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Previous Article | Next Article 
Journal of Virology, May 2003, p. 5178-5191, Vol. 77, No. 9
0022-538X/03/$08.00+0 DOI: 10.1128/JVI.77.9.5178-5191.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Department of Biological Sciences, Boehringer Ingelheim (Canada) Ltd., Laval, Canada H7S 2G5
Received 26 September 2002/ Accepted 16 January 2003
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The life cycle of papillomaviruses is tightly coupled to the cellular differentiation program that occurs in the epithelium (for a recent review, see reference 52). These viruses infect the basal cell layer where they establish their small double-stranded DNA genome, 7.9 kbp in length, as a circular extrachromosomal element in the nucleus of infected cells. Maintenance of the viral genome in the infected cell is central to the life cycle of papillomaviruses and their associated pathologies. Hence, interfering with this process has been considered a valuable strategy for the development of antiviral therapeutic agents.
Maintenance of the viral genome in infected cells requires the activity of E1 and E2, the two viral proteins necessary for replication of the HPV genome in conjunction with the host cell DNA replication machinery (reviewed in reference 12). E1 is the replicative helicase of papillomavirus (reviewed in reference 56) and shares extensive amino acid sequence and functional homology with the related helicase, large T antigen, of simian virus 40 (SV40) and polyomavirus (14, 36). As an initiator protein E1 acts both as a DNA binding protein to recognize the viral origin of DNA replication and subsequently as a helicase to unwind the origin and the DNA ahead of the replication fork (22, 31, 49). Binding of E1 to an 18-bp inverted-repeat element within the origin (27, 39, 53) is facilitated by its interaction with E2 (5, 6, 7, 21, 22, 33, 34, 40, 44, 46, 48, 50, 65) a dimeric transcription-replication factor that binds with high affinity to sites located in the origin (reviewed in reference 38). The preferred binding site for bovine papillomavirus (BPV) E1 in complex with E2 is the hexanucleotide sequence 5'-ATTGTT-3'; four to six related sites are present in an overlapping fashion in the origin of many papillomaviruses (9). The E1-E2-DNA complex is comprised of an E2 dimer and two molecules of E1 (11) and serves as a starting point for the assembly of larger oligomeric E1 complexes, either hexamers or double hexamers, that have unwinding activity (20, 47). In vitro, E1 binds to DNA with little sequence specificity in absence of E2 but can nevertheless assemble into replication-competent oligomers at high protein concentrations. Hence, E2 serves both as a specificity factor to direct E1 to the origin and as a loading factor to recruit additional E1 monomers and favor their assembly. A role for heat shock proteins in stimulating the assembly of oligomeric E1 complexes has been reported (30).
Structure function studies on BPV and HPV E1 have been useful in dissecting the domain organization of this replicative helicase. The C-terminal domain of HPV11 E1 (amino acids 353 to 649; Fig. 1) was shown to be sufficient for oligomerization into hexamers that have ATPase and unwinding activity (58, 63). This enzymatic domain of HPV E1 is also responsible for interaction with the host polymerase alpha primase and, in a mutually exclusive manner, with the transactivation domain of E2 (2, 15, 37, 59, 66, 67). An additional interaction between E1 and E2 has been reported that involves the DNA binding domains (DBDs) of both proteins (4, 10, 11, 29, 41). This second interaction occurs only between the two proteins of BPV, and not those of HPV, and appears to be relevant only in the context of the BPV origin where an E1 binding site is juxtaposed close to an E2 binding site (5, 43). In this case the interaction between the E1 and E2 DBDs serves to trigger the interaction between the E2 transactivation domain and the E1 enzymatic domain (23).
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-helix, respectively, that together form a positively charged DNA-binding surface. The DNA-binding loop interacts on one strand with the first three nucleotides, ATT, of the hexanucleotide binding site 5'-ATTGTT-3', while the remaining three nucleotides on the opposite strand, AAC, are bound by the DNA-binding helix. The DNA-binding loop makes all the base contacts including the major ones with the thymidine at position 2 of the E1 binding site (19). Some distortion in the DNA occurs upon binding of a DBD dimer that becomes more pronounced upon binding of a second dimer and which ultimately may facilitate unwinding (19). The crystal structures also revealed a DBD dimer interface that was validated by mutagenesis. Specifically, two amino acid substitutions, V202R and A206R, in -helix 3, which forms part of this dimer interface, were shown to result in mutant proteins that bind DNA as monomers (19). The requirement and role of this dimer interface during replication is currently unknown. It has been hypothesized that each monomer within the initial E1 dimer may nucleate the assembly of an hexameric helicase, resulting in the formation of a replication-competent double hexamer bound at the origin (18). For HPV11 E1 previous studies indicated that the region corresponding to the minimal DBD identified in BPV E1 was required but not sufficient for binding to the origin (54, 58). In these studies, the C-terminal oligomerization/enzymatic domain was additionally required, perhaps because oligomerization of the protein stabilizes its interaction with the origin. Studies using purified recombinant HPV11 E1 helicase suggested that this protein binds DNA with even less sequence specificity than BPV E1 (17). However, this last study was performed with an E1 protein that was purified as preformed oligomers (17, 42) and which may bind to DNA differently than monomeric E1 or the isolated minimal DBD.
To determine if the region of HPV11 E1 corresponding to the minimal DBD identified in BPV E1 is capable of binding DNA in absence of the C-terminal enzymatic domain, we produced this domain of HPV11 E1 and characterized its DNA-binding activity using a quantitative assay based on fluorescence anisotropy. For comparison and to determine the generality of our findings, we similarly produced and characterized the minimal E1 DBD from BPV and from HPV18, a high-risk virus. We found that the HPV11 DBD binds DNA with an affinity and sequence specificity comparable to those of the analogous domain of BPV, but that the HPV18 DBD has a higher affinity for nonspecific DNA. For HPV11, we provide evidence that dimerization of the DBD increases its affinity for DNA and occurs only on binding to two appropriately positioned E1 binding sites. In contrast, the HPV11 E1 helicase purified as preformed hexamers binds to DNA with little sequence specificity. Finally, we have found that a deleterious amino acid substitution in the dimer interface, when introduced in the context of a functional E1 helicase, affects the ability of the protein to bind to the origin and support transient HPV DNA replication. These results are discussed in light of a model for the assembly of double hexamers at the origin.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To express the A251R mutant HPV11 E1 DBD, the plasmid encoding the wild-type protein was mutagenized with the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions and with the following pair of complementary oligonucleotides: 5'-CATAGCATAGCAGATCGATTTCAAAAGTTAATTG-3' and 5'-CAATTAACTTTTGAAATCGATCTGCTATGCTATG-3'.
Protein expression and purification. Expression vectors for the various GST-His-E1 DBD were introduced into Escherichia coli BL21 (Novagen). Bacterial cultures were grown in Luria-Bertani media at 37°C to an optical density of 0.5 (at 595 nm) and then induced for 6 h by the addition of 1 mM IPTG (isopropyl-ß-D-thiogalactopyranoside). Cells were then harvested and frozen at -80°C for later purification.
For each protein, a thawed pellet from a 500-ml culture was resuspended in 5 ml of lysis buffer (50 mM Tris [pH 7.6], 250 mM NaCl, 5 mM EDTA, 5 mM dithiothreitol [DTT], 10% glycerol, 0.1% NP-40, antipain [10 µg/ml], leupeptin [2 µg/ml], pepstatin [1 µg/ml], aprotinin [2 µg/ml]) and sonicated three times for 30 s each using a Tekmar Sonic Disruptor. The resulting cell lysate was cleared by centrifugation at 30,000 x g for 30 min and then incubated with 0.4 ml of glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) for 3 h at room temperature. Beads were washed with 50 bed volumes of HS buffer (50 mM Tris [pH 8.0], 1 M NaCl, 5 mM EDTA, 5 mM DTT, 10% glycerol) followed by 50 bed volumes of LS buffer (same as HS buffer but containing 0.2 M NaCl). Fusion proteins were either eluted at room temperature for 30 min in 1 bed volume of elution buffer (25 mM Tris [pH 8.0], 0.2 M NaCl, 1 mM EDTA, 5 mM DTT, 10% glycerol, 20 mM reduced glutathione [pH 8.0]) or cleaved with thrombin to release the polyhistidine-tagged DBD from the GST-moiety. For thrombin cleavage, bead-bound GST-His-DBD fusion proteins were resuspended in 1 bed volume of cleavage buffer (25 mM Tris [pH 8.0], 0.2 M NaCl, 1 mM EDTA, 5 mM DTT, 2.5 mM CaCl2, 10% glycerol) and incubated for 16 h at 4°C with 2.5 units of biotinylated thrombin (Novagen). The supernatant was then recovered, and thrombin was removed by incubation with 100 µl of streptavidin agarose beads (Novagen) for 30 min at 4°C. The soluble cleaved DBD was then recovered after centrifugation of the supernatant at 14,000 x g for 15 min at 4°C and stored at -80°C. Protein concentrations were determined by absorbance readings at 280 nm in 6 M guanidine hydrochloride using the following calculated molar extinction coefficients of each of the following proteins: GST fusion proteins, HPV11 72.7 mM-1 cm-1, HPV18 70.9 mM-1 cm-1, and BPV1 60.1 mM-1 cm-1; purified DBDs, HPV11 31.5 mM-1 cm-1, HPV18 29.8 mM-1 cm-1, and BPV1 18.9 mM-1 cm-1.
Expression and purification of the truncated HPV11 E1(72-649) helicase, lacking the N-terminal 71 amino acids of the enzyme, was described previously (63).
Electrophoretic mobility shift assays (EMSA). Binding reactions were performed at room temperature in a total volume of 10 µl and in the following buffer: 20 mM Tris (pH 7.6), 100 mM NaCl, 1 mM EDTA, 0.1% NP-40, bovine serum albumin (1 mg/ml), 1 mM DTT, and 10% glycerol. Reactions contained 7 nM radiolabeled probe and either 100, 50, 25 or 0 nM concentrations of GST-His-DBD protein. After 20 min samples were analyzed by electrophoresis on a 0.5x Tris-borate-EDTA (TBE) polyacrylamide (8%) gel and visualized by autoradiography.
To prepare the DNA probes, 2.5 µg of overlapping oligonucleotides were annealed by heating at 70°C for 5 min in 100 µl of 1 x One-Phor-All Buffer Plus (Amersham Pharmacia Biotech) and then slowly cooled to room temperature. To facilitate labeling, oligonucleotides were designed to generate duplex DNAs with four protruding nucleotides at both 5' ends. For labeling, 1 µg of annealed oligonucleotides was radiolabeled with 30 U of Klenow fragment in a 120-µl reaction mixture using the same buffer as for annealing but supplemented with 0.1 mM concentrations of dGTP, dATP, and dTTP and 100 µCi of [-33P]dCTP at 3,000 Ci/mmol. Labeling reactions were performed at room temperature for 2 h, and probes were purified using MicroSpin G-25 columns (Amersham Pharmacia Biotech) according to the instructions supplied by the manufacturer.
One probe was designed such as to contain two inverted E1 binding sites (E1BS) separated by 3 bp. A similar probe in which the two E1BS were each inactivated by two mutations was used as a control. The sequence of these two pairs of oligonucleotides was as follows (with the E1 binding sites underlined): 2E1BS, 5'-CCGGGCGGGATTGTTGCTAACAATGGGCG-3' and 5'-CCGGCGCCCATTGTTAGCAACAATCCCGC-3'; control probe, 5'-CCGGGCGGGTTTCTTGCTAAGAAAGGGCG-3' and 5'-CCGGCGCCCTTTCTTAGCAAGAAACCCGC-3'.
Fluorescence anisotropy DNA binding assay. Binding assays were performed in 96-well HTRF plates (Packard) using 10 nM fluorescein-labeled probe and the indicated concentrations of protein in 150-µl reaction mixtures using the following buffer: 20 mM Tris (pH 7.6), 50 mM NaCl, 1 mM EDTA, 0.02% NP-40, and 1 mM DTT. Fluorescence readings were taken on a POLARstar Galaxy plate reader (BMG LabTechnologies GmbH) with excitation and emission filters at 485 and 520 nm or on a Victor 2 1420 Multilabel HTS Counter (Wallac) using the 485 nm/535 nm filter sets. Background fluorescence from buffer was subtracted, and polarization and anisotropy values were defined as follows: P = (I|| - I[perp])/(I|| + I[perp]) and A = (I|| - I[perp])/(I|| + 2I[perp]), where I|| and I[perp] are the fluorescence intensities recorded in the parallel and perpendicular orientations respective to the orientation of the excitation polarizer.
Fluorescent DNA probes. High-pressure liquid chromatography-purified, fluorescein-labeled oligonucleotides were purchased from Genset (Paris, France), with the fluorophore attached at the 5' end by a six-carbon linker. Blunt duplex DNA probes were prepared by annealing each fluorescein-labeled oligonucleotide to a complementary oligonucleotide. The sequence of the different fluorescein-labeled oligonucleotides were as follows (the E1 binding sites are underlined; "F" indicates the position of the fluorescein moiety):
1 bp 2E1BS, 5'-F-GATTGTTGCTAACAATGGGC-3'; 2 bp 2E1BS probe, 5'-F-GGATTGTTGCTAACAATGGGC-3'; 4 bp 2E1BS probe, 5'-F-CGGGATTGTTGCTAACAATGGGC-3'; 2 bp proximal E1BS probe, 5'-F-GGATTGTTGCTAAGAAAGGGC-3'; 2 bp distal E1BS probe, 5'-F-GGTTTCTTGCTAACAATGGGC-3'; 0 E1BS probe, 5'-F-GGTTTCTTGCTAAGAAAGGGC-3'.
Competitor oligonucleotides. Unless stated otherwise, all duplex oligonucleotides that were used in competition experiments were designed similarly to those used in EMSA, so as to carry a 4-nucleotide overhangs at each 5' end. Oligonucleotides were designed to contain 1, 2, or no E1BS. Duplex oligonucleotides containing two and no E1BS, respectively, were the same as those used in EMSA (see above). The duplex DNA containing a single E1BS was made by annealing the following two oligonucleotides (with the E1 binding sites underlined and the inactivating mutations written in bold): 5'-CCGGGCGGGTTTCTTGCTAACAATGGGCG-3' and 5'-CCGGCGCCCATTGTTAGCAAGAAACCCGC-3'. In control experiments, we verified that the presence or absence of the 4 nucleotide overhang had no effect on the affinity of the different DBDs for their targets binding site, yielding identical Ki values (data not shown).
To investigate the effect of the position of the E1BS relative to the end of the DNA duplex (see Fig. 4A), blunt duplex oligonucleotides were used in which the two E1BS were located 0 to 6 bp away from the duplex end. The sequence of the top strand of these oligonucleotides was as follows (with the E1 binding sites underlined):
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Kd determination.
Kd values were obtained from direct binding isotherms, with each data point prepared as duplicates or quadruplicates, and fitted by nonlinear least-squares regression with the program GraFit 3.09b (Erithicus Software Ltd.) to the standard equation describing the following tight binding equilibrium, D + E 
DE (where D is duplex DNA, E is E1 DBD, and DE is DNA-DBD complex): 
A = Amax [(DT + ET + Kd) - {(DT + ET + Kd)2 - (4DTET)}1/2]/(2DT), where A is measured anisotropy gain at total concentrations of DBD (ET) and fluorescent probe (DT), Amax is anisotropy gain once probe is bound (Abound - Afree), and Kd is dissociation constant. No computational corrections for emission intensity were performed since the quantum yield did not change significantly upon binding of the DBD.
IC50 measurements and Ki determinations.
The 50% inhibitory concentrations (IC50) of various DNA competitors was measured in the fluorescence polarization assay. Competitors were titrated in binding reactions to reach 100% inhibition (each concentration point was performed in duplicate or quadruplicate, as indicated) in reactions performed at the indicated concentrations of protein and fluorescent probe. IC50 were determined by a nonlinear least-square regression fit of the inhibition curve using the SAS program package (software release 6.12; SAS Institute Inc., Cary, N.C.). Ki values were then calculated by using the standard equation that portrays the equilibrium EI I + E + D DE (where I is inhibitor):
P0 = Pmax ([Kd(1 + IC50/Ki) + DT + ET] - { [Kd(1 + IC50/Ki) + DT + ET]2 - (4DTET)}1/2)/DT, where Pmax is the polarization gain once probe is bound (Pbound - Pfree), P0 is the polarization gain in absence of inhibitor at the working concentrations of DBD (ET) and probe (DT) and Ki the dissociation constant of the inhibitor.
ATPase activity. ATPase activity of in vitro-translated E1 proteins was measured as described previously (63). Briefly, radiolabeled E1 proteins that were synthesized in vitro were immunoprecipitated using an anti-E1 antibody coupled to protein A beads. The ATPase activity of bead-bound immunopurified E1 was then assayed using an ammonium molybdate-malachite green colorimetric assay. ATPase activities above background levels were normalized to the amount of 35S-labeled E1 protein present in each immunoprecipitate, as determined by SDS-PAGE analysis and autoradiography.
Cross-linking of in vitro-translated E1. Oligomerization of in vitro-translated E1 was determined using a cross-linking assay as described previously (58). Briefly, 35S-labeled E1 protein was incubated in presence or absence of 50 ng/µl single-stranded DNA (60-mer, corresponding to nucleotides 7902 to 34 of the HPV11 origin) and then cross-linked with the sulfhydryl-reacting reagent bismaleimidohexane (BMH) (Pierce). Cross-linking was performed by diluting the binding reactions 13-fold with phosphate buffer (0.1 M pH 7.0) containing 100 µM BMH and then stopped after 1 min by addition of DTT to a final concentration of 2.5 mM. Cross-linked E1 proteins were then immunoprecipitated with an anti E1 antibody and analyzed by gel electrophoresis on 3% Weber-Osborn polyacrylamide gel (60) and autoradiography.
E1 DNA-binding coimmunoprecipitation assay. The DNA-binding activity of in vitro-translated E1 was assayed as described previously (58). Briefly, in vitro-translated E1 protein was incubated with a 33P-radiolabeled DNA probe comprised of two DNA fragments, one containing and the other lacking the minimal HPV11 origin of DNA replication. Binding reactions were allowed to proceed at the indicated temperature for 90 min. When indicated, ATP and MgCl2 were supplemented to the binding reactions at a final concentration of 5 and 3 mM, respectively. We previously noted that ATP-Mg is essential for binding when reactions are performed at 37°C. DNA-protein complexes were immunoprecipitated using an anti-E1 antibody coupled to protein-A Sepharose beads, and washed extensively. The coimmunoprecipitated DNA present in these complexes was then analyzed on a 5% polyacrylamide TBE gel and visualized by autoradiography.
Transient HPV DNA replication assay. This assay was performed essentially as described previously (58) but with the following modifications. Briefly, approximately one million CHO-K1 cells were transfected using Lipofectamine (Gibco BRL) with three plasmids encoding, respectively, E1 (pCR3-E1; 500 ng), E2 (pCR3-E2; 50 ng), and the minimal origin of DNA replication of HPV11 (pN9; 500 ng). At 4 h posttransfection, cells were treated with trypsin and seeded in 96-well plates (20 x 103 cells/well). Cells were harvested 48 h posttransfection, and total genomic DNA was isolated with the QIAmp blood kit (Qiagen). Replicated pN9 plasmid DNA was then detected by PCR amplification of an origin-containing fragment using DpnI-digested total genomic DNA as template and with the same primers as described previously (58). As a control, a fragment of the pCR3-E1 plasmid devoid of Dpn1 restriction sites was amplified in the same PCR using the following pair of primers that hybridize within the E1 open reading frame: 5'-ACCACATGTGCCGATTGGGTGGTTGCAGGA-3' and GCTGAAGGGTCACAGTCCACCGGGATGTT-3' (corresponding to nucleotides 1525 to 1554 and 2286 to 2258 of the HPV11 genome). The PCR conditions consisted of an initial denaturation step at 95°C for 5 min, followed by 21 rounds of sequential denaturation at 95°C for 30 s and extension at 72°C for 1.5 min, and ending with a final extension at 72°C for 10 min. In control experiments, we found that this low number of PCR cycles insures that amplification reactions for both fragments remain in the linear range (data not shown). PCR products were separated on a 1% TBE agarose gel and visualized by staining with the intercalating dye SYBRGreen I (Molecular Probes). The amount of replicated HPV (pN9) DNA was quantified by exposure on a Storm 860 Phosphorimager (Molecular Dynamics) and normalized to the amplified E1 signal. Transfection and detection of replicated pN9 plasmid were performed in quadruplicates.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We next set out to characterize the DNA-binding activity of the E1 DBD from HPV11, HPV18 and BPV in the absence of GST as a fusion partner, since GST is itself a dimer. The GST moiety was removed from the different fusion proteins by proteolytic cleavage. The resulting hexahistidine-tagged DBDs (Fig. 1B) were found to be monomeric in solution under the conditions used in DNA-binding reactions, when analyzed by size exclusion chromatography or analytical ultracentrifugation (data not shown). To better characterize the DNA-binding activity of these monomeric E1 DBDs, we then set out to develop a quantitative assay based on fluorescence anisotropy, which unlike EMSA allows for measurements to be performed in solution and at equilibrium (26, 32). These studies are presented below.
Measurement of the DNA binding activity of HPV11 E1 by fluorescence anisotropy. As a first step towards developing a DNA-binding assay based on fluorescence anisotropy, we measured the binding of the HPV11 E1 DBD to four different fluorescein-labeled duplex oligonucleotides. Three of the probes contained two inverted E1BS separated by three bp, but positioned 1, 2, and 4 bp, respectively, away from the fluorescein-labeled end of the duplex (Fig. 2A). The fourth probe was a control probe with both E1BS mutated. As can be seen in Fig. 2B, titration of the HPV11 E1 DBD resulted in a dose-dependent change in anisotropy for all four probes. The change in anisotropy was maximal for the probe with E1BS positioned 2 bp away from the fluorescein-labeled end of the duplex. From a series of binding isotherms similar to that shown in Fig. 2B, a dissociation constant (Kd) of 10 nM was obtained for the binding of the E1 DBD to this probe. In contrast, the DBD bound to the probe with E1BS located 1 bp away from the end of the duplex with a higher Kd of 50 nM and gave rise to a maximal change in anisotropy that was of intermediate value. Interestingly, binding of the E1 DBD to the probe with the two E1BS positioned 4 bp away from the end of the duplex consistently generated a biphasic curve. It is conceivable that the lower gain in anisotropy (20 mP) seen at low protein concentrations is due to specific binding of the DBD on the two E1BS, whereas the larger anisotropy change is due to nonspecific binding of additional DBD molecules to the probe. As anticipated the E1 DBD bound only weakly to the control probe indicating that its binding to DNA is sequence-specific. Collectively, these results indicate that the presence of the two E1BS and their distance relative to the fluorescein-labeled end of the duplex are critical parameters to detect binding of the HPV11 E1 DBD to the probe. A spacing of 2 bp was found to be optimal and hence was used in subsequent experiments.
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i) Position of the E1BS. To determine the effect of the position of the E1BS on affinity, we measured binding of the HPV11 DBD to duplex oligonucleotides carrying two E1BS sites positioned from 0 to 6 bp away from one end of the duplex (Fig. 4A). As anticipated the E1 DBD bound more weakly to sites located only 0 to 2 bp from the end of the duplex, than to sites located more distally. Hence the proximity of the E1BS to the end of the duplex does weaken binding. Therefore, oligonucleotides in which the E1BS are positioned at least 4 bp from the end of the duplex were used for all subsequent competition experiments. Finally, the fact that the affinity of the protein for sites located 2 bp away from the end of the duplex was comparable to the Kd value measured in Fig. 2 with a similar fluorescent probe, suggested that the fluorescein moiety does not have a substantial effect on binding.
ii) Affinity of the HPV11 wild type and A251R DBD. Next we used competition experiments to compare the affinities of the HPV11 DBD and A251R mutant derivative for DNA duplexes carrying two, one, or no E1BS (Fig. 4B). As expected the wild type protein showed an approximately 10-fold-higher affinity for two inverted E1BS, spaced by three nucleotides, than to a single site. In contrast, but as expected for a dimerization-defective protein, the A251R DBD had a similar affinity for one or two E1BS, which was comparable to that of the wild type protein for a single site. The affinity of both the wild type and mutant DBD for a single E1BS was only about two- to threefold higher than for nonspecific DNA.
iii) Affinity of the oligomeric E1 helicase. Next, we wished to compare the DNA-binding affinity of the DBD to that of a longer E1 protein with helicase activity. For these experiments we used a truncated form of E1 lacking the first 71 amino acids, E1(72-649), which we showed previously is hexameric in solution and retains both helicase and ATPase activity (63). Titration of purified E1(72-649) in presence of a fluorescent probe containing two E1BS resulted in a substantial change in anisotropy but the shape of the binding curve was inconsistent with a single binding site (data not shown), perhaps because of the oligomeric nature of the protein. Although this complicates deriving a true Kd value for this interaction and calculating Ki values for competitor oligonucleotides, it did not prevent the determination of the relative affinity of E1(72-649) for duplex oligonucleotides carrying either 1, 2 or no E1BS. In competition experiments, we found that E1(72-649) has a similar affinity for one or two E1BS (IC50 = 12 to 14 nM) and a slightly weaker affinity for nonspecific DNA (IC50 = 49 nM) (Fig. 4B). Hence, our hexameric E1 preparation binds DNA with little sequence-specificity, in contrast to the DBD.
Identification of a consensus binding sequence for HPV11 E1. To identify a consensus binding sequence for a dimer of the HPV11 E1 DBD, we performed a systematic mutational analysis of the hexanucleotide E1BS, 5'-ATTGTT-3'. In these experiments, we used as competitors a series of mutated DNA duplexes containing two E1BS (Fig. 5A). Each position of the E1BS was mutated to the other three possible nucleotides and each mutation was introduced in both E1BS simultaneously. The three-nucleotide spacer region between the two E1BS was also mutagenized in a similar way. Results shown in Fig. 5B indicated that positions 1, 2 and 4 of the E1BS could not be mutated without affecting binding. In contrast some mutations at positions 3, 5 and 6 were tolerated. Specifically, a less than a twofold loss in binding affinity was observed when the T residue at position 3 was mutated to A or G or when the two T residues at positions 5 and 6 were changed to a C. From these results, the following consensus E1BS was obtained: 5'-AT(A/G/T)G(C/T)(C/T). As for the spacer region, we found it to be very tolerant to mutations. All three nucleotides could be changed to any of the other three possible nucleotides with a <2-fold loss in affinity (Fig. 5B).
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The consensus binding sequence for a dimer of the E1 DBD described above is present in two overlapping copies in the origin of HPV11 and of other HPV types (9). In the HPV11 origin these two copies are comprised, respectively, of E1BS 1 and 3, and E1BS 2 and 4 (Fig. 6A). Competition experiments were used to measure the affinity of the HPV11 E1 DBD for a combination of either E1BS 1 and 3 or E1BS 2 and 4 or for the origin. As indicated in Fig. 6B, the binding sequence comprised of E1BS 1 and 3 had the weakest affinity, comparable to that for a single consensus E1BS. In contrast, the combination of E1BS 2 and 4 was nearly as good a competitor as the origin sequences or two consensus E1BS. This was somewhat surprising since E1BS 2 carries a G at position 4, which should be detrimental to binding. However, it has been observed for BPV that the presence of a deleterious G at position 4 can be compensated for by a thymidine at position 3 (19), as is the case for E1BS 2. As suggested by Enemark et al. (19), such compensatory phenomenon is consistent with some mutations having more of a structural effect on DNA such as affecting its ability to become distorted.
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(i) Affinity for 2, 1 or no E1BS. As indicated in Fig. 7, under standard assay conditions (i.e., 50 mM NaCl), the affinities of the three DBDs for two E1BS were comparable. In contrast, the HPV18 DBD showed a substantially higher affinity for a duplex DNA containing either one or no E1BS than the other two DBDs. In an attempt to reduce the higher nonspecific DNA-binding of the HPV18 DBD, these experiments were repeated at a higher salt concentration (100 mM NaCl). As anticipated, increasing the salt concentration reduced the affinity of all three DBDs for any given duplex DNA. However, the HPV18 DBD still retained its higher affinity for nonspecific DNA compared to the other two DBDs.
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Effect of the A251R substitution on the replication functions of HPV11 E1. The quantitative binding affinity measurements presented above suggest that the A251R substitution affects specifically the dimerization of the HPV11 DBD but not the affinity of a DBD monomer for DNA. To further characterize the effect of this substitution on the replication activities of E1, we tested its effect on the ATPase, oligomerization and origin-binding activities of the protein in vitro. These experiments were performed with in vitro-translated E1(72-649). We showed previously that this truncated protein, lacking the N-terminal 71 amino acids, is as active as wild type E1 in supporting cell-free HPV DNA replication but unlike the full-length protein binds more readily to the viral origin in vitro (1, 58). We also tested the effect of the A251R substitution on the ability of full-length E1 to support transient HPV DNA replication in vivo. These results are presented below.
(i) ATPase activity. ATPase activity of in vitro-translated E1 was measured using a colorimetric assay and following immunoprecipitation of the protein from the translation reaction, as described previously (63). In this assay, the A251R mutant protein was found to be almost as active as the wild type protein (Fig. 8A). The previously characterized and catalytically inactive K484R mutant E1 protein (63) was used as negative control in this experiment.
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(iii) Origin binding. To test for origin binding we used a previously described DNA coimmunoprecipitation assay (58) in which in vitro-translated E1 is first incubated with two radiolabeled DNA fragments, one containing and the other lacking the origin. E1-DNA complexes are then immunoprecipitated with an anti-E1 antibody and the coprecipitated DNA analyzed by gel electrophoresis and autoradiography. As can be seen in Fig. 8C, the A251R mutant protein showed a reduced ability to bind to the origin compared to wild type E1, a result suggesting that the DBD-dimer interface is required for stable binding to the origin in this assay. The previously characterized K484R and Y380A mutant E1 proteins (58) were used as negative controls in this experiment. As observed previously, we found that the K484R mutant protein retains some activity at 23° that is lost at 37°C (58).
(iv) Transient HPV DNA replication. A cotransfection assay was used to measure the ability of the A251R mutant E1 to support transient HPV DNA replication. In this assay, constructs expressing E1 and E2 were transfected into CHO cells along with a plasmid carrying the HPV11 minimal origin of DNA replication. 48 h post transfection, the amount of replicated DNA (Dpn1 resistant) was quantified by PCR. In this assay, the A251R mutation greatly reduced the ability of E1 to support HPV DNA replication (Fig. 8D), consistent with the results presented above that this mutation has a deleterious effect on origin-binding in vitro. The two DNA-binding defective E1 proteins (K286A/R288A and A292L/R293E [58]) were used as negative controls in this experiment.
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DISCUSSION |
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In contrast to hexameric E1, the HPV11 DBD shows a greater than 20-fold higher affinity for two appropriately positioned E1BS (Ki = 4 nM) than for nonspecific DNA (Ki = 96 nM). The affinity of the DBD for a single E1BS (Ki = 48 nM) however is only two- to threefold higher than for nonspecific DNA, and in that respect the DBD behaves similarly to hexameric E1. These results suggested that dimerization of the DBD is critical to achieve both sequence-specific and high-affinity binding. Characterization of a mutant HPV11 DBD carrying a deleterious substitution, A251R, in the dimer interface provided additional evidence for the role of dimerization. In particular the following observations suggested that dimerization occurs only on binding of the DBD to two appropriately positioned E1BS. First, in solution, the DBD is monomeric at concentrations of up to 4 mg/ml (data not shown), the highest concentration tested, indicating that it does not dimerize readily in absence of DNA. Second, the binding of the DBD to a fluorescent probe containing a single E1BS generated a maximal change in anisotropy that was smaller than that observed with a probe containing two E1BS, but similar to what is observed for the binding of the dimerization-defective A251R DBD to either probes. The simplest interpretation of these results is that the lower gain in anisotropy results from the binding of a single DBD to the probe whereas the larger one results from dimerization of the DBD on two E1BS. Third, the affinity of the wild type DBD for a single E1BS (Ki = 45 nM) is comparable to that of the A251R mutant (Ki = 59 nM) and both proteins have comparable affinities for nonspecific DNA (KI = 113 and 135 nM, respectively). If the wild type DBD could dimerize readily in absence of E1BS, we would have expected its affinity for nonspecific DNA to be significantly higher than that of the A251R mutant protein. Fourth, the length of the spacer region between two E1BS is critical for the affinity of the wild type DBD (Fig. 5) but has little effect on that of the A251R mutant protein. Altogether, the results presented above indicate that dimerization of the DBD increases both sequence-specificity and affinity for its target site. In agreement with this, we found that the HPV11 DBD has a substantially higher affinity for two E1BS when artificially made into a dimer by fusion with GST, than as a monomeric protein (Ki of 2 and 80 nM, respectively; compare Fig. 7).
Our comparison of the HPV11 and BPV DBDs revealed that their affinities for DNA are very similar (Fig. 7), with both showing a 10- to 20-fold higher affinity for two appropriately positioned E1BS than for nonspecific DNA. Both proteins also show very little preference for a single E1BS compare to nonspecific DNA. This is particularly striking for the BPV DBD which shows a <2-fold difference in affinity for duplex DNAs containing one or no E1BS (Fig. 7), even as a dimeric GST-fusion protein (Fig. 7). Again, these results reinforce the notion that dimerization of the DBD on two E1BS is critical for formation of a stable DBD-DNA complex. From a biological standpoint, the fact that formation of a stable dimer occurs only on two E1BS and that the DBD monomer has little affinity for a single E1BS would help ensure that monomeric E1 does not get trapped on binding sites located outside of the origin.
The similarity between the HPV11 and BPV E1 DBD also extends to their sequence requirement. Indeed, our mutational analysis of the E1BS indicated that the sequence-specificity of the HPV11 E1 DBD is nearly identical to that previously published for the BPV E1 DBD (9). This is despite the fact that the study for BPV was performed in the presence of the E2 DBD, although earlier studies have suggested that the sequence recognition of BPV E1 is not affected by its interaction with E2. As mentioned in the introduction, the interaction between the respective DBD of E1 and E2 does not appear to exist for HPV11 and, accordingly, we have been unable to detect an interaction between the HPV11 E1 DBD and E2 by EMSA on an origin-containing DNA probe (data not shown). To further characterize the sequence-specificity of the HPV11 DBD, we examined the effect of mutations within the spacer region between two inverted E1BS. These studies revealed that a spacing of any 3 bp was required for high affinity binding.
In contrast to the BPV and HPV11 E1 DBDs, we found that the analogous domain of HPV18 has a higher affinity for nonspecific DNA and shows less of a sequence-preference, even as a GST-fusion protein. This suggests that the HPV18 DBD either can dimerize more readily on DNA or that each monomer makes stronger contacts with the DNA backbone, or both. Further studies will be required to distinguish between these possibilities. Nevertheless, these findings reveal that there can be subtle differences in how the E1 proteins from different papillomavirus types interact with their cognate origin. It was previously shown for HPV18 that two E2 binding sites alone are sufficient to function as a minimal origin of replication (55). It is likely that the lack of sequence specificity of HPV18 E1 contributes to promoting DNA replication in absence of consensus E1BS for this HPV type.
In this study, we examined for the first time the effect of a deleterious amino acid substitution in the DBD dimer interface on the replicative activities of E1. We found that the A251R substitution does not affect the ATPase activity of E1 nor its ability to oligomerize in presence of single stranded DNA. However, this substitution did reduce binding of E1 to the origin in vitro and its ability to support transient HPV DNA replication in vivo, but did not abolish completely either of these activities. Hence these results suggest that the interaction between DBDs is important but not essential for E1 to bind to the origin in vitro or for DNA replication in vivo. Enemark et al. (18) hypothesized that each monomer within the initial E1 dimer could nucleate the assembly of an hexameric helicase. Our findings that the A251R substitution affects neither the ability of the protein to assemble into hexamers in a cross-linking assay nor the interaction of the monomeric DBD with DNA per se, but specifically its ability to dimerize, are entirely consistent with this proposal. Hence it might be instructive to consider our results in light of a model of the assembly of double-hexameric E1 complexes that involves the interaction between DBDs (Fig. 9). Based on our findings that the DBD dimerizes only upon binding to two appropriately positioned E1BS, we propose that a major function of the E1BS within the viral origin is to favor dimerization of E1, through the DBD, such as to stabilize its initial binding to the origin. Key to this process is the fact that monomeric E1 is recruited to the origin by E2, such that the local E1 concentration at the origin is sufficiently high to favor dimerization. For HPV11, a dimer of E1 would probably assemble first on the high affinity E1BS 2 and 4 followed by the assembly of a second one on the lower affinity E1BS 1 and 3. Formation of these two E1 dimers would then act as a starting point for the assembly of double-hexamers, in a process that is likely to require additional E1-E1 interactions, such as those involved in oligomerization of the C-terminal enzymatic domain, and be stimulated by ATP. For the A251R mutant protein, interaction within the initial dimer cannot occur and as a result, binding of the first four E1 molecules to the origin would neither be cooperative nor involve all four E1BS. This mutant protein could still assemble into hexamers as suggested by this study, but these would be prevented from interacting with each other to form a double hexamer. If this model were correct, the fact that the A251R mutant protein has residual activity would indicate that the interaction between E1 monomers leading to the assembly of double hexamers is not essential for DNA replication but contributes to its overall efficiency. The transient HPV DNA replication assay used in these experiments probably measures no more than two rounds of DNA synthesis. It is conceivable that the interaction between DBDs may be more critical for the long-term maintenance of the viral genome in infected cells, which requires multiple rounds of DNA replication.
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primase. J. Biol. Chem. 274:2696-2705.
