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Journal of Virology, February 2006, p. 1584-1587, Vol. 80, No. 3
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.3.1584-1587.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Papillomavirus E1 Protein Binds to and Stimulates Human Topoisomerase I

Randolph V. Clower, John C. Fisk, and Thomas Melendy^*

University at Buffalo, Department of Microbiology and Immunology and Witebsky Center for Microbial Pathogenesis, 210 Biomedical Research Building, School of Medicine and Biomedical Sciences, Buffalo, New York 14214

Received 21 September 2005/ Accepted 8 November 2005

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The papillomavirus (PV) E1 helicase plays a direct role in recruiting cellular DNA replication factors, such as replication protein A or polymerase -primase, to replicate PV genomes. Here, E1 is shown to bind to human topoisomerase I and stimulate its relaxation activity up to sevenfold. The interaction between E1 and topoisomerase I was mapped to the E1 DNA binding domain and C terminus. These findings imply a mechanism for the recruitment of topoisomerase I to PV DNA replication forks and for stimulating topoisomerase I to allow for efficient relaxation of the torsional stress induced by replication fork progression.

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Papillomaviruses (PVs) are small, nonenveloped, circular DNA viruses (6, 10, 14). The PV E1 protein binds to the PV origin (along with the PV E2 protein), assembles into a hexameric ATP-dependant DNA helicase, and recruits cellular replication factors that work in concert with E1 to replicate the viral genomes (17, 20, 21, 24). E1 has been shown to bind and recruit the cellular factors replication protein A (RPA) and DNA polymerase -primase (2, 6, 12, 13, 15).

One of the essential cellular factors required for PV DNA replication is human topoisomerase I (topoI) (11, 27). topoI is an ATP independent protein that modifies the topology of supercoiled DNA, acting as a swivel to decrease torsional stress on DNA (3, 4, 22). Topoisomerases are important during many cellular processes, such as DNA replication, segregation of daughter chromosomes, modulating nucleosomal structure, transcription, DNA supercoiling, recombination, DNA repair, and genomic stability (8, 18, 25, 26). During the initial stages of DNA replication, the strands of the DNA helix must be separated and the resulting overtwisting of the adjacent DNA must be relieved. Helicases and topoisomerases have been proposed to act together as a "swivelase," instrumental to overcoming these topological obstacles (8). The protein complex that is produced when a helicase and a topoisomerase interact, such as E1-topoI, may be a key step in initiation and play a major role in elongation of DNA replication (8, 19).

The PV E1 protein and human topoI interact. To ascertain whether the PV E1 protein interacts with human topoI, enzyme-linked immunosorbent assay (ELISA)-based protein interaction assays were performed as described previously (12). Purified topoI was immobilized in ELISA plate wells, GST-E1 (human papillomavirus type 11 [HPV-11] or bovine papillomavirus [BPV-1]) was added, and binding was clearly detected (Fig. 1a). Glutathione S-transferase (GST)-YY1, GST, and bovine serum albumin (BSA) did not interact with topoI (Fig. 1a and data not shown). Previous results have demonstrated that GST-E1 also does not interact with other proteins such as SSB or the RPA 32/14 subcomplex (12). topoI was also shown to bind to immobilized GST-E1 and not GST or BSA (Fig. 1b). GST coprecipitations also demonstrated an interaction between E1 and topoI. Glutathione beads were incubated with GST-E1 and 293 cell extract (or purified topoI). Following incubation, the beads were washed in buffer containing 500 mM NaCl and subjected to Western blot analysis (Fig. 1c). These results demonstrate that E1 binds to topoI not only as purified proteins but also within the context of crude cell extracts.

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FIG. 1. E1 binds topoisomerase I. (a) ELISAs were performed using 3 pmol of topoI as the immobilized protein. Increasing concentrations of the indicated proteins (BPV-1 GST-E1, HPV-11 GST-E1, GST-YY1) were added to the wells. Binding was detected using a polyclonal -GST antibody followed by a secondary horseradish peroxidase antibody. After developing, the absorbance was measured at 450 nm. GST also did not bind topoI. The results are representative of three separate experiments; the variation (range) is demonstrated by error bars. (b) ELISAs were performed using 3 pmol of GST-E1, BSA, or GST, as the immobilized protein. Increasing concentrations of topoI were added to the wells. Binding was detected using a monoclonal -topoI antibody followed by a secondary horseradish peroxidase antibody. After developing, the absorbance was measured at 450 nm. The results are representative of three separate experiments; the variation (range) is demonstrated by error bars. (c) GST-E1 coprecipitation experiments were performed in which 10 or 50 µg of GST-E1 or GST was incubated with glutathione Sepharose beads in the presence of 5 mg of 293 cell extract (S100) or 50 µg of purified topoI (the asterisk indicates which panel is from a separate blot). The beads were washed with high-salt Tris-buffered saline (500 mM NaCl) three times and subjected to sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis. Western analysis was performed using a -topoI monoclonal antibody (the asterisk also indicates the use of a polyclonal antibody).

E1 stimulates topoI activity. To determine whether the E1-topoI interaction modulates topoI activity, topoI relaxation assays were performed in the presence and absence of GST-E1 essentially as described previously (22). Increasing amounts of GST-E1 led to a substantial increase in topoI activity (greater than sixfold) (Fig. 2a and b). GST-E1 in the absence of topoI had no intrinsic relaxation activity (Fig. 2a, lane 7). GST, GST-YY1, and additional BSA did not stimulate the relaxation activity of topoI (Fig. 2b). PV origin (BPV-1 and HPV-11) and nonorigin DNA templates were both tested for E1 stimulation of relaxation. All templates showed similar degrees of stimulation by E1, indicating that the presence of a PV origin had no effect on the stimulation of topoI (Fig. 2c and data not shown).

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FIG. 2. E1 stimulates topoisomerase I relaxation activity. (a) topoI relaxation assays were performed in the presence of increasing levels of GST-E1. Plasmid template pUC119 (30 ng/µl; lane 2) was incubated with 5 pg/µl of topoI (+) for 30 min at 37°C (lane 1). GST-E1 was added to the reaction at 3, 7.5, and 15 ng/µl in lanes 3 through 5. GST-E1 (30 ng/µl) alone had no effect on the plasmid DNA topology (lane 7). GST (30 ng/µl) also had no effect on topoI activity (lane 6). BSA was added to each reaction at 0.1 mg/ml. Arrows show positions of relaxed or nicked (form II/IV) and supercoiled (form I) plasmid DNA. (b) The degree of topoI stimulation was measured by the disappearance of the supercoiled band. topoI stimulation (of DNA relaxation), produced by E1 (and other proteins), was calculated by comparison with a topoI standard curve. Standard curves were created by adding increasing amounts of topoI to relaxation assays in order to produce a quantitative measure of the amount of topoI required to generate a specific amount of relaxed form IV DNA. The addition of E1 to a topoI relaxation assay produced a stimulation of DNA relaxation that was subsequently compared to the standard curve, which then produced a numerical value (n-fold stimulation) correlating to a concentration of topoI needed to achieve the equivalent amount of form I DNA. The concentration of GST-E1 is shown, and all other proteins were assayed at the same molar equivalent. The results are representative of three or more separate experiments; the variation (range) is demonstrated by error bars. (c) The PV origin of replication does not affect the stimulation of topoI by E1. Relaxation assays were performed as described above using a PV origin (pUCAlu, a plasmid containing the BPV-1 origin; nucleotides 7891 through 7952) or a nonorigin (pUC119) template (7, 14). The degree of topoI stimulation on the various templates was measured as described above. The results are representative of three or more separate experiments; the variation (range) is demonstrated by error bars.

Mapping the E1-topoI interaction. To determine the domains within E1 that interact with topoI, GST-E1 truncation mutants were used in ELISAs. HPV-11 GST-E1 containing amino acids (aa) 1 through 403 of E1 showed a positive interaction with topoI (Fig. 3a, open squares), albeit to a slightly lower level than full-length E1. Conversely, the smaller GST-E1 truncation mutant (aa 1 through 165) showed no significant binding to topoI (Fig. 3a, x). The region between E1 amino acids 165 and 403 encompasses the HPV-11 E1 DNA binding domain (DBD), which consists of amino acids 181 through 353 (1, 23). The homologous BPV-1 E1 DBD (consisting of corresponding amino acids 142 through 308) (5, 9, 16) was also purified and tested for topoI binding using ELISA. The BPV-1 E1 DBD demonstrated a positive interaction, suggesting that the E1 DBD contains a bona fide topoI binding site (Fig. 3a, open triangles, binding site A).

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FIG. 3. DBD and C terminus of E1 bind topoI. (a) ELISAs were performed using 3 pmol of immobilized topoI as described above. As second proteins, GST-E1, GST-E1 aa 1 through 403, BPV-1 GST-E1 DBD, GST-E1 aa 1 through 165, and GST alone are depicted. (b) ELISAs were performed using 3 pmol of immobilized topoI. As second proteins, GST-E1, GST-E1 aa 589 through 649, GST-E1 aa 310 through 649, GST-E1 aa 346 through 649, and GST alone are shown. All results represent three separate experiments; the variation (range) is depicted by error bars. (c) E1 binds topoI through two regions (A and B). Graphic depictions of linear representations of E1 showing the binding results in panels a and b. Binding site A is the E1 DBD, and binding site B consists of amino acids 589 through 649 (shaded regions).

Three amino-terminal HPV-11 GST-E1 truncation mutants were also purified and tested for topoI binding using ELISAs. HPV-11 GST-E1 truncation mutants of aa 310 through 649, aa 346 through 649, and aa 589 through 649, which contain all or part of the core helicase domain (1, 7, 13), all demonstrated a similar positive interaction with topoI (Fig. 3b). From these data, we conclude that the minimal topoI binding site within the C terminus of E1 is within aa 589 through 649 (binding site B). The schematic map of E1 depicts the two domains (sites A and B) within E1 that bind to topoI (Fig. 3c).

E1 truncation mutants and topoI relaxation activity. Plasmid relaxation assays were implemented to assess which of the binding domains affect topoI activity. For each E1 truncation, control reactions were performed in parallel to establish basal topoI relaxation activity and full-length E1's ability to stimulate topoI. As demonstrated above, full-length E1 stimulated topoI approximately sixfold while GST and BSA alone had little effect (Fig. 4a, lanes 8, 2, and 4, respectively). The fragment from aa 1 through 165 E1, which does not bind to topoI, did not stimulate topoI relaxation activity (Fig. 4a, lane 7). The E1 fragment from aa 1 through 403, which binds to topoI, stimulated relaxation activity approximately 3.5-fold (Fig. 4a, lane 10; see also Fig. 4b for quantitation). However, the BPV-1 E1 DBD (Fig. 3c, binding site A) showed no appreciable stimulation of topoI (Fig. 4a, lane 6). This result appears to contrast with the result for the fragment from aa 1 through 403, for which the E1 DBD appeared to be the relevant binding domain. It is possible that aa 1 through 403 may contain amino acid residues outside the E1 DBD that play an ancillary role in the E1-topoI interaction.

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FIG. 4. topoI stimulation by E1 truncations. (a) topoI plasmid relaxation assays were performed in the presence of a second protein: GST, BSA, HPV-11 GST-E1 (aa 589 through 649), BPV-1 GST-E1 DBD (aa 142 through 308), HPV-11 GST-E1 (aa 1 through 165), BPV-1 GST-E1, HPV-11 GST-E1, or HPV-11 GST-E1 (aa 1 through 403) as labeled (lanes 2 and 4 through 10). Plasmid template pUC119 (30 ng/µl) was incubated with 5 pg/µl of topoI (+) for 30 min at 37°C, where indicated. Second proteins were added to the reaction at the same molar equivalent as full-length GST-E1. The reaction shown in lane 1 was performed without a second protein. Lane 3 shows pUC119 with no topoI. Lane 11 shows plasmid with GST-E1 and no topoI. BSA was added to each reaction at 0.1 mg/ml. Arrows show positions of relaxed (form II/IV) and supercoiled (form I) plasmid DNA. (b) Stimulation of topoI relaxation of DNA produced by the proteins and E1 truncations indicated was calculated as described in the legend to Fig. 2. The concentration of GST-E1 is shown, and all other proteins were assayed at the same molar equivalent. Higher levels of the proteins did not show appreciably greater stimulation (data not shown). The results are representative of three or more separate experiments; the variation (range) is shown by error bars.

Binding site B (the C-terminal E1 fragment, aa 589 through 649) stimulated topoI relaxation activity about half as efficiently as full-length E1 (3.5-fold) (Fig. 4a, lane 5). The two other C-terminal fragments tested (aa 310 through 649 and aa 346 through 649) also stimulated topoI relaxation activity to a similar degree as binding site B (data not shown). All truncations were assayed at the same molar equivalent of full-length E1 (Fig. 4b). The E1 aa 1 through 403 and C-terminal fragments could increase the stimulation of topoI up to 4.5-fold at concentrations at a greater than 1:1 molar ratio (data not shown).

Discussion. In this study, we demonstrated that human topoI binds two distinct regions within the PV E1 protein, the DBD and the C terminus. Using ELISAs and topoI relaxation assays, we demonstrated that of the two minimal regions within E1 that bind topoI, the C-terminal region stimulates topoI relaxation activity as well as the fragment from aa 1 through 403 (which contains binding site A, the DBD), although to a lesser degree. The BPV-1 E1 DBD itself did not stimulate topoI relaxation, indicating that an additional region located within the aa 1 through 403 fragment may be necessary for stimulation. It is unclear at this time how E1 stimulates topoI. Two possibilities are that E1 stimulates the catalytic activity of topoI or that E1 may affect the turnover rate of topoI on DNA. In either case, it is reasonable to suggest that the E1-topoI interaction may be an important aspect of PV DNA replication.

Now that a potential role for the novel E1-topoI interaction has been determined, it will be interesting to ascertain whether topoI stimulates the recruitment of E1 to PV origin sequences and whether E2 plays any role in this interaction.

 
We thank Jen-Sing Liu, Shu-Ru Kuo, and all members of the Witebsky Center for Microbial Pathogenesis at the University at Buffalo for invaluable scientific discussion. We also thank Te Chung Lee for GST-YY1 protein and J. J. Champoux for the human topoI baculovirus vector. We also thank Dennis McCance for supplying E1 vectors and Meghan Barnhart for her work on this project. Thank you to Yan Hu for purified human topoI preparations. Also, thanks to Philippe Clertant for his help on this project. Thanks to Ed Niles and Mark Sutton for critical reading of the manuscript.

This work was supported by NIH grant R29 GM56406 to T.M. R.V.C. was also supported in part by NIH training grant AI07614.

 
* Corresponding author. Mailing address: University at Buffalo, Department of Microbiology & Immunology, The School of Medicine and Biomedical Sciences, 213 Biomedical Research Building, 3435 Main Street, Buffalo, NY 14214. Phone: (716) 829-3789. Fax: (716) 829-2158. E-mail: TMelendy{at}buffalo.edu.

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Journal of Virology, February 2006, p. 1584-1587, Vol. 80, No. 3
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.3.1584-1587.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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