Purification and biochemical characterization of the E1 replication initiation protein of the cutaneous human papillomavirus type 1

Saifuddin Sheikh, Gerald Van Horn, Asma Naqvi, Laura Sheahan and Saleem A. Khan

Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA

Correspondence
Saleem Khan
khan{at}pitt.edu


   ABSTRACT
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
The E1 and E2 proteins encoded by papillomaviruses are required for viral DNA replication. Although E1 is the replication initiator protein, previous studies have shown that the full-length E1 protein binds to the origin weakly and with low sequence specificity. The E2 protein facilitates binding of the E1 protein to the origin, triggering the initiation of replication. The E1 protein contains ATPase, helicase and DNA unwinding activities. In vivo studies with mucosal human papillomavirus (HPV) types 11 and 18 have shown that while E1 is absolutely essential for replication, the E1 binding site is dispensable. However, both the E2 protein and E2 binding sites are required for their replication. In contrast to these HPVs, transient replication of HPV type 1, which infects cutaneous tissue, requires only the viral E1 protein and E1 binding site. To understand the basis for these differences, we have overexpressed and purified the HPV-1 E1 and E2 proteins and studied their biochemical properties. The purified E1 protein was shown to have an ATPase activity with a very low Km value, similar to that of the SV40 large T antigen. The E1 protein bound to the HPV-1 origin in the absence of the E2 protein and without the use of any cross-linking agents. Our results suggest that the ability of the HPV-1 E1 protein to initiate DNA replication in vivo in the absence of the E2 protein may be due to its stable interaction with the HPV-1 origin.


   Introduction
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Human papillomaviruses (HPVs) are double-stranded DNA viruses that cause benign and malignant lesions of epithelial cells. HPVs are further subdivided into mucosotropic or cutaneous types depending on the nature of the tissue they infect. The mucosal HPV types, such as 6 and 11, are classified as low-risk viruses, while others, such as 16 and 18, are classified as high-risk viruses that cause cancers, predominantly cancer of the cervix (zur Hausen & de Villiers, 1994). In vivo replication of bovine papillomavirus type 1 (BPV-1) and several HPVs is known to require both the viral E1 and E2 proteins (Chiang et al., 1992; Chow & Broker, 1994; Del Vecchio et al., 1992; Lambert, 1991; Stenlund, 1996; Ustav & Stenlund, 1991). The origin of replication (ori) of various papillomaviruses (PVs) contains one E1 binding site (E1BS) and several E2 binding sites (E2BSs) (Chow & Broker, 1994; Gilette & Boroweic, 1998; Gilette et al., 1994; Holt & Wilson, 1995; Holt et al., 1994; Stenlund, 1996; Sun et al., 1996). Recent studies with BPV-1 have suggested that the E1BS may contain several overlapping E1 binding sequences that may participate in a step-wise assembly of a multimeric E1 protein–ori complex (Chen & Stenlund, 2000, 2001). Studies with BPV-1 and several HPVs have shown similarities as well as differences in the replication properties of PVs. For example, while the in vivo replication of mucosotropic HPVs 11 and 18 requires both the E1 and E2 proteins, plasmids containing two or more E2BSs but lacking an E1BS are competent for replication (Chow & Broker, 1994; Lu et al., 1993; Sverdrup & Khan, 1994, 1995). Thus, the E1BS is not absolutely required for their replication, implying that the E2 protein must target E1 to the DNA through its interaction with the E2BSs contained within the ori (Berg & Stenlund, 1997; Bonne-Andrea et al., 1997; Chow & Broker, 1994; Frattini & Laimins, 1994a, b; Gilette et al., 1994; Winokur & McBride, 1996; Yang et al., 1991). Since the E2BSs are conserved in the ori of various PVs, the E1 and E2 proteins from one PV type can support replication from a heterologous PV ori (Chow & Broker, 1994; Stenlund, 1996). In contrast to the results obtained with HPVs 11 and 18, replication of the cutaneous HPV type 1 is unique in that the E1 protein alone can support in vivo replication of ori plasmids (Gopalakrishnan & Khan, 1994). Furthermore, the HPV-1 E1 protein can support replication of plasmids containing an E1BS but lacking E2BSs (Gopalakrishnan & Khan, 1994; Gopalakrishnan et al., 1995). These results support the idea that HPV-1 E1 is capable of a specific interaction with the ori in a stable manner, resulting in the initiation of replication.

The E1 proteins of PVs have ATPase, helicase, origin binding and unwinding activities and it is assumed that they play a similar role during replication of different PVs (Jenkins et al., 1996; Mansky et al., 1997; Muller et al., 1997; Rocque et al., 2000; Santucci et al., 1995; Sedman & Stenlund, 1998; Seo et al., 1993a, b; Stenlund, 1996; Thorner et al., 1993; Wilson & Ludes-Myers, 1991; Yang et al., 1993). Biochemical studies with the full-length E1 proteins of BPV-1 and HPVs 6b, 11, 16, 31b and 33 have shown that either they do not bind to the ori, or they bind very weakly and with low specificity (Bream et al., 1993; Chen & Stenlund, 2001; Dixon et al., 2000; Leng et al., 1997; Liu et al., 1995, 1998; Masterson et al., 1998; Muller & Sapp, 1996; Muller et al., 1997; Rocque et al., 2000; Sanders & Stenlund, 2000). However, in the presence of the viral E2 protein, ori binding by E1 becomes more efficient and specific (Berg & Stenlund, 1997; Mohr et al., 1990; Sarafi & McBride, 1995; Woytek et al., 2001). Thus, the detection of full-length E1–ori complexes by electrophoretic mobility-shift assays (EMSAs) requires the use of cross-linking agents, which is consistent with the weak DNA binding activity of E1 (Chen & Stenlund, 1998, 2001; Gonzalez et al., 2000; Liu et al., 1995, 1998). On the other hand, truncated derivatives of the BPV-1 E1 protein containing its DNA binding domain bind to the ori stably, and this binding can be detected by EMSA in the absence of any cross-linking agents (Berg & Stenlund, 1997; Chen & Stenlund, 1998, 2001; Gonzalez et al., 2000; Leng et al., 1997; Liu et al., 1995, 1998). The binding of the full-length E1 proteins of BPV-1 and some HPVs to specific sequences within the ori has also been demonstrated by footprint analysis (Chen & Stenlund, 1998, 2001; Frattini & Laimins, 1994a, b; Gilette & Boroweic, 1998; Holt et al., 1994; Muller et al., 1997; Sanders & Stenlund, 2000). The following model has emerged from a number of recent studies. The E2 protein targets E1 to the ori, resulting in the formation of an E1–E2–ori complex. In the case of BPV-1, this complex is subsequently converted to a stable E1–ori complex, which lacks the E2 protein, has a larger size and contains a higher multimeric form of E1 (Berg & Stenlund, 1997; Gilette & Boroweic, 1998; Lusky et al., 1994; Sedman & Stenlund, 1995; Stenlund, 1996). This multimeric E1–ori complex is postulated to be the substrate for the initiation of BPV-1 replication. However, in the case of HPV-11, the E1–E2–ori complex does not appear to be a precursor for an E1–ori complex and it is possible that the E1–E2–ori complex may be competent for initiation (Chao et al., 1999).

Previous studies in our laboratory have shown that the HPV-1 E1 protein is sufficient for the transient replication of ori plasmids, suggesting that a stable E1–ori complex is formed that is competent for the initiation of replication. To find biochemical support for this model, we have purified the HPV-1 E1 and E2 proteins as fusion proteins, with the FLAG peptide as an epitope tag. EMSAs showed that the E1 protein bound to the ori region containing the E1BS in the absence of any cross-linking agents. In vitro-translated, native E1 protein also bound to the ori. In the presence of both the E1 and E2 proteins, an E1–E2–ori complex was also observed. The E1 protein was shown to have a DNA-independent ATPase activity with a Km value that was comparable with that of the SV40 large T antigen. Our results suggest that the E1 protein of HPV-1 (and possibly other related HPVs) may be capable of supporting in vivo replication of ori plasmids in the absence of E2 due to a stable interaction with the HPV-1 origin.


   Methods
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Plasmids and cells.
The Sf-21 insect cell line was used to express the HPV-1 E1 and E2 proteins. The cell line was maintained in Grace's complete suspension culture medium containing 4 % foetal bovine serum and 1 % Pluronic F-68. To facilitate purification of the HPV-1 E1 and E2 proteins, plasmids encoding in-frame fusions of two tandem copies of the FLAG peptide (DYKDDDDK) at the N-terminal ends of these proteins were generated. Synthetic oligonucleotides with BamHI ends and containing the initiation ATG codon and encoding two tandem copies of the FLAG epitope were ligated into the unique BamHI site of a derivative of the mammalian expression vector pSG5, which contains two NotI sites flanking the BamHI site (Gopalakrishnan et al., 1999). The E1 and E2 open reading frames of HPV-1 were obtained by PCR amplification of HPV-1 DNA using primers containing BamHI ends such that they generated E1 and E2 cDNAs starting from their second codons. The PCR-amplified DNAs were cloned into the BamHI site of the above vector. The FLAG–E1 and FLAG–E2 proteins were found to be biologically active in a transient HPV replication assay and their levels of activity were similar to those of the native E1 and E2 proteins (data not shown). For protein overexpression in insect cells, the FLAG–E1 and FLAG–E2 cDNAs were isolated as NotI fragments from the above plasmids and ligated into the NotI site of the baculovirus expression vector pFASTBAC1 (Life Technologies). This places the E1 or E2 gene downstream of the baculovirus polyhedrin promoter. The ligation mixtures were used to transform E. coli DH10 BAC cells, which were plated on nutrient agar containing X-Gal, IPTG, kanamycin, tetracycline and gentamicin according to the manufacturer's instructions. Three white colonies were picked, plasmid minipreparations were made and the presence of the appropriate E1 or E2 gene was confirmed by PCR.

Expression and purification of the E1 and E2 proteins.
Bacmid DNAs were transfected into Sf-21 cells using Cellfectin (Life Technologies) and expression of the FLAG–E1 and FLAG–E2 proteins was confirmed by Western blot analysis, as described below. To isolate the recombinant baculovirus expressing the E1 and E2 proteins, supernatant from cells grown for 48 h after transfection with bacmid DNAs was collected. The titre of the viral stock was determined by a plaque assay, as recommended by the supplier. Various conditions were tested for optimal expression of the E1 and E2 proteins by isolating protein lysates from infected cells, followed by SDS-PAGE and Western blotting using the anti-FLAG M2 monoclonal antibodies. The HPV-1 FLAG–E1 and FLAG–E2 proteins were optimally expressed at 24 and 72 h, respectively. The optimum m.o.i. was 1 for E1 and 2 for the E2 protein. Large-scale cultures of insect cells expressing the HPV-1 E1 and E2 proteins were obtained from the National Cell Culture Center (Minneapolis, MN, USA). For purification of the FLAG–E1 protein, the cell pellet was thawed and suspended in buffer A [50 mM Tris/HCl, pH 8·0, 1 mM EDTA, 1 mM DTT, 0·15 M NaCl, 10 % glycerol and Complete (Boehringer Mannhein) protease inhibitor (1 tablet in 50 ml extraction buffer); the tablet contains EDTA, aprotinin, leupeptin and Pefabloc SC]. The cells were lysed by sonication at a continuous cycle for about 8 min with 30 s intervals after each 2 min. The supernatant containing the E1 protein was collected by centrifugation (at least 3 h at 37 000 r.p.m. in an SW41 rotor), and the protein was precipitated by the addition of ammonium sulfate to a final concentration of 40 %. At this cut-off range, most of the E1 protein was precipitated. The precipitated proteins were resuspended in buffer A and loaded directly on to a DEAE–Sepharose column (pre-equilibrated with buffer A) to remove the DNA associated with the proteins. The column was washed with two bed volumes of buffer A. The wash fractions containing unbound proteins free of DNA were pooled. The A280/A260 ratio was close to 1 for the E1 protein preparations. Western dot blots were performed to identify the fractions containing the E1 protein. Fractions containing E1 were pooled and dialysed for at least 2 h at 4 °C with one change of buffer. The pooled fraction was centrifuged to remove any debris and loaded on to 2–3 ml of an affinity column containing anti-FLAG M2 monoclonal antibody coupled to Sepharose, according to the manufacturer's protocol (Sigma). The effluent was passed through the column at least three times to enhance retention of the E1 protein on the column. The column was then washed several times with buffer B (as buffer A but containing 1 M NaCl in place of 0·15 M) to remove proteins that were bound non-specifically to the resin. The FLAG–E1 protein was then eluted with buffer A containing 10 mM FLAG peptide, and 0·5 ml fractions were collected. Aliquots of these fractions were analysed by SDS-PAGE to identify the fractions containing the E1 protein. Western blot analysis was used to confirm the presence of the E1 protein in these fractions. The fractions containing the purified E1 protein were pooled, concentrated and dialysed in buffer A without the protease inhibitor. The FLAG–E1 protein was stored in small aliquots at -80 °C. The procedure for the purification of the FLAG–E2 protein has been described elsewhere (Van Horn et al., 2001). The MBP–E2 fusion gene was constructed by PCR amplification of the E2 cDNA using primers with EcoRI ends followed by ligation into the pMALC2 expression vector (New England Biolabs). The MBP–E2 fusion protein was expressed in E. coli by IPTG induction,as suggested by the manufacturer. Induced cells from a 500 ml culture were resuspended in 15 ml of buffer C (50 mM Tris/HCl, pH 8·0, 1 mM EDTA, 1 mM DTT, 100 mM NaCl and 10 % glycerol) and lysed by treatment with lysozyme (1 mg ml-1) for 30 min at 4 °C. The cell lysate was subjected to two freeze/thaw cycles and sonication at a continuous cycle twice for 90 s bursts. The cell debris was removed by centrifugation for 20 min at 15 000 r.p.m. in an SS34 rotor. The supernatant was collected and passed through an affinity column containing amylose–Sepharose resin. The column was washed with two column volumes of buffer C and the MBP–E2 protein was eluted with buffer C containing 15 mM maltose. The fractions were checked for the presence of the MBP–E2 protein by SDS-PAGE on a 10 % gel followed by staining with Coomassie brilliant blue. The amount of E1 and E2 protein in purified fractions was estimated using the Bio-Rad protein assay kit, with BSA as the standard.

Determination of the ATPase activity of the E1 protein.
ATP hydrolysis was measured in a 50 µl reaction mixture containing 50 mM Tris/HCl, pH 8·0, 10 mM MgCl2, 1 mM DTT and 0·03–10 µM [{alpha}-32P]ATP (3000 Ci mmol-1). After incubation at 37 °C for 60 min, the reaction was stopped by addition of EDTA to a final concentration of 100 mM. An aliquot of the reaction was spotted on to a polyethyleneimine–cellulose thin-layer plate, which was developed with 0·5 M potassium phosphate buffer (pH 3·5). After TLC, the plates were dried and subjected to autoradiography for 30 min at 25 °C. The products were quantified using a Phosphorimager (Molecular Dynamics).

In vitro synthesis of the E1 protein.
The pSG5 expression vector containing the HPV-1 E1 gene downstream of the T7 RNA polymerase promoter (Gopalakrishnan & Khan, 1994) was used for the in vitro synthesis of native, unfused E1 protein using the TNT Quick Coupled Transcription/Translation System (Promega). Reactions were carried out according to the manufacturer's protocol with some modifications. Briefly, 20 µl rabbit reticulocyte lysate (TNT Master Mix) was mixed with 1 µg DNA and 1 µl [35S]methionine (1175 Ci mmol-1; ICN) in a total volume of 25 µl. Reactions were incubated at 30 °C for 90–120 min. The samples were run on 10 % polyacrylamide gels (29·2 : 0·8, acrylamide : bis-acrylamide). Before drying, gels were soaked in 30 % methanol and 10 % acetic acid for 15 min. The gels were soaked for an additional 15 min in the Enhancer solution (NEN). The gels were rinsed three times with water, placed on a piece of Whatman paper, dried for 2 h at 90 °C and subjected to autoradiography. Non-radioactive E1 protein was synthesized as above except that the labelled methionine was replaced with 1 µl 1 mM cold methionine supplied with the TNT kit.

DNA binding experiments.
The 171 bp optimal HPV-1 ori (ori171) containing one putative E1BS, one high-affinity E2BS and one low-affinity E2BS was isolated by digesting the pori171 plasmid (Gopalakrishnan & Khan, 1994) with HindIII and BamHI. A 60 bp oligonucleotide containing the E1BS and the surrounding AT-rich region (ori60) was chemically synthesized (Life Technologies). A 244 bp origin mutant fragment was isolated by digesting the pori312 plasmid with HindIII and HpaI (Gopalakrishnan & Khan, 1994). This fragment contains HPV-1 nt 7593–7815/1-3 and disrupts the putative E1BS at the HpaI site. An unrelated 166 bp fragment containing an AT-rich region that includes the origin of replication of plasmid pT181 (Koepsel et al., 1986) was used as a negative control. EMSAs were performed by labelling the ori DNAs using 50 µCi [{gamma}-32P]ATP (3000 Ci mmol-1) and T4 polynucleotide kinase (Sambrook et al., 1989). DNA binding reactions contained 25 mM Tris/HCl, pH 8·0, 7 mM MgCl2, 1 mM DTT, 5 % glycerol (v/v), 45 or 200 ng poly(dI·dC), labelled DNA and FLAG–E1 and/or FLAG–E2 (or MBP–E2) protein. Some EMSA reactions also included NP-40 at a final concentration of 0·1 % (v/v). The reactions were incubated at room temperature for 20 min and the DNA–protein complexes analysed by electrophoresis on 5·5 or 6 % native polyacrylamide or 1·2 % agarose gels (Sambrook et al., 1989). For EMSA with native in vitro-synthesized E1, rabbit reticulocyte lysates containing in vitro-translated, non-radioactive E1 protein were incubated with the labelled DNA probes as above and subjected to electrophoresis on 1·2 % agarose gels.


   Results
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Overexpression and purification of the HPV-1 E1 and E2 proteins
The HPV-1 E1 and E2 proteins containing the FLAG epitope at their N-terminal ends were purified by affinity chromatography. Analysis of the proteins by SDS-PAGE followed by staining showed the presence of a major band in each case (Fig. 1). The sizes of E1 (~70 kDa) and E2 (~50 kDa) were consistent with that predicted from their coding sequences. Additional faint bands were presumed to represent protein breakdown products. Western blot analysis using the anti-FLAG M2 antibody showed a single band corresponding to the E1 and E2 proteins containing the FLAG epitopes. The purified FLAG–E1 and FLAG–E2 proteins were used for biochemical analysis.



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Fig. 1. SDS-PAGE and Western blot analysis of the purified HPV-1 E1 and E2 proteins. (A) Purified E1 protein was analysed by 7·5 % SDS-PAGE. One lane was stained with silver while the other was analysed by Western blot using the anti-FLAG monoclonal antibody. (B) Purified E2 protein was analysed on a 10 % polyacrylamide gel and stained with Coomassie blue. The Western blot analysis was carried out using the anti-FLAG monoclonal antibody. The positions of the size markers are indicated.

 
ATPase activity of the HPV-1 E1 protein
The ATPase activity of E1 was tested by incubating 30 ng of the purified E1 protein with varying concentrations of [{alpha}-32P]ATP and analysing the products by TLC. The E1 protein exhibited ATPase activity (Fig. 2A), which was not significantly stimulated in the presence of non-specific single-stranded DNA (M13) or specific double-stranded DNAs (ori171 and ori312) (not shown). The Km value for the ATPase activity of HPV-1 E1 protein was approximately 4 µM, which is similar to that of the SV40 large T antigen and the recently reported values for the HPV-6 and HPV-11 E1 proteins (Table 1). The presence of the viral E2 protein did not have any appreciable effect on the ATPase activity of the HPV-1 E1 protein (Fig. 2B).



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Fig. 2. ATPase activity of the HPV-1 E1 protein. (A) Hydrolysis of ATP by E1 (30 ng) in the presence of the indicated concentrations of [{alpha}-32P]ATP. (B) Effect of the E2 protein on the ATPase activity of E1. The reaction mixtures contained 10 µCi [{alpha}-32P]ATP at a final concentration of 3·33 µM. Purified HPV-1 E1 protein was assayed for ATPase activity in the presence of increasing concentrations (50–200 ng) of HPV-1 E2. Equal amounts of BSA were also used as controls.

 

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Table 1. Comparison of the ATPase activities of large T antigen and papillomavirus E1 proteins

The Km value of HPV-1 E1 protein was determined from a Lineweaver–Burk plot and is the average of three independent experiments. The Km values of the other E1 proteins and the SV40 and polyomavirus large T antigen have been previously published (Clark et al., 1981; Jenkins et al., 1996*; Rocque et al., 2000{ddagger}; Santucci et al., 1995; White et al., 2001{dagger}§; Wilson & Ludes-Myers, 1991). ND, Not determined.

 
Origin binding activity of the HPV-1 E1 and E2 proteins
To test whether the HPV-1 E1 protein is capable of binding to the origin, EMSA was performed using an oligonucleotide (ori60) containing the genetically defined minimal ori of HPV-1 (Gopalakrishnan & Khan, 1994; Gopalakrishnan et al., 1995). This sequence included an imperfect 18 bp palindromic sequence that resembles the putative E1BS based on the results from other PVs (Holt & Wilson, 1995; Holt et al., 1994). EMSA showed the presence of a single DNA–protein complex in the presence of the E1 protein (Fig. 3A). The E1 protein also bound to ori171 DNA, which represents the optimal HPV-1 ori and contains both E1 and E2 binding sites (Fig. 3B). However, E1 also bound to a mutant ori fragment in which half of the putative E1BS of HPV-1 was deleted (Fig. 3C). No significant binding of E1 to an AT-rich, non-specific DNA fragment containing the origin of replication of plasmid pT181 was observed (Fig. 3D). We also tested the ability of native in vitro-translated HPV-1 E1 protein (without the FLAG tag) to bind to the origin. Plasmid pSGE1 was translated in vitro by using rabbit reticulocyte lysate and the synthesis of the E1 protein was confirmed by labelling the protein with [35S]methionine, SDS-PAGE and autoradiography (Fig. 4A). Incubation of unlabelled, in vitro-translated native E1 with the ori171 DNA generated a specific DNA–protein complex that was absent when the in vitro translation reaction was programmed with the vector pSG5 template (Fig. 4B). The above results showed that the E1 protein is able to bind stably to the HPV-1 origin in the absence of any cross-linking agents. The binding of E1 to ori171 was also studied in the presence of the E2 protein. Individually, both the E1 and E2 proteins bound to the DNA generating one predominant complex, E1CX and E2CX, respectively (Fig. 5A). In the presence of both the E1 and E2 proteins, an additional, slower-migrating complex, E1-E2-CX, was also observed (Fig. 5A). This complex could either represent binding of both the E1 and E2 proteins to the DNA, or a larger E1–ori complex that has been observed previously in the case of BPV-1 E1. To distinguish between these possibilities, we utilized an MBP-tagged E2 protein along with FLAG–E1 in EMSA. If the larger complex contained only the E1 protein, it should migrate to a similar position regardless of whether the FLAG–E2 or MBP–E2 protein is used. EMSA showed that the larger E1-E2-CX complex migrated more slowly in the presence of MBP–E2 compared with FLAG–E2 (Fig. 5B). These results showed that the larger complex contained both the HPV-1 E1 and E2 proteins and presumably represents an E1–E2–ori complex.



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Fig. 3. Binding of HPV-1 E1 protein to the ori regions. (A) Binding of E1 to ori60 containing an E1BS and the surrounding AT-rich region. (B) Binding of E1 to ori171 containing an E1BS as well as E2BSs. (C) Binding of E1 to an HPV-1 ori mutant that contains a 9 bp deletion in the putative E1BS. (D) Binding of E1 to a 166 bp non-specific DNA fragment. EMSA was carried out on a 6 % non-denaturing polyacrylamide gel. E1CX, E1–ori complex; F-E1, FLAG–E1 fusion protein; NS, non-specific DNA.

 


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Fig. 4. In vitro-translated, native HPV-1 E1 protein binds to the origin. (A) SDS-PAGE of in vitro-translated HPV-1 E1 protein. E1 protein was synthesized using rabbit reticulocyte lysate. Plasmid pSG5 (vector), pSGE1 (expressing the HPV-1 E1 protein) and a plasmid expressing the control luciferase protein were translated in the presence of [35S]methionine. Positions of the molecular mass markers are indicated. (B) Electrophoretic mobility-shift assay using the in vitro-translated native E1 protein and the HPV-1 ori. Rabbit reticulocyte lysates programmed with either the vector (pSG5) or the pSGE1 plasmid expressing the HPV-1 E1 protein were used. E1CX, E1–ori complex.

 


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Fig. 5. DNA–protein complexes formed in the presence of the E1 and E2 proteins. (A) Binding of FLAG–E1 and FLAG–E2 proteins to the ori171 DNA containing both the E1BS and E2BSs. (B) Binding of the FLAG–E2 and MBP–E2 proteins to the ori171 DNA in the presence of FLAG–E1. The DNA–protein complexes were resolved on a 5·5 % non-denaturing polyacrylamide gel. F-E1, FLAG–E1 fusion protein; F-E2, FLAG–E2 fusion protein; M-E2, MBP–E2 fusion protein; E1CX, (FLAG–E1)–ori complex; F-E2-CX, (FLAG–E2)–ori complex; E1-E2-CX, (FLAG–E1)–(FLAG–E2)–ori complex; M-E2-CX, (MBP–E2)–ori complex; E1-FE2-CX, (FLAG–E1)–(FLAG–E2)–ori complex; E1-ME2-CX, (FLAG–E1)–(MBP–E2)–ori complex.

 

   Discussion
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
The E1 protein encoded by PVs is required for the initiation of virus replication. Studies with BPV-1 and several HPVs have shown both similarities as well as differences in their replication properties. We overexpressed and purified the HPV-1 E1 protein as a fusion protein with the FLAG epitope at its N-terminal end. The FLAG–E1 protein was similar to the wild-type E1 protein in its in vivo replication activity (data not shown). The HPV-1 E1 protein possesses ATPase activity, with a specific activity of ~0·12 µmol min-1 mg-1. The HPV-1 E1 had a high affinity for ATP and its Km for ATP (4 µM) was similar to that of the SV40 large T antigen. The Km value for HPV-1 E1 was much lower than some of the values reported previously for other PV E1 proteins (Table 1). However, a more recent study showed that the Km values for the HPV-6 and HPV-11 E1 proteins were 12 and 6 µM, respectively (White et al., 2001). These values are close to that obtained for the HPV-1 E1 protein in our study. The apparent differences in the Km values previously reported for the HPV E1 proteins with those of White et al. may be due to several factors, including their oligomeric or phosphorylation states and the purification procedures used. The E2 protein did not affect the ATPase activity of E1, similar to the previous observations with other PV E1 proteins (Santucci et al., 1995). The HPV-1 E1 protein was also capable of hydrolysing GTP and CTP, indicating that it does not have an absolute specificity for ATP (data not shown). The ATPase activity of the HPV-1 E1 protein was not stimulated by either single- or double-stranded DNA (data not shown). During the purification of the E1 protein by affinity chromatography, we included a step involving DEAE–Sepharose column chromatography to remove the contaminating DNA from the protein preparation. Furthermore, the A280/A260 ratio of the E1 preparation was consistent with minimal or no DNA contamination. The ATPase activity of the SV40 large T antigen and the BPV-1 E1 protein is stimulated by DNA, whereas that of the polyoma virus large T antigen and HPV types 6b and 11 E1 proteins is not appreciably affected in the presence of DNA (Hughes & Romanos, 1993; Jenkins et al., 1996; Santucci et al., 1995). So far, we have been unable to demonstrate a robust DNA helicase activity for the HPV-1 E1 protein (data not shown).

Since the HPV-1 E1 protein alone is sufficient to support robust replication of ori plasmids (Gopalakrishnan & Khan, 1994; Gopalakrishnan et al., 1995, 1999; Van Horn et al., 2001), we tested whether it was capable of stable interaction with the HPV-1 ori by EMSA. E1 was found to form a single DNA–protein complex in the presence of DNA fragments containing the putative E1BS (Fig. 3A, B). Furthermore, in vitro-translated native HPV-1 E1 protein also bound to the HPV-1 ori (Fig. 4B). In a similar experiment, in vitro-translated HPV-18 E1 protein did not bind to the HPV-18 origin (L. Sheahan and S. A. Khan, unpublished data). While an ori mutant fragment that is deleted for half of the E1BS also bound to the E1 protein, a non-specific DNA fragment did not stably bind to this protein (Fig. 3C, D). These results suggest that sequences in addition to the putative E1BS may be required for E1 binding. Since in vivo replication in the presence of E1 alone is specific to the HPV-1 ori (Gopalakrishnan & Khan, 1994), HPV-1 E1 must be able to recognize specifically the HPV-1 ori during initiation. The FLAG–E2 protein also generated a single DNA–protein complex in the presence of the optimal ori (Fig. 5). In the presence of both the E1 and E2 proteins, a novel, slower-migrating complex (E1-E2-CX) was also observed, presumably corresponding to an E1–E2–ori complex (Fig. 5A). In the case of the BPV-1 E1 protein, it has been shown that the E2 protein stimulates binding of E1 to the ori, initially generating an E1–E2–ori complex, which is subsequently converted to an E1–ori complex that contains a larger multimeric form of E1 and is competent for initiation (Berg & Stenlund, 1997; Chen & Stenlund, 1998, 2001; Gilette & Boroweic, 1998; Lusky et al., 1994). We did not observe such an E1–ori complex in the presence of E2, similar to the results obtained with the HPV-11 E1 protein (Chao et al., 1999; Rocque et al., 2000). It is possible that both the E1–ori and the E1–E2–ori complexes that assemble in the presence of both the E1 and E2 proteins are capable of supporting the initiation of HPV-1 replication.

In EMSA, the E2 protein did not significantly stimulate the ori-binding activity of E1 or the multimeric state of E1 in E1–ori complexes (Fig. 5 and data not shown). In the case of BPV-1 and HPV types 11, 16 and 31b, the E2 protein has a strong stimulatory effect on the ori binding activity of E1, and E2 also enhances the specificity of the E1–ori interaction (Berg & Stenlund, 1997; Chen & Stenlund, 1998; Frattini & Laimins, 1994a, b; Gilette et al., 1994; Masterson et al., 1998; Seo et al., 1993a, b). Since HPV-1 E1 can stably interact with the ori in the absence of E2, it is possible that E2 may have a more limited role in targeting E1 to the ori in this case. However, it is known that the E2 protein stimulates E1-dependent replication of HPV-1 ori plasmids in vivo (Gopalakrishnan & Khan, 1994; Gopalakrishnan et al., 1995). Thus, it is possible that the E2 protein may have a more important role in the establishment of a nucleosome-free ori region, recruitment of host factors to the ori, etc. during HPV-1 replication.

Our observations suggest that the E1 protein of HPV-1 can stably interact with the ori. Recent studies have shown that the BPV-1 E1 protein recognizes the hexanucleotide sequence AACAAT, or its variants, which are present in multiple copies in the ori of BPV-1 as well as several other HPVs (Chen & Stenlund, 2001; Holt et al., 1994). Interestingly, the HPV-1 ori has six such putative E1 binding sequences, more than in any other PV (Chen & Stenlund, 2001). Thus, it is possible that the E1 protein of HPV-1 stably interacts with the ori due to the presence of a larger number of high-affinity E1 binding sequences. This prediction is consistent with the lack of an absolute requirement for the E2 protein or E2BSs in HPV-1 replication (Gopalakrishnan & Khan, 1994). Since HPVs that infect cutaneous tissues replicate to much higher levels and produce higher levels of virions than the mucosotropic HPVs, it is possible that an efficient E1–ori interaction in the case of HPV-1 (and similar HPV types) may play a critical role in the high-level replication of the virus in the productive phase during terminal differentiation of epithelial cells.


   ACKNOWLEDGEMENTS
 
We thank the National Cell Culture Center, Minneapolis, MN, USA, for the large-scale production of insect cells expressing the HPV-1 E1 and E2 proteins. We thank Christin Glorioso for assisting in some of the experiments. This work was supported by grant GM51861 to S. A. K. from the National Institutes of Health.


   REFERENCES
Top
ABSTRACT
Introduction
Methods
Results
Discussion
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Received 18 April 2002; accepted 25 September 2002.