small, double-stranded DNA viruses, whichinfect a wide variety of vertebrate species and induce proliferativeepithelial lesions of skin and mucosa in their host. Studies on BPV1DNA replication have shown the requirement of two virally encodedproteins, the viral helicase E1 and regulatory protein E2 as well as thesrcin region containing E1 and E2 binding sites together with A/T-rich region for papillomavirus DNA replication (Ustav and Stenlund,1991; Ustav et al.,1991). The E1 protein binds with low speci fi city tothe srcin of replication, and speci fi c and ef  fi cient recognition isachieved bycooperative bindingof E1 and E2 to adjacent sites. So, theE2 protein serves as a loading factor and helps to recruit the viralhelicase E1 to the srcin in the initiation of viral DNA replicationwithin the cells (Sanders and Stenlund, 2001; Sedman and Stenlund,1995; Sedman et al.,1997).The E2 open reading frame of BPV1 encodes a family of proteinswhichformthecentralregulatorysystemofthevirus,controllingbothviral geneexpression and replication. The E2 proteinis a transcriptionactivator which regulates transcription of viral early promotersthrough binding to E2 binding sites (Spalholz et al., 1985). E2participates in initiation of viral DNA replication by loading viralhelicase E1 onto the srcin of DNA replication (Sedman and Stenlund,1995; Ustav and Stenlund, 1991), and in virus genome episomalmaintenance by tethering viral genomes to mitotic chromosomesduring cell division (Ilves et al.,1999; Lehman and Botchan,1998). E2is a modular protein consisting of three different structural as well asfunctionaldomains; the N-terminal transactivation domain (TAD) (aa1 – 200), the C-terminal DNA-binding dimerization domain (DBD) (aa310 – 410),and fl exibleunstructured “ hingeregion ” whichfunctionsasa linker between the two functional domains.In addition to the full-length E2 protein, the BPV-1 genomeencodes two truncated E2 proteins. The full-length E2 is coded by theentireE2ORF(bp2608 – 3840ofBPV-1genome);theE2Cis translatedfrom transcript from promoter P 3080  with initiator codon correspond-ingtoMet162inE2;andE8/E2istranslatedfromalternativelysplicedmRNA-s with splice donor at 1235 and splice acceptor at 3225, thusfusing 11 aa from E8 ORF to aa 205 of E2 ORF. The two truncated E2proteins maintain the DNA-binding-dimerization domains of E2, butlack the activation domain, and therefore act as repressors of E2dependent transcription. The E2 repressors antagonize the full-lengthE2 function by competing for E2 DNA binding sites (Barsoum et al.,1992; Lambert et al., 1987; Lim et al., 1998). The promoters for full-length E2 as well as for repressors are themselves controlled by E2(Baker and Howley, 1987; Li et al., 1989). Despite the E2 repressorsshorter half-life (Hubbert et al.,1988) they predominate in the steadystate (Kurg et al., 2006). However, the ratio of the repressors toactivatorschanges throughthecell cycle(Yangetal.,1991)suggestingthatthebalanceofdifferentE2proteinsisakeyeventintheregulationof virus life cycle. The truncated E2 proteins are able to formheterodimers with full-length E2 through the common C-terminalDBD (McBride et al., 1989), and the E2 heterodimers with single Virology 386 (2009) 353 – 359 ⁎  Corresponding author. Fax: +372 7374900. E-mail address: (R. Kurg).0042-6822/$  –  see front matter © 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.virol.2009.01.025 Contents lists available at ScienceDirect Virology  journal homepage:  activation domain are the most abundant form of E2 containing thefull-length E2 protein in virus transformed cells (Kurg et al., 2006).Using the E2 single-chain heterodimer as a model, we have shownthat BPV1 E2 heterodimers can function as activators of transcriptionand replication (Kurg et al., 2006).Mostof our knowledge about the role of E2 inpapillomavirus DNAreplication is based on studies with the full-length E2 homodimer. Inthis study, we have investigated in detail the localization andreplicationfunctionofE2heterodimerswithsingleactivationdomain.Our data show that the single activation domain of E2 is suf  fi cient tolocalize the E2 protein into cellular chromatin, for interaction withviral helicase E1 and for initiation of DNA replication from differentpapillomavirus srcins. E2 heterodimer in the context of entire BPV1genome is able to initiate the papillomavirus DNA replication, but notto maintain it for a long time. Results Localization of E2 heterodimers is determined by the activation domain Our previous study has shown that E2 heterodimers with singleactivation domain are the preferential form for BPV1 E2 protein invirus-transformed cells as their amount is four times higher than thatof the full-length E2 homodimer (Kurg et al., 2006). E2 heterodimerconsists of full-length and truncated E2 proteins which cellularcompartmentalizationis different: thefull-lengthE2proteinismostlyassociated with cellular chromatin while truncated E2C protein is insoluble nucleoplasm fraction (Kurg et al., 2005). In order to study thesubnuclear localization of E2 heterodimers with single activationdomain,wehaveusedbiochemicalfractionationapproachasdepictedin (Fig. 1B, (Kurg et al., 2005)). First we carried out fractionation of  BPV1-transformed C127 cells, which maintain and replicate the BPV1genome as an episome and express all three E2 proteins at low,physiological levels (Hubbert et al., 1988; Kurg et al., 2005). Toexamine the localization of E2 dimers in virus transformed cells, theUV-treated C127-BPV1 cells were biochemically fractionated asshown in Fig. 1B, and E2 proteins were immunoprecipitated withE2-speci fi c 3F12 antibody (Kurg et al., 1999). UV treatment of cellscovalently crosslinks E2 dimers through amino acid Trp360 (Prakashet al., 1992) allowing us to follow the fractionation pro fi le of E2hetero- and homodimers in virus transformed cells. The ef  fi ciency of UV-treatment is approximately 50% explaining why we can see both,E2 monomers and E2 heterodimers on the Western blot. As shown onFig. 1C, E2 homo- and heterodimers with single activation domainfractionated similar to the full-length E2 protein: the E2 homodimeras well as the E2:E2C and E2:E8/E2 heterodimers all fractionated into0.4 M salt-sensitive chromatin fraction (lanes 4, 5) while E2Chomodimer was found in soluble fraction (lane 2). These data showthat E2 heterodimers composed of full-length E2 protein and E2repressor localize into the salt-sensitive nuclear chromatin fractionsimilar to full-length E2 homodimer.To determine whether one activation domain is suf  fi cient tolocalize the E2 protein into cellular chromatin, we analyzed thefractionation pro fi le of   “ single-chain heterodimer ”  of E2 (scE2)constructed by fusing the coding region of C-terminal DBD in frameto the full-length E2 (Fig. 1A). scE2 heterodimer could bind to E2binding sites similar to the full-length E2 suggesting that thismolecule is structurally and functionally intact and can be used as amodel for E2 heterodimer with single activation domain. Using “ single-chain E2 heterodimer ”  we have seen only formation of  Fig.1.  Subnuclear localization ofE2 proteins. (A) Schematic representation ofE2 proteins used in this study. (B) Protocolfor biochemical fractionation.(C) Subnuclearlocalization of E2 proteins expressed in BPV1 transformed C127 cells. The UV-treated C127-BPV cells were biochemically fractionated, and E2 proteins were immunoprecipitated from all fractionsandanalyzedbyimmunoblottingwithE2speci fi cantibody.Localization ofE2homo-andheterodimers isindicatedontheright.Non-speci fi csignalwithcellularprotein isshownbyasterisk.Lane1,15ngofbacteriallypuri fi edE2protein.(D)U2OScells,transfectedwith100ngofexpressionplasmidsforE2orscE2,werefractionated24hafterelectroporationandsubjected to biochemical fractionation. Equal volume of each fraction was subjected to SDS-PAGE and immunoblotted with HRP-conjugated E2-speci fi c antibody 5E11. Non-speci fi csignal with cellular protein is shown by asterisk.354  R. Kurg et al. / Virology 386 (2009) 353 –  359  intramolecular dimers, but not any kind of intermolecular complexesin several biochemical assays (gel-shift assay, Western blot after UV crosslinking) used (Kurg et al., 2006). In addition, different biophy-sical analyses have also con fi rmed almost identical behavior for E2DBD homodimer and single-chain species (Dellarole et al., 2007). Weexpressed E2 and scE2 in two different cell lines, hamster CHO andhuman U2OS cells. As shown on Fig. 1D, lanes 8 and 9, scE2fractionated similar towild-type E2 protein into salt-sensitive nuclearfraction. Similar results were obtained expressing E2 and scE2 in CHOcells (data not shown). These data suggest once more that oneactivation domain is suf  fi cient to localize the E2 protein into cellularchromatin. The E2 heterodimer with single activation domain activates replication from different srcins E2heterodimerwithsingleactivationdomainisabletosupportthereplication of BPV1 srcin-containing plasmid pUCAlu (Kurg et al.,2006). Single-chain E2 heterodimer supports also replication of BPV1URR-containingplasmidintransientassay,whenE1andE2expressingplasmids are transfected into CHO cells together with URR-containingplasmid (Fig. 2A). The BPV1 minimal srcin of replication consists of two E2-binding sites, E2BS11 and E2BS12, the E1 protein binding siteand the A/T rich region (Ustav et al.,1991). The presence of both E2-binding sites within the srcin is necessary for the full replicationactivityinvivo; E2BS11 andE2BS12 playseparatebutsynergisticrolesin the initiation of viral DNA replication that are dependent on theirlocation within the srcin (Gillette and Borowiec, 1998; Sanders andStenlund,2000;SandersandStenlund,2001).InordertotestwhetherE2 heterodimers function on both, E2BS11 and E2BS12, expressionvectors for E1 and wild-type E2 or scE2 were co-transfected togetherwith OriA, (containing only E1 distal E2BS11), and OriB (proximalE2BS12) (Fig. 2B) into CHO cells. As shown on Fig. 2A, scE2 heterodimer is able to support DNA replication of both plasmidssimilar to wild-type E2 protein.To analyze the ability of native E2 heterodimers to supportpapillomavirus DNA replication in vivo, we have also used E2 doublemutant 390/388. Alanine substitutions at positions 390/388 severelyreduce the interaction of DBDs of E2 and E1 and disrupt theinteractionbetweentheE2activationdomainandE1helicasedomain,and therefore are incapable of functioning ef  fi ciently from theproximal E2-binding site in OriB. At the same time these mutationshave no effect on replication of OriA containing the distal E2BS11(Gillitzer et al., 2000) (Fig. 3C). We co-transfected the expression plasmid for E2 or E2 double mutant 390/388 or E2C or E2C doublemutant 390/388 together with OriA and OriB and with E1 expressionvector into the CHO cells. As expected, both full-length proteins were Fig. 2.  Replication function of E2 heterodimer with single activation domain. CHO cellswere transfected with 100 ng of reporter plasmid containing either BPV1 URR region(A), or E2BS11 or E2BS12 together with E1 binding site (B), 500 ng of expressionplasmid for E1, and 250 ng of expression plasmid for E2 or scE2 and harvested 24, 48and 72 h after electroporation. Episomal DNA was isolated, digested with DpnI andlinearizing enzyme, and analyzed by Southern blotting using the radiolabeled probespeci fi c to the reporter plasmid. Fig. 3.  E2:E2C heterodimer activates DNA replication in vivo. (A) CHO cells were transfected with 100 ng of reporter plasmid containing either OriA or OriB, 500 ng of expressionplasmidforE1,and250ngofexpressionplasmidforwild-typeE2sorE2mutants,andharvested36,60and84hafterelectroporation.EpisomalDNAwasisolated,digestedwithDpnIandlinearizingenzyme,andanalyzedbySouthernblottingusingtheradiolabeledprobespeci fi ctothereporterplasmid.Thelinearized OriAcontaining reporterplasmidasamarkeris shown on the left. (B) The average values of the ratio of OriB/OriA signals from two independent experiments. (C) Schematic representation of the  fi rst steps of the initiation of replication of OriB in case of different E2 dimers. Black dots indicate mutation 388/390 in E2.355 R. Kurg et al. / Virology 386 (2009) 353 –  359  able to support replication of OriA (Fig. 3A, lanes 2, 3; upper band)while only the wild-type E2, but not E2 mutant 390/388, was able tosupport ef  fi ciently the replication of OriB (lower band). E2C and E2Cmutant 390/388 were inactive in supporting replication of both Oris(lanes 4, 5). Next, co-transfection of the expression plasmids for E2mutant390/388andE2CtogetherwithOriAandOriBwascarriedout.Co-expression of E2 mutant 390/388 and E2C results in the formationof E2(390/388) homodimers, E2(390/388):E2C heterodimers andE2C homodimers from which the latter is not able to supportpapillomavirus DNA replication, and E2(390/388) homodimer isable to support replication of OriA only (Fig. 3A; lane 3, upper band).Thus, ef  fi cient replication of OriB can be achieved only by E2(390/388):E2C heterodimers. Indeed, as shown on Fig. 3A, the heterodimerconsisting of E2 mutant 390/388 and E2C was able to supportreplication of both, OriA and OriB (lane 7) suggesting that E2heterodimercontainingoneactivationdomainandonlyonefunctionalDBD is active for replication of OriB. When both proteins, E2 and E2C,carried mutation, the replication was initiated much more ef  fi cientlyfrom OriA compared to OriB (Fig 3A, lane 8), like in case of E2(390/388) alone (lane 3). The replication signals were quanti fi ed usingPhosphoImager SI (Molecular Dynamics) and the ratio of signals of OriBandOriAisshowngraphicallyinthediagraminFig.3B.Thesedatashow that the native E2 heterodimers are able to support DNAreplication of papillomavirus srcin within the cells. The E2 protein with single activation domain interacts with E1 The role of E2 in DNA replication is to target viral helicase E1 to itscognate binding site by cooperative binding with E1. The cooperativebinding is mediated by an interaction between the activation domainof E2 and E1. To determine whether E1 is able to form a complex withE2 heterodimers with single activation domain, we co-transfected theCOS-7 cells with expression plasmids for E1 and E2 proteins, andimmunoprecipitated E1:E2 complex with anti-E1 antibodies. The E1protein used in this study contains N-terminally fused HA epitope tagand we have used anti-HA antibodies in this assay. As shown in Fig. 4,scE2 heterodimer was able to co-immunoprecipitate with E1 with thesameef  fi ciencyasthefull-lengthE2protein(lanes3 and4).However,under competitive conditions, when both, E2 and scE2 heterodimers,were expressed together with the E1 protein, E1 interacted betterwith scE2 heterodimer (lane 5). The E2 and E1 proteins wereexpressed at comparable levels (Fig. 4, lower panel) and E2 proteinswere not precipitated from the cell lysate when E1 was not present(lane2).Theantibodywascapturedfromallpreparationsequallywellas tested in Western blot against mouse IgG (data not shown). Thesedata suggest that E1 is able to form a complex with E2 heterodimersimilar to full-length E2 homodimer, and furthermore, interacts withmonomeric E2 activation domain better than with dimeric E2. E2 heterodimer with single activation domain initiates BPV1 genomereplication In order to follow the ability of scE2 heterodimer to supportpapillomavirus DNA replication in the context of the whole BPV genome, we have replaced the open reading frame of E2 with scE2sequence in the context of the entire BPV1 genome using BPV mutantgenomes E2C − and, double mutant E2C − E8/E2 − where initial ATGcodons of E2C or E2C and E8/E2 have been mutated, respectively. Themutated BPV1scE2 genomes encode only E2 proteins with two DBDs,and presumably only intramolecular dimers are formed. For instance,BPVscE2_E2C − genome encodes scE2 heterodimer, and pre-formedE8/E2 homodimer, while BPVscE2_E2C − E8/E2 − encodes only scE2proteinbecauseE2C andE8/E2 openreading frames aremutated. E2Cis the major E2 repressor protein and disruption of this gene altersthe relative abundance of E2 transactivator to E2 repressor resultingin ten fold increase in E2 transcriptional activity and ten to twentyfold increase in viral copy number (Lambert et al., 1990). In ourexperiment, we have used BPV E2C − genome because the replicationsignal of wild-type BPV genome in C127 cells was very low andremained sometimes under detection level. The mutated BPV genomes were transfected into C127 cells and low molecular weightDNA was isolated at days 3, 6, 8 and 11 after electroporation. Asshown on Fig. 5, transient replication signals were detected in allcases up to 8 days after transfection using either wild-type ormutated BPV genomes. But on day eleven, transient replicationsignals of BPV_E2C − E8E2 − genome and both BPVscE2 genomes Fig.4.  E1isabletoformacomplexwithE2heterodimerwithsingleactivationdomain.E2and HA-tagged E1 proteins were co-expressed in COS-7 cells, and immunoprecipitatedfrom cell lysates with HA-speci fi c antibodies. Equal amount of immunoprecipitatedmaterial was subjected to SDS-PAGE and analyzed with HRP-conjugated E2-speci fi cantibody 5E11 and HA-speci fi c antibody 3F12 (Sigma). Fig. 5.  Replication of BPV genome. E2 is expressed as full-length or scE2 within thecontext of BPV genome with E2 repressor E2C or both E2C and E8/E2 knockouts. C127cells transfected with 3  μ  g of respective BPV genome and harvested 3, 6, 8 or 11 daysafter electroporation. Episomal DNA was isolated, digested with DpnI and linearizingenzyme,andanalyzedbySouthernblottingusingtheradio-labeledprobespeci fi ctothereporter plasmid.356  R. Kurg et al. / Virology 386 (2009) 353 –  359  disappeared while replication signal of BPV_E2C − genome stillincreased (Fig. 5, compare lanes 8, 12, 16 and 4). As shown byLambert et al., the disruption of both E2 repressor proteins, E2C andE8/E2 severely reduces the copy number of BPV genomes afterseveral passages of transformed cells (Lambert et al., 1990). Weobserved the same phenomena with BPV_E2C − E8E2 − genome11 days after transfection of cells (Fig. 5). The replication signals of both BPVscE2 genomes also decreased in course of time and wereundetectable in day eleven after transfection. Our earlier studies haveshown that scE2 heterodimer does not support the partitioning of viral genomes in dividing cells (Kurg et al., 2006) and this may be theexplanation for reduced replication signals of BPVscE2 genomes afterseveral cell divisions. However, these data suggest that E2 hetero-dimer with single activation domain alone is able to initiatepapillomavirus DNA replication in the context of entire viral genome. Discussion The E2 ORF encodes a family of proteins which form the centralregulatory system of the virus. The full-length E2 protein acts aspositive regulator of replicationwhile shorter forms of E2, lacking theactivationdomain,serveasrepressorsofreplicationandtranscription.It has been thought for years that heterodimers of full-length andrepressor forms are unstable and act as negative regulators. In ourprevious study (Kurg et al., 2006), we showed that in virally infectedcells, the full-length E2 protein forms heterodimers with repressorforms of E2 and these heterodimers are the preferential form for full-length E2 protein in virus transformed cells. In addition, the forcedheterodimers of full-length E2 with repressor form could mediatetransactivation and replication of papillomavirus DNA (Kurg et al.,2006). In the present study, we go further and provide evidence thatE2heterodimerswithsingleactivationdomainareabletoinitiateBPV replication in the context of papillomavirus genome in the absence of other E2 proteins.Inthepresentstudy,weshowthatE2heterodimerconsistingofthefull-lengthandtruncatedE2proteinassociateswithcellularchromatinsimilar to the full-length E2 suggesting that this localization isdetermined by the single activation domain. This result was notsurprising as E2 heterodimer is able to support papillomavirus DNAreplication and transcription, and for these activities E2 requiresseveral different cellular interactionpartners.However, E2homo- andheterodimer interact differently with some proteins. E2 requires onlyone activation domain for complex formation with viral helicase E1andtwodomainsforinteractionwithcellularBrd4protein(Kurgetal.,2006). It has been previously reported that E2 binds to Brd4 mostef  fi cientlyasadimer(Cardenas-Moraetal.,2008)andthisinteractionis important for E2-mediated transcriptional regulation (Ilves et al.,2006). E2 point-mutation R37A disrupting the N-terminal dimeriza-tion surface (Hernandez-Ramon et al., 2008) had a major effect ontransactivationanddidnotaffectreplication(Abroietal.,1996).Theseresults are consistent with our previous study showing that E2heterodimerswithsingleactivationdomainareweakertransactivatorsthan E2 dimer with two activation domains (Kurg et al., 2006).Our data show that E2 heterodimers form a complex with E1 andare able to load the viral helicase E1 to the srcin of replication. ThescE2 heterodimer forms complex with E1 in solution as ef  fi ciently aswith full-length E2 protein or even better, and is a preferred partnerfor E1 at competitive conditions. These data are supported by earlierstudies. First, Lim et al. showed that pre-formed E2:E2C heterodimersenhance the binding of E1 to the srcin of replication in vitro asef  fi cientlyasE2homodimerandcanactivateDNAreplicationinacell-free system (Lim et al.,1998). Second, two recent works have shownthat disruption of N-terminal dimerization of BPV1 and HPV16 E2proteins does not affect replication suggesting that monomerictransactivation domain is functional in replication (Hernandez-Ramon et al., 2008; Sanders et al., 2007). The use of monomericactivation domain together with dimericactivation domain in loadingE1 molecules may be important for introduction of conformationalheterogeneity in loaded E1 molecules which in turn is required forsequential ATP hydrolysis by double hexameric E1 (Enemark and Joshua-Tor, 2006). We suggest that E2 homo- and heterodimers workin concert in viral DNA replication. Ef  fi cient formation of E1:E2complex is only one step in initiation of papillomavirus DNAreplication, E2 has additional activities in papillomavirus DNAreplication within the cells. E2 is able to alter chromatin structurewithin the srcin and through this facilitate the initiation of DNAreplication (Li and Botchan, 1994). For this activity virus needsprobably full-length E2 homodimers as at least two E2 binding sitesare required for its chromatin remodeling activity in yeast model(Lefebvre et al.,1997). The full-length E2 homodimer is also requiredfor long-term persistence of viral genomes as the E2 heterodimerswith single activation domain are crippled in the segregation of viralgenomes (Kurg et al., 2006).Genetic studies showing that at least one of two E2 repressors of BPV1 is required for the long-term maintenance of viral genomes andfor thevirustransformingactivity (Lambertet al.,1990; Lehmanetal.,1997) demonstrate the important role of truncated E2 proteins in theviral life cycle. Disruption of E2C results in ten fold increase in E2transcriptional activity and ten to twenty fold increase in viral copynumber. However, disruption of both E2 repressor proteins, E2C andE8/E2, reduces severely the copy number of BPV genomes (Lambertet al., 1990). Our results presented in this study are consistent withtheseworksandsupporttheconceptthattruncatedformsofE2haveacritical regulatory role on E2 function, and suggest that a very wellorchestratedcontrolofpapillomavirusmaintenanceandcopynumberis achieved by the balance of different E2 proteins.The intriguing question is what is the possible role for E2heterodimers in virus life cycle and is it essential? Our recent workshave shown that E2 heterodimers are able to initiate virus DNAreplication,butnot to segregateandmaintainthe virusgenome (Kurget al., 2006). This is supported by the work of Yang et al. where thedynamic change in the balance of E2 repressors to activators withinthe cell cycle was measured. In asynchronous and G 1  cells, the E2repressors dominate, but in late S phase and G 2 /M, the activator ispresent in about equal levels to that of the repressors (Yang et al.,1991). We also show that the E2 protein can be replaced with scE2heterodimer in the context of BPV genome and these scE2 genomesreplicate in transient assays in the absence of other E2 proteins. OnepossibilityisthatE2heterodimersareessentialforquickampli fi cationof virus genomes taking place within limited number of cell cycles,and full-length E2 homodimer for maintenance of viral genomes individing cells. This hypothesis is supported by the work of  SzymanskiandStenlund (1991) whohaveanalyzedthe transcriptional activityof BPV promoters in their natural genomic context. In the absence of E2,the level of P 3080 -speci fi c messages was more than 10-fold greaterthan the level of any other promoter suggesting that BPV earlypromoter P 3080  expressing E2C repressor protein is the mostproductive promoter upon initial viral infection while P 89  and P 2443 expressingfull-lengthE2havelowbasalactivity.Inaddition,BPVearlypromoters display differential sensitivities to the E2 protein, and theP 3080  was the least responsive to E2 (Szymanski and Stenlund, 1991)suggesting that synthesis of E2 and E2C are differently regulated byE2. So, the level of E2C is high and full-length E2 is low upon initialviral infection, but after some time the level of full-length E2 isincreasing due to positive autoregulation of E2 expression. Theabundance of E2 and E2C mRNAs is quantitatively different betweenthe transiently transfected and stably transformed cells (Szymanskiand Stenlund, 1991). Presumably the level of E2 heterodimer withsingle activation domain is also initially high, and the ratio of E2homo- and heterodimers is changing during the course of the viralinfection. These  fi ndings, together with our results that E2 hetero-dimers with single activation domain are able to initiate BPV  357 R. Kurg et al. / Virology 386 (2009) 353 –  359
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