Chaperone Proteins Abrogate Inhibition of the Human Papillomavirus (HPV) E1 Replicative Helicase by the HPV E2 Protein

Human papillomavirus (HPV) DNA replication requires the viralorigin recognition protein E2 and the presumptive viral replicativehelicase E1. We now report for the first time efficient DNAunwinding by a purified HPV E1 protein. Unwinding depends ona supercoiled DNA substrate, topoisomerase I, single-stranded-DNA-bindingprotein, and ATP, but not an origin. Electron microscopy revealedcompletely unwound molecules. Intermediates contained two single-strandedloops emanating from a single protein complex, suggesting abidirectional E1 helicase which translocated the flanking DNAin an inward direction. We showed that E2 protein partiallyinhibited DNA unwinding and that Hsp70 or Hsp40, which we reportedpreviously to stimulate HPV-11 E1 binding to the origin andpromote dihexameric E1 formation, apparently displaced E2 andabolished inhibition. Neither E2 nor chaperone proteins weredetected in unwinding complexes. These results suggest thatchaperones play important roles in the assembly and activationof a replicative helicase in higher eukaryotes. An E1 mutationin the ATP binding site caused deficient binding and unwindingof origin DNA, indicating the importance of ATP binding in efficienthelicase assembly on the origin.

INTRODUCTION

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Introduction

Human and animal papillomaviruses are prevalent pathogens. Efficientorigin (ori)-dependent replication of viral DNA requires thevirus-encoded E1 and E2 proteins as well as cellular replicationproteins (11, 27, 46, 59, 68). As such, these DNA viruses mayserve as a model for higher eukaryotic DNA replication, as dosimian virus 40 (SV40) and polyomavirus. The papillomavirusori consists of several E2 binding sites (BS) flanking one E1BS. E1 recruits the DNA polymerase ${alpha}$ /primase (6, 12, 41) andthe single-stranded-DNA-binding protein RPA (25). The humanpapillomavirus (HPV) E1 protein is required during initiationand elongation and is thought to be the replicative helicase(33). However, HPV E1 proteins are poor helicases in stranddisplacement assays (26, 65), and there has been no report ofDNA-unwinding activity. In contrast, the bovine papillomavirus1 (BPV-1) E1 exhibits helicase activity in both assays (51,69).

We previously reported that purified HPV-11 E1 protein expressedin insect Sf9 cells binds to ori with low affinity and specificityand also binds to DNA nonspecifically (33). Electron microscopy(EM) shows that E1 binds ori primarily as a hexamer and, ata low frequency, as a dihexamer (34). The human heat shock proteinsHsp70, Hdj2, and Hdj1 greatly stimulate E1 binding to ori. Hdj1and Hdj2 encode members of the Hsp40 family of proteins thatnormally function as cochaperones of the Hsp70 proteins andgreatly stimulate the ATPase activity of Hsp70 (for a review,see reference 21). However, in the case of the HPV-11 E1-oriassociation, their effects are independent and additive. Moststrikingly, EM has revealed that Hsp40 but not Hsp70 promotesE1 dihexamer formation on ori (34). The BPV-1 E1 has been reportedto be a hexameric helicase (48). However, EM revealed a bilobedcomplex, which was presumed to be a dihexamer as well, but thesize and the frequency of this complex were not reported (20).Two helicases functioning at divergent orientations are necessaryfor bidirectional DNA replication, but it has not been establishedwhether E1 proteins function as a dihexameric, bidirectionalhelicase or whether the dihexamer separates into two independenthexameric helicases as the DNA flanking the ori unwinds.

From bacteria to higher eukaryotes, chaperone or heat shockproteins play crucial roles in protein folding, trafficking,and protein complex assembly and disassembly (21). The Escherichiacoli chaperones DnaJ and DnaK serve important roles in the assemblyof replicative helicases for bacteriophages ${lambda}$ , P1, and P7 (2,3, 31, 66, 72). Human Hdj2 and Hsp70 are homologs of DnaJ andDnaK, respectively. In particular, the domain of Hsp40 whichinteracts with HPV-11 E1 has been mapped to a span of 20 aminoacids within the highly conserved J domain (34). Moreover, incubationof HPV-11 E1, ori, and chaperones decreases the lag time inthe onset of DNA replication and increases cell-free replicationdespite the presence of abundant chaperone proteins in the cellextracts (34). Along with SV40 T antigen, this provided thefirst indication that cellular chaperones have a role in DNAreplication in higher eukaryotes.

The SV40/polymavirus T antigen functions as a dihexameric helicase(15, 17, 42, 52, 61, 64). Interestingly, its amino terminusis homologous to the J domain of DnaJ and interacts with Hsc70(for a review, see reference 54). This domain is required forefficient SV40 DNA replication in vivo (7), and its truncationleads to incorrect oligomerization (62). Recently, Hsp40 andHsp70 have also been reported to enhance the binding of UL9,the origin-binding protein of herpes simplex virus type 1, tooriS and the resultant ori opening (57). hTid-1, a human homologof E. coli DnaJ, also binds to UL9 and promotes multimer formationfrom dimers (19). In addition, Hsp70 also interacts with Orc4pof Saccharomyces cerevisiae, dissociating the oligomerized Orc4pamino-terminal domain (23), a function which we previously proposedfor Hsp70 in stimulating HPV-11 E1 binding to ori (34). Collectively,these observations strongly suggest that chaperone proteinsplay a role in the assembly and activation of replicative machineryin both prokaryotes and eukaryotes.

The papillomavirus E2 protein is also a multifunctional protein.Among its many functions, it is the primary ori recognitionprotein. It binds to the E2 BS with high specificity and highaffinity and is crucial for assembly of the preinitiation complexby recruiting and targeting E1 to ori (27, 36, 44, 50, 68).In vivo, E2 prevents nucleosome formation around ori (30). Therefore,only plasmids containing the E2 BS function in transient replication(10, 35, 46). In addition, E2 also associates with the nuclearmatrix and PML (a component of nuclear domain 10), where viralDNA replication takes place (56, 71). On the other hand, a stableassociation of E2 to E2 BSs that flank the E1 BS may act asa DNA clamp, preventing DNA unwinding. This latter possibilitymay explain why E2 is absent from the replication complex postinitiation(33). However, because HPV E1 DNA-unwinding activity has notbeen demonstrated, neither the E2 inhibition of DNA unwindingby E1 nor the mechanism of E2 release has been investigated.

In this study, we report that bacterially purified HPV-11 E1protein exhibited a robust supercoiled DNA-unwinding activity.Using biochemical assays and EM examination, we characterizedthe requirements of the unwinding reaction, the nature of unwindingintermediates and products, and the effects of E2 protein andchaperone proteins. The implications of these findings are presented.

MATERIALS AND METHODS

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Materials and Methods

Plasmids and proteins. p7874-99 and p7874-20 are HPV-11 ori plasmids based on the pUC19vector, containing HPV-11 nucleotides 7874 to 7933/1 to 99 and7874 to 7933/1 to 20, respectively. p7730-99 (234M) containsmutations in E2 BS copies 2, 3, and 4 in the ori (spanning nucleotides7730 to 7933/1 to 99) (10). Epitope-tagged HPV-11 E1 and E2proteins were expressed and purified from E. coli. To expressthe EE-E1 protein (27), E. coli BL21(DE3) (Stratagene, La Jolla,Calif.) harboring pRSET-EE-E1 (32) was induced at mid-log phasewith 0.3 mM isopropylthio-ß-galactopyranoside (IPTG)for 24 h at 18°C. Cells were disrupted by sonication inlysis buffer (20 mM Tris-HCl [pH 7.0], 250 mM NaCl, 1 mM dithiothreitol).The soluble fraction was first passed through a Q-Sepharosecolumn (Bio-Rad, Hercules, Calif.) and eluted with 20 mM Tris-HCl(pH 7.5)-800 mM NaCl. The eluant was then applied to an anti-EEimmunoaffinity column, washed with 1 M NaCl-20 mM Tris-HCl (pH7.5), and eluted with 100 mM triethylamine, as described previously(27, 34). The eluted protein was dialyzed overnight againstTris-HCl (pH 7.0)-50 mM NaCl-10% glycerol buffer and kept at-80°C. A P479S mutation of HPV-11 E1 was generated by site-directedmutagenesis with PCR amplification. This mutated protein wassimilarly tagged with the EE epitope and expressed and purifiedfrom E. coli as described for the wild-type E1 protein.

The induction of the HPV-11 E2 protein from pRSET-11E2, whichwas tagged with an epitope from a cytomegalovirus-encoded protein(32), in E. coli BL21(DE3)pLysS (Stratagene), was conductedas described previously for the E1 proteins except for the additionof 0.2 mM IPTG. To purify the E2 protein, the soluble fractionwas diluted with buffer Q (20 mM Tris-HCl [pH 7.0], 10 mM 2-mercaptoethanol)to a final concentration of 50 mM NaCl and applied to a 10-mlQ-Sepharose column. The flowthrough was then applied to a 1-mlMacro-Prep High S column (Bio-Rad) and eluted with a 100 to500 mM NaCl gradient in buffer Q. The fractions were analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresisand Western blot, and those containing E2 protein were pooled.The human Hsp70 (1) and Hdj2 (9, 34) proteins were expressedin and purified from E. coli as described previously (34). Arecombinant baculovirus expressing human topoisomerase I wasa gift from Jim Champoux, and protein was purified as describedpreviously (53). An E. coli expression vector for His-taggedhuman RPA was a gift from Mike O'Donnell (70), and the proteincomplex was purified by nickel affinity chromatography (Qiagen,Valencia, Calif.). All proteins were purified to near homogeneity,as determined by Coomassie blue staining and Western blottingwith antibodies (Fig. 3A and data not shown). For gel assays,the E. coli single-stranded-DNA-binding protein (SSB) was obtainedfrom Amersham Pharmacia Biotech. For EM, SSB was purified asdescribed previously (8).

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FIG. 3. HPV-11 E1 P479S mutant protein is defective in multiple activities. Wild-type HPV-11 E1 proteins purified from Sf9 cells (27) and from E. coli were tested for comparison. (A) Wild-type (WT) E1 and mutated E1 P479S protein were analyzed by SDS-PAGE and stained with Coomassie blue (left) and also by Western blot with monoclonal antibody against the epitope tag (right). (B) Autoradiogram of ATPase assays. The positions of [ ${gamma}$ -³²P]ATP and free ³²P_i are marked. (C) Unwinding of ori plasmid p7874-99 as revealed in an ethidium bromide-stained chloroquine-agarose gel. Lane 1, supercoiled input DNA (*) in reaction buffer. Lane 2, reaction without E1. Lane 3, reaction with wild-type E1 purified from E. coli. U-form DNA is marked (<). Lane 4, reaction with E1 P479S. (D) Autoradiogram of cell-free replication of ori DNA in the presence of purified HPV-11 E2 protein, wild-type HPV-11 E1, or E1 P479S and [ ${alpha}$ -³²P]dCTP. Form I product (*) and replication intermediates (bracket) are marked. (E) Autoradiogram of EMSA of wild-type HPV-11 E1 or E1 P479S and ³²P-labeled ori-containing fragment (nucleotides 7874 to 99) in the presence and absence of Hdj2 or Hsp70. DNA-protein complexes are marked (bracket).

DNA unwinding and chloroquine-agarose gel electrophoresis. The conditions for unwinding were modified from those describedpreviously (16). A standard reaction mixture of 60 µlcontained 500 ng of supercoiled DNA, 300 ng to 1.0 µgof E1 protein, 25 ng of topoisomerase I, 300 ng of human RPAor E. coli SSB, 4 mM ATP, 40 mM creatine phosphate, 23.3 µgof creatine phosphate kinase (creatine phosphate kinase) perml, 16.7 µg of bovine serum albumin per ml, 7 mM MgCl_2,and 5 mM dithiothreitol. Variations in conditions were as describedin Results. After incubation at 37°C for different lengthsof time as indicated in each figure legend, reactions were terminatedby adding 6.8 µl of stop mix, containing 13.5 mM EDTA,30 µg of tRNA per ml, 0.3% N-lauroylsarcosine, and 0.45mg of proteinase K per ml, and incubation was continued for30 min at 37°C. After extraction with phenol-chloroform,DNA was separated by electrophoresis in 1.2% agarose gels containing0.25 µg of chloroquine per ml (16). The gels were stainedwith ethidium bromide and documented with a Bio-Rad Gel Doc2000 system.

Cell-free DNA replication and EMSA. Cell-free replication of p7874-99 was conducted with human 293cell extracts (27). Replication products were labeled with [ ${alpha}$ -³²P]dCTP(Amersham Pharmacia Biotech), purified, and separated by agarosegel electrophoresis. Electrophoretic mobility shift assays (EMSAs)were performed as described previously (33, 34). The EcoRI-HindIIIrestriction fragment spanning HPV-11 ori (nucleotides 7874 to7933/1 to 99) was labeled with [ ${gamma}$ -³²P]ATP (Amersham PharmaciaBiotech) and T4 polynucleotide kinase (Life Technology, Inc.)and used as a substrate. Data were acquired with a PhosphorImager(Molecular Dynamics, Sunnyvale, Calif.).

ATPase assay. ATPase activity was detected by release of ³²P_i from [ ${gamma}$ -³²P]ATPas described previously (49). A 20-µl amount of reactionmixture contained 100 ng of HPV-11 E1 protein, 25 mM Tris-HCl(pH 8.0), 1 mM dithiothreitol, 10 mM MgCl₂, 250 µg ofbovine serum albumin per ml, 50 µM [ ${gamma}$ -³²P]ATP (1.5 x 10⁴cpm/pmol) (Amersham Pharmacia Biotech), and 50 ng of single-strandedM13 DNA. After incubation for 60 min at 37°C, 1 µlwas spotted onto a polyethyleneimine-cellulose thin-layer chromatographyplate (Sigma), which was developed with 1.0 M formic acid-0.5M LiCl for 45 min. Data were captured with a PhosphorImager(Molecular Dynamics, Sunnyvale, Calif.).

Electron microscopy. Unwinding assays were conducted with 400 ng of ori plasmid p7874-99,500 ng of E1, 25 ng of human topoisomerase I, and 180 ng ofE. coli SSB in 60 µl of buffer containing 20 mM HEPES(pH 7.8), 80 mM NaCl, 4 mM ATP, and 2 mM MgCl₂; 300 ng of E2,Hsp70, or Hdj2 was added as specified. For binding assays, oriplasmid and proteins were incubated in buffer containing 2 mMATP or ATP- ${gamma}$ -S (33) for 20 min at 37°C. Reactions were terminatedby adding EDTA to 10 mM on ice and then glutaraldehyde to 0.6%for 5 min at 20°C. The samples were chromatographed over2 ml of BioGel A5m (Bio-Rad) columns equilibrated with TE buffer(10 mM Tris-HCl [pH 8.0], 1 mM EDTA). Fractions containing theDNA-protein complexes were collected, and aliquots were mountedon glow discharge-treated carbon supports as described previously(24). U-form DNA purified from a chloroquine-agarose gel wasconcentrated by ethanol precipitation, redissolved in TE, incubatedwith SSB (3 µg/ml), and prepared for EM as described above.

For immunogold EM, the DNA-protein complex-containing fractionsfrom the A5m BioGel columns were pooled. After the NaCl concentrationwas adjusted to 80 mM, aliquots were incubated for 1 h at 37°Cwith primary rabbit polyclonal antibodies to E2 (11) or monoclonalantibodies to Hsp70 (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.) or Hdj2 (Neomarker, Fremont, Calif.) (1:1,000 to 1:5,000dilution) and then for another 1 h with secondary goat anti-rabbitor anti-mouse immunoglobulin antibodies conjugated to 5- or10-nm gold particles (Amersham Pharmacia Biotech), respectively.The reactions were stopped, and the mixtures were chromatographedand prepared for EM as described above. All samples were analyzedwith a Philips CM12 electron microscope. The images were scannedwith a Nikon LS-4500AF film scanner, and contrast was adjustedwith Adobe Photoshop software.

RESULTS

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Results

Requirements for DNA unwinding by HPV-11 E1. The supercoiled HPV-11 ori plasmid p7874-99, which containsthree copies of the E2 BS flanking the single E1 BS, was usedin unwinding reactions (Fig. 1A, lanes 1 to 8) based on theassay for the SV40 T antigen (16). The reactions were optimizedby reiterative experiments with set amounts of DNA substrate,various amounts of purified E1, and lengths of reaction timeat several concentrations of topoisomerase I and RPA (data notshown). DNA from the reactions was then purified, electrophoreticallyseparated in a chloroquine-agarose gel, and revealed by ethidiumbromide staining.

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FIG. 1. Requirements for DNA unwinding by HPV-11 E1 protein. Agarose gels were stained with ethidium bromide. The presence or absence of specific reagents (see Materials and Methods) is indicated by + or - above each lane. (A) Substrates were supercoiled plasmid DNA, p7874-99, which contains the HPV-11 ori cloned in pUC19 (lanes 1 to 8), or the vector (lanes 9 to 11). Incubation was for 15 min at 37°C. Similar results were obtained when incubation was for 10 min (data not shown). (B) Substrates were circular (lanes 1 to 6) or ScaI-linearized p7874-99 (lanes 7 and 8). All reagents were as described for panel A except in lane 5, in which topoisomerase I (Topo I) was reduced from 25 ng to 10 ng. Lane 1, input supercoiled DNA in reaction buffer. Preincubation of plasmid DNA with E1 (lane 3) or topoisomerase I (lane 6) was for 10 min at 37°C. The missing enzyme was then added, and incubation was continued for another 5 min at 37°C. Increasing the second incubation to 10 min yielded the same result (data not shown). Relaxed circular DNA (#), form I supercoiled DNA (*), U-form DNA (> or <), and linear DNA (arrowhead) are marked. Lane M, double-stranded size markers.

In the absence of E1, the addition of topoisomerase I convertedthe input, fast-migrating form I DNA (_*) to slow-migrating relaxedcircular DNA (#) and a ladder of closed circular DNA with reducedsuperhelicity (collectively termed covalently closed [cc] DNAtopoisomers) (compare lanes 1 to 2 and 10 to 11). Unwindingtook place efficiently in the presence of E1, human topoisomeraseI, human single-stranded-DNA-binding protein RPA, ATP, and creatinephosphate kinase, a component of an ATP-regenerating system.A fraction of the fast migrating form I DNA was converted toa slightly slower migrating unwound form (U-form DNA) (lane6) (marked with > or < in this and other figures), asdescribed previously (16, 43), the balance being the cc DNAtopoisomers. Reactions reached a plateau by 10 to 15 min at37°C (data not shown). An ori was, however, not necessary(Fig. 1A, lanes 9 to 11).

The ori-independent DNA unwinding is consistent with the nonspecificassociation of E1 with DNA and with its ability to support ori-independentreplication in the cell-free system when E1 or DNA is presentat high concentrations (27, 33). Both conditions were also usedin this study. Our result also agrees with reports that BPV-1E1 unwinds DNA without an ori in the absence of a nonspecificinhibitor DNA (51, 69).

The E. coli single-stranded-DNA-binding protein SSB was lesseffective than the human RPA (Fig. 1A, compare lanes 5 and 6).Omission of one or more of the components abolished unwindingat the sensitivity limit of this detection method (lanes 1 to4, 7, 8, 10, and 11) with one exception. Upon prolonged exposure,a faint U-form band was detected in the reaction from whichcreatine phosphate kinase was omitted (data not shown). Thiswas confirmed by EM (see below). A reduction of E1 protein tobelow 300 ng abolished unwinding (data not shown). Neither preincubationof E1 with the supercoiled DNA nor reduction of topoisomeraseI by 2.5-fold significantly affected the amount of U-form DNAproduced (Fig. 1B, lanes 3 to 5, and data not shown). Thus,under our reaction conditions, binding of E1 to DNA was notrate limiting, nor was topoisomerase I in excess relative toE1 and DNA.

Because single-stranded DNA binds ethidium bromide poorly relativeto double-stranded DNA, the percentage of DNA converted to theU form can best be approximated by comparing the remaining ccDNA topoisomers to those formed in the absence of E1 (for instance,Fig. 1A, compare lanes 2 and 4 to 6 and lane 10 to 9). Althoughthe efficiency of generating U-form DNA varied with differentE1 protein preparations, in some reactions, there was clearlymore U-form DNA than remaining cc DNA topoisomers, indicatinga conversion efficiency of over 50% (Fig. 1A, lanes 6 and 9;Fig. 1B, lane 5). These properties are similar to those reportedfor BPV-1 E1 (51, 68) except that unwinding with HPV-11 E1 proteinwas fast and highly efficient and did not require radioactiveprobes to reveal the U-form DNA.

Because of the poor helicase activity of HPV E1 proteins instrand displacement assays, we suspected that the nature ofthe substrate might be important. We tested this supposition.Indeed, linear ori DNA and ori DNA prerelaxed with topoisomeraseI were poor substrates, as we detected no U-form DNA (Fig. 1B,lanes 8 and 6), which we showed to contain single-stranded circularand linear DNA (see below). However, a low level of partialunwinding of linear DNA probably did occur, as suggested bya reproducibly observed reduction in the substrate (comparelanes 7 and 8, and data not shown). We believe that unwindingof linear DNA was incomplete and that the products had heterogeneousmigration rates and hence escaped detection.

U-form DNA contains single-stranded DNA. U-form DNA was described previously as unwinding intermediatescontaining single-stranded regions of various lengths (16, 17,51, 60, 67). We purified U-form DNA from an agarose gel, incubatedit with E. coli SSB to extend the single-stranded regions, andthen examined it by EM. We observed no partially unwound moleculesor interlocked single strands. Rather, 60 to 70% of the moleculeswere circular or linear, SSB-coated single strands, with theremainder being double-stranded open circles (Fig. 2A, and datanot shown). Since the slower-migrating open circles (and thecc DNA topoisomers) are not likely to contaminate the fast-migratingU-form DNA to a significant extent (Fig. 1, compare bands markedwith #, >, and <), we suggest that the double-strandedopen circles were generated by reannealing of single strandsduring purification of the U-form DNA from the agarose gel.We also suggest that single-strand breaks are introduced intothe products by topoisomerase I or by physical strand breakageduring DNA purification prior to agarose electrophoresis. Thus,any interlocked single-stranded circles would have been convertedto separate single-stranded circles and linear molecules. Someof the single strands subsequently renature during purificationfrom the agarose gel. Since we only examined DNA recovered fromthe U-form DNA band, unwinding intermediates that migrated moreslowly in the agarose gels were not recovered and hence escapeddetection.

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FIG. 2. Electron micrographs of U-form DNA and protein-DNA complexes in unwinding reactions. Row A, circular and linear single strands recovered from the U-form DNA band from an unwinding reaction of p7874-99 under complete reaction conditions. The purified U-form DNA was coated with E. coli SSB and cross-linked with glutaraldehyde. Rows B to D, protein-DNA complexes present in the unwinding reactions with conditions modified for EM. Row B, DNA bound by an E1 dihexamer without discernible DNA unwinding. DNA bound by an E1 hexamer was more frequently observed (not shown). Row C, unwinding intermediates containing two rabbit ear-like, SSB-coated single-stranded DNA loops. Row D, completely unwound circular single-stranded DNA coated with SSB. Linear single strands were also observed (not shown). Images are shown in reverse contrast. Identical complexes were also visualized when E2, Hsp70, and Hsp40 were also present, but the relative frequencies of observation were affected (Table 1). Bar, 100 nm.

E1 mutation in the ATP binding site causes defects in ori binding and DNA unwinding. To substantiate our interpretation that E1 was responsible forgenerating the single-stranded U-form DNA, we expressed andpurified an epitope-tagged HPV-11 E1 mutation, P479S, from E.coli. Figure 3A shows a Coomassie blue-stained SDS-PAGE gelof the purified wild-type and mutated E1 proteins, along witha Western blot made with an antibody to the epitope tag. Itcan be seen that both proteins were purified to near homogeneity.This mutated HPV-11 E1 protein exhibited reduced ATPase activity(Fig. 3B), produced no U-form DNA in ethidium bromide gels (Fig.3C), and had a very low activity in cell-free replication (Fig.3D). These observations support the conclusion that the wild-typeHPV E1 unwinds DNA.

To understand the molecular basis for these properties, we examinedthe ability of E1 P479S to bind ori DNA by EMSA (Fig. 3E). Thewild-type HPV-11 E1 protein formed a faint smear of slowly migratingcomplexes with a radiolabeled ori fragment, and the presenceof Hsp40 and Hsp70 greatly stimulated complex formation (Fig.3E, lanes 1 to 5), as described previously (34). E1 P479S generatedfew or no detectable DNA-protein complexes in the absence orpresence of either chaperone (Fig. 3E, lanes 6 to 9). EM confirmedthat this mutated E1 protein did not form a discernible proteincomplex on ori DNA (data not shown).

E1 complexes function as a bidirectional helicase. We next used EM to examine DNA-protein complexes present inunwinding reactions conducted under modified conditions. E.coli SSB was used because SSB-coated single strands were moreextended than RPA-coated molecules (unpublished observations).Creatine phosphate kinase and bovine serum albumin were omittedto reduce the amount of proteins, facilitating the visualizationof protein-DNA complexes, especially unwinding intermediates,as the efficiency of unwinding was greatly reduced (Fig. 1).The majority of the DNA was present as relatively relaxed circlesgenerated by topoisomerase I (Fig. 1). Some were bound by asingle hexamer and, less frequently, by a dihexamer withoutvisible unwinding (Fig. 2B). No ori DNA was associated withmore than one E1 hexamer or dihexamer. Completely unwound SSB-coatedsingle strands were also observed (Fig. 2D).

Importantly, we observed complexes at the early stages of unwinding(Fig. 2C) that contained two rabbit ear-like, single-strandedDNA loops emanating from a large protein complex. The E1 dihexamerwas no longer discernible due to the presence of additionalproteins and the overlay of SSB-coated single-stranded DNA.Nevertheless, the presence of double loops is entirely consistentwith an interpretation that the functional helicase is a dihexamerand that DNA was translocated inwardly while being unwound.Other partially unwound complexes were uninformative due toa nonoptimal orientation on the EM grids or coalesence of SSB-coatedsingle strands. It is notable that in no case did we observeunwinding intermediates in which a bubble of two SSB-coatedsingle-stranded arms was flanked by one or two protein complexes.These structures would have suggested one or two unidirectionalhelicases that operate at one or both forks of two expandingsingle-stranded arms on an otherwise double-stranded circularDNA.

E2 inhibits DNA unwinding by E1. As we expected, addition of purified E2 protein, which was activein supporting HPV ori replication (Fig. 3D) and in binding toori in EMSA (data not shown), reduced the amount of U-form DNAin a dose-dependent manner (Fig. 4). Inhibition was observedwith an ori plasmid containing either one copy (p7874-20) orthree copies (p7874-99) of the E2 BS (Fig. 4, left panel), inagreement with observations made with the BPV-1 system (36).Inhibition was also observed with p7730-99 (234M), in whichall three E2 BSs are site mutated while maintaining a wild-typeE1 BS (Fig. 4, right panel). This mutated ori no longer bindsE2 in vitro, nor does it replicate in a transient or cell-freesystem (11, 27). Thus, protein-protein interactions may alsocontribute to the inhibition. EM confirmed that, in the presenceof 300 ng of E2 protein, partially and completely unwound DNAmolecules were reduced from 80% to 20% among the DNA-proteincomplexes scored (Table 1).

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FIG. 4. HPV-11 E2 inhibition of DNA unwinding by E1 is dose dependent but E2 BS independent. DNA substrates used were p7874-20, p7874-99, and p7730-99 (234M), which contain 1, 3, and 0 copies of the E2 BS, respectively. The presence (+) or absence (-) of HPV E1 and E2 and human topoisomerase I and the amounts of E2 protein (in nanograms) added are indicated. All other components were as described in Materials and Methods. Form I supercoiled DNA (*) and U-form DNA (> or <) are marked. Lane M, double-stranded size markers.

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TABLE 1. Modulation of E1 helicase by E2 and chaperones as visualized by EM^a

E2 protein does not affect E1 assembly on ori but remains associated with the E1-ori complex. To investigate the mechanism by which the E1 helicase is inhibitedby E2, protein-DNA complexes in binding reactions were examinedby EM. In reactions containing both E1 and E2 proteins, thepercentage of E1 dihexamer varied from 5 to 15% of the protein-oricomplexes. The remaining complexes contained a single E1 hexamer(Fig. 5A, left panel). This distribution was similar to thatin our previous report on E1-ori complexes formed in the absenceof E2 (34). Furthermore, the frequency of observing protein-DNAcomplexes among all DNA molecules visualized was similar oronly slightly reduced compared to that in the absence of E2protein. However, because of the small size of the E2 protein(43 kDa), which binds to the E2 BS as a dimer, we were not ableto ascertain the presence of bound E2.

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FIG. 5. Immunogold labeling of proteins associated with E1-ori. The binding reactions were conducted with E1 protein and ori DNA in the presence of E2, Hsp70, or Hdj2. Row A, left panel, E1 hexamer-ori complex assembled in the presence of E2 protein; middle and right panels, complexes assembled in the presence of E1 and E2 and then probed with primary antibodies (Ab) against E2 and secondary immunogold conjugates. The large complexes shown were coated with antibodies but not with gold particles. Row B, complexes assembled in the presence of E1 and E2 and then probed with primary antibodies against E2 and secondary immunogold conjugates. The complexes carried 5-nm gold-secondary antibody conjugates. Rows C and D, E1-ori complexes assembled in the presence of Hdj2 and Hsp70, respectively, were treated with primary antibodies against the respective chaperone and 10-nm gold-secondary antibody conjugates. Examples shown carried gold particles. Images are shown in reverse contrast. Bar, 100 nm.

To determine whether E2 remains associated with the E1-ori complexto account for its inhibitory effect on the E1 helicase, weperformed immunogold EM with a rabbit polyclonal antibody againstE2 and a secondary antibody reactive with the rabbit antibody.This secondary antibody had been electrostatically conjugatedto 5-nm gold particles. In the absence of E2 protein or theprimary antibody, immunogold particles were attached to about2% or less of the E1-ori complexes. In contrast, among 126 protein-oriDNA complexes scored in a binding reaction containing E1, E2,the ori plasmid, the primary antibody, and the secondary immunogoldconjugates, 19% of the protein-ori complexes were associatedwith immunogold particles (Table 2, Fig. 5B). Most of the complexeswithout attached immunogold particles were larger than thoseobserved in the absence of any antibody, suggesting that thesecomplexes were bound by antibodies lacking gold particles (comparemiddle and right panels of Fig. 5A to left panel). Thus, thepercentage of immunogold labeling likely represents an underestimationas a result of dissociation of some of the gold conjugates duringpreparation for EM. Furthermore, the gold particles significantlyretard the rate of association between the conjugates and thetarget complexes relative to secondary antibodies that had lostthe gold particles, further reducing the efficiency of immunogolddecoration.

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TABLE 2. Hsp70 and Hdj2 displace E2 from E1-orl complexes as visualized by immunogold EM^a

Because the protein-DNA complexes reacted with antibodies wererather large, we were not able to discern simultaneously E1hexamers or dihexamers in the complexes. Even though we couldnot rule out the possibility that some of these complexes containedonly E2, others must also contain E1 to account for the inhibitoryeffect of the E2 protein on the E1 helicase (Fig. 4 and Table1). We suggest that, at the concentrations used, E2 remainsassociated with the E1-ori complexes without affecting E1 assemblyon the ori.

Chaperone proteins abolish E2 inhibition of E1 helicase. To determine whether chaperones might play a role in activatingthe E1 helicase inhibited by the E2 protein, we added 300 ngof Hsp70 or Hdj2 to the p7874-99 ori plasmid unwinding reactionsin the presence of 300 ng of the E2 protein. The conditionsused were those modified for EM, as described previously. EMexamination showed that, among the DNA-protein complexes scored,the inhibitory effect of the E2 protein on DNA unwinding waslargely alleviated by either or both chaperone proteins (Table1). However, the overall unwinding remained very inefficient,as the majority of the DNA visualized was still free DNA relaxedby topoisomerase I. This is because the reaction was conductedin the absence of creatine phosphate kinase, where the ATP supplyis limiting. Furthermore, the assembly of E1 on the ori DNArequires ATP (Fig. 3), which was also consumed by Hsp70.

To obtain more quantitative results on the effects of chaperoneson p7874-99 unwinding by E1 in the presence of E2, we conductedunwinding reactions in the presence of all the necessary componentsand analyzed the products by chloroquine-agarose gel electrophoresis.Addition of 200 ng of Hsp70 or Hdj2 to the unwinding reaction,which was partially inhibited by 300 ng of HPV-11 E2, indeedpartially restored the unwinding activity, based on the amountsof U-form DNA observed. However, when both chaperones were present,the increase in U-form DNA was marginal relative to that achievedin the presence of either chaperone alone (Fig. 6A, comparelanes 3 through 7), as also observed by EM (Table 1). We attributedthis result to a highly stimulated ATPase of Hsp70 when Hdj2was also present (28). A depletion of ATP would then impedeE1 assembly on the ori DNA (33) and the E1 helicase activity(Fig. 1).

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FIG. 6. Effects of chaperone proteins on HPV E1 helicase inhibited by HPV E2. (A) Unwinding reactions were conducted in the presence (+) or in the absence (-) of 200 ng of E2, Hsp70, or Hdj2. All reactions contained 4 mM ATP. (B) Unwinding reactions were conducted in the presence (+) or in the absence (-) of 200 ng of E2 and 50 ng of Hsp70, Hdj2, or both. The concentration of ATP was 4 or 20 mM, as indicated. Lane M, size markers.

To substantiate this interpretation, we modified the reactionconditions. As shown in Fig. 6B, increasing the ATP concentrationto 20 mM in the otherwise standard reaction conditions significantlyincreased the amount of U-form DNA (Fig. 6B, compare lane 4to 3). Although we did not test additional ATP concentrations,this experiment clearly showed that the 4 mM ATP in the standardreaction was limiting for the amount of E1 protein used. Theinhibitory effect of 200 ng of E2 protein persisted under thiscondition (Fig. 6B, lane 5). In the presence of 20 mM ATP, theaddition of only 50 ng of Hsp70 or Hdj2 was sufficient to abolishmuch of the E2 inhibition (Fig. 6B, lanes 6 and 7). When bothchaperones were present, the amount of U-form DNA exceeded thatobserved in the absence of E2 protein (Fig. 6B, compare lane8 to 4). Thus, chaperone proteins indeed abolish E2 inhibitionas long as ATP is not limiting. In vivo, the supply of ATP willbe continuously replenished, and ATP should not be a limitingfactor as it is in vitro.

Chaperone proteins displace E2 from the E1-ori complex. To investigate the mechanism by which chaperones reactivatethe E1 helicase in the presence of the E2 protein, we proceededto examine the E1-ori (p7874-99) complexes formed in a seriesof binding reactions in the presence of either chaperone aloneor when E2 was also added with immunogold by EM (see Materialsand Methods). The data are summarized in Table 2. In the presenceof chaperones but in the absence of primary antibodies to eitherchaperone, only 2 to 4% of the protein-ori complexes carriedimmunogold conjugates targeted to the chaperones. In anothercontrol, in the absence of chaperones but in the presence ofprimary antibodies to chaperones, only 1% of the protein-oricomplexes carried immunogold particles. These control experimentsdemonstrate a lack of nonspecific association of the primaryand second antibodies with the protein-DNA complexes, in agreementwith our previous report (34). In contrast, in binding reactionscontaining chaperones and primary antibodies, 18% and 36%, respectively,of the protein-ori complexes carried immunogold conjugates (Table2), indicating that both chaperones can independently associatewith the E1-protein complexes. Examples are presented in Fig.5C and D.

Since the immunogold conjugates obscured the E1-ori complexes,we analyzed the nature of protein-ori complexes after incubationwith either chaperone in the absence of any antibodies. OnlyHdj2 induced efficient formation of dihexameric E1 complexeson over 90% of the E1-ori complexes observed, in agreement withour previous data (34). Because Hdj2 coimmunoprecipitates withE1 and also remains associated with E1-ori in EMSA (34), thehigh efficiency of dihexameric E1-ori complexes would suggestthat most of these complexes also contain the Hdj2 protein.Thus, these data support our interpretation that immunogoldEM indeed underestimated the presence of the target proteinin the protein-ori complexes.

We then examined the effect of chaperones on the associationof the E2 protein with the E1-ori complexes and, conversely,the effect of the E2 protein on the association of either chaperonewith the E1-ori complexes. We incubated E1, the p7874-99 oriplasmid, E2, and either chaperone in parallel experiments, whichwe then probed with primary antibodies to E2, Hsp70, or Hdj2along with the appropriate secondary immunogold conjugate. Interestingly,the frequencies of chaperone association with E1-ori complexeswere not affected in the presence of the E2 protein (Table 2).In contrast, the frequency of E2 association with E1-ori complexeswas significantly reduced to near background levels in the presenceof either chaperone (Table 2). Collectively, these data suggestthat chaperone proteins displace E2 protein from the E1-oricomplex and that this E2 displacement is the most likely mechanismfor E1 helicase reactivation. The alternative possibility, thatchaperones shields E2 from primary antibody, cannot explainthe reactivation of the DNA unwinding (Fig. 6, Table 1).

Finally, we examined the protein-ori complexes in an unwindingreaction containing both E2 and chaperones by immunogold EM.Three parallel experiments were conducted under the reactionconditions modified for EM. To each we added one of the primaryantibodies, followed by the appropriate secondary immunogoldconjugates. Only 2% (n = 265), 4% (n = 361), or 2% (n = 357)of the unwinding intermediates carried E2, Hsp70, and Hdj2 immunogoldconjugates, respectively, within the range of background labeling,when the protein or the primary antibody was omitted. We concludethat neither E2 nor chaperone remains associated with E1-oricomplexes in active unwinding complexes.

DISCUSSION

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Discussion

In this study, we demonstrate for the first time that HPV E1possesses a highly efficient DNA-unwinding activity on supercoiledsubstrates, generating U-form DNA (Fig. 1), which contains completelyunwound single-stranded molecules (Fig. 2). EM conducted undermodified conditions with reduced efficiency of unwinding revealedunwinding intermediates with two single-stranded loops extendingfrom a large protein complex on the DNA substrate (Fig. 2).This structure suggests that the HPV E1 dihexamer functionsas an integral helicase. We propose that each E1 hexameric subunitis an active helicase and that DNA on both sides of ori is unwoundwhile being translocated in an inward direction.

A bidirectional helicase is required for bidirectional HPV replication(4). Interestingly, unlike the SV40 T antigen, which has stronghelicase activities in unwinding circular or linear DNA andin strand displacement assays, DNA unwinding by HPV E1 is dependenton supercoiled DNA, as no U-form DNA was generated from eithera prerelaxed circular DNA or a linear DNA substrate detectableby ethidium bromide staining (Fig. 1 and 2). Thus, energy storedin supercoils helps the E1 dihexamer untwist duplex DNA to initiateunwinding. This observation would explain the poor helicaseactivities exhibited by HPV E1 proteins in strand displacementassays. We also showed that the E2 protein partially inhibitedthis E1 activity, but inhibition was abolished by Hsp70 andHdj2 (Fig. 4 and 6 and Table 1). A systematic EM examinationof the binding and unwinding reactions provided strong evidencethat chaperone proteins displace E2 from the E1-ori complexesand are themselves released during unwinding (Fig. 5, Table2).

In work by Fouts et al. (20), a bilobed BPV-1 E1 bound to oriwas observed, but the bidirectional unwinding intermediatescontained two single-stranded arms rather than two loops. Bothtypes of unwinding intermediates have been reported for theSV40 T antigen (15, 17, 42, 52, 61, 64). We believe that single-strandedarms were converted from single-stranded loops when the dihexamerdissociated during subsequent manipulations in those studies.The origin-binding protein of herpes simplex virus type 1, UL9,also forms binary complexes consisting of two dimers and inducesbidirectional DNA unwinding from oriS (39). This mode of DNAunwinding by a replicative helicase implies that bidirectionalDNA replication would occur on two expanding central loops.DNA translocation through centrally located replication machineryis far more favorable in terms of energy consumption and speedrelative to sliding the large replication machinery along theDNA strands. The commonly depicted Cairn's or ${theta}$ form replicationintermediates with divergently expanding arms on which bidirectionalreplication takes place would then be the result of deproteinationof the replication complexes.

The mutated HPV-11 E1 P479S in the putative ATP binding siteshows dramatically reduced ATPase activity (Fig. 3) (65). Itexhibits no discernible DNA-unwinding activity and supportscell-free HPV ori replication very poorly, in agreement withan analogous BPV E1 mutation (38, 55). Our EMSA and EM assaysfurther show that the defect stems from its poor ability tobind ori DNA (Fig. 3 and data not shown). Thus, binding of ATPmay have induced a conformational change in E1, allowing itto bind and assemble as a hexamer or dihexamer on the ori. Thisinterpretation is in agreement with our previous observationthat HPV-11 E1 binds to ori only in the presence of ATP, althoughATP hydrolysis is not necessary (33, 34), a property sharedwith SV40 T antigen (22, 42). In contrast, the BPV E1 proteinbinds to BPV-1 ori in the absence of Mg²⁺ and ATP, althoughATP greatly stimulates binding (47, 50). The HPV-11 E1 P479Swas previously reported to exhibit reduced ATPase activity (65)but nearly wild-type activity in transient replication (58).The discrepancy may have arisen from the use of PCR amplificationof a DNA fragment to demonstrate replication in that study ratherthan Southern blot hybridization to reveal full-length, newlyreplicated DNA (11, 59) or cell-free replication, as in thisstudy.

Replicative helicases are substrates of cyclin-dependent kinasesor other kinases that regulate cell cycle progression and initiationof DNA replication. For instance, the MCM proteins essentialfor initiating cellular DNA replication are phosphorylated (29,40). Thus, the issues of whether and how phosphorylation mayaffect helicase activity have been of considerable interest.SV40 T antigen purified from E. coli does not function properlyfor lack of phosphorylation (45). In particular, phosphorylationby cdk at T124 of T antigen purified from insect cells is critical,and a T124A mutation was shown to cripple the protein for dihexamerformation and ori unwinding (5, 43, 63). Both the BPV-1 andHPV-11 E1 proteins are also substrates of cyclin E/cdk2 andother cdk complexes in vitro and in vivo (14, 37). In particular,the cyclin E-cdk2 complex is critical for efficient HPV-11 orireplication in vitro (32, 37). Our results do not rule out thepossibility that E1 phosphorylation by cdk may modulate itsDNA-unwinding activity. However, they clearly demonstrate thatphosphorylation is not essential for dihexamer formation orfor efficient DNA unwinding. E1 phosphorylation must then affectits interactions with other proteins. This issue remains tobe investigated.

The papillomavirus E2 protein targets E1 to the viral ori. OurEM data suggest that, at the concentrations used, HPV E2 doesnot promote E1 dihexamer formation, nor does it disrupt E1 hexameror dihexamer formation on ori. Rather, E2 remains bound to theE1-ori complexes (Fig. 5) and inhibits the HPV E1 helicase (Fig.4). Since inhibition was observed with plasmids containing 0,1, or 3 copies of the E2 BS, we further suggest that unwindingof DNA on both sides of the ori might be a concerted reactionor that interactions between E2 and E1 or cellular proteinsalso adversely affect the E1 helicase. Indeed, E1 and E2 interactionshave been previously demonstrated for HPV-11 and BPV-1 proteinsin vitro (13, 35, 50, 68), consistent with their function inrecruiting E1 to the ori. In contrast to our observations, inhibitionof the BPV-1 E1 helicase by BPV-1 E2 has been attributed tointerference with E1 oligomer assembly on the ori (36). Also,unlike our results, in the presence of ATP and Mg²⁺, BPV-1 E2dissociates from the E1-ori complex (47). The reasons for thedistinctions between the HPV and BPV proteins are not understood.

Hdj2 associates with purified HPV-11 E1 expressed in insectcells and promotes dihexamer formation on ori (34). These observationswere reproduced in the present study with bacterially expressedE1 protein and were further substantiated by immunogold EM.We have now demonstrated by immunogold EM that Hsp70 also associateswith the E1-ori complex. Our EM data further suggest that chaperonescan displace E2 and reactivate an E2-inhibited E1 helicase andthat chaperones are then released during DNA unwinding (Fig.6, Tables 1 and 2). It is intriguing that, although chaperonesstimulate E1 hexamer and dihexamer formation on the ori, theyare not required for E1 helicase activity unless E2 is alsopresent (Table 1, Fig. 6). Nevertheless, the structure of theunwinding intermediates clearly suggests that DNA unwindingis associated with E1 dihexamers (Fig. 2). Either the E1 helicaseactivity originates from the low percentage of dihexamers thatformed in the absence of the chaperones, or there is anothermechanism by which dihexameric E1 assembles on the ori. Possibilitiesinclude an interaction of E1 with topoisomerase or RPA and therecruitment of a second hexamer upon initial limited DNA openingby the first hexamer. We further propose that, when E1 proteinconcentration is low, as it is in vivo, chaperone proteins mayindeed assist in the assembly of dihexamers on ori.

Based on previous and present reports, we propose a workingmodel for the role of chaperone proteins in the assembly andactivation of the E1 helicase on the HPV ori (Fig. 7). The E2protein targets E1 to the ori by virtue of its highly specificassociation with E2 BSs and its interaction with E1. Hsp70 mayfacilitate E1 binding to the ori by dissociating previouslyformed oligomers into monomers, which can then reassemble onthe ori as hexamers. Hsp40 promotes E1 dihexamer formation onthe ori. The E1 dihexamer then unwinds DNA bidirectionally bytranslocating DNA on both sides of the ori in an inward direction,generating two centrally located, single-stranded loops. Theloops are stabilized by RPA, while topoisomerase I removes theresultant positive supercoiling. However, the high-affinityassociation between E2 and E2 BSs may prevent the translocationand unwinding of DNA sequences beyond the E2 BSs that flankthe E1 dihexamer. An interaction between E2 and E1 or possiblya cellular protein(s) could also inhibit E1 activity (not illustratedfor simplicity).

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FIG. 7. Proposed model to illustrate the functions of E2 and chaperone proteins in the assembly and activation of the HPV E1 helicase on the origin. The E1 BS is flanked by E2 BSs. For simplicity, not illustrated is the E1/E2 interaction, which helps recruit E1 to the ori and may also contribute to inhibition of E1 helicase. The binding of E1 to ori, the activity of Hsp70, and DNA unwinding by E1 dihexamer all require ATP. The two single-stranded loops are shown to emerge from the center of the dihexamer. Not shown is the possibility that the two E1 hexamers each encircle the opposing single strands (as depicted in reference 20). Hsp70 and Hdj2 can independently displace E2 from the E2 BS. The stoichiometry of Hjd2 and E1 during the assembly of E1 dihexamer is not known. Upon completion of the unwinding in vitro or coupled DNA replication in vivo, chaperone proteins may promote E1 dihexamer disassembly from the substrate or template, and the process is then repeated on another ori DNA molecule.

In either case, DNA unwinding beyond the ori region occurs onlywhen E2 is released from the complex. Our data show that therelease can be accomplished by chaperone proteins, althoughother mechanisms cannot be excluded. Chaperones may also promoterecycling of E1 onto new substrates by dissociating E1 dihexamersinto monomers after each round of unwinding and replication(33). These activities of chaperones show similarities but alsodifferences with the known functions of E. coli DnaJ and DnaKduring replication of bacteriophages ${lambda}$ , P1, and P7 (2, 3, 18,66, 72).

To our knowledge, our study is one of a few examples that demonstratehow heat shock proteins can play a role in chaperoning the assemblyand activation of the unwinding activity of a replicative helicasein higher eukaryotes. We are not certain which members of thefamily of chaperones and cochaperones preferentially interactwith E1 and E2 proteins in vivo. The mechanisms by which chaperonespromote the assembly or disassembly of protein complexes arenot understood at present. Finally, because E1 protein is theonly enzyme encoded by papillomaviruses, an in-depth understandingof its function as well as a robust helicase assay are prerequisiteto the development of efficient antiviral compounds for thislarge family of medically important human pathogens.

ACKNOWLEDGMENTS

This research was supported by USPHS grants CA CA83679 to L.T.C.and T.R.B. and GM 31819 to J.D.G.

We thank Jim Champoux, Mike McDonnell, Richard Morimoto, T.Mohanakumar, and Douglas Cyr for sharing expression vectorsfor various cellular proteins.

The first two authors contributed equally to this work.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 1918 University Blvd, MCLM 510, Birmingham, AL 35294-0005. Phone: (205) 975-8300. Fax: (205) 975-6075. E-mail: LTChow{at}uab.edu.

REFERENCES

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