Clinical Cancer Research Vol. 9, 5423-5428, November 1, 2003
© 2003 American Association for Cancer Research
Experimental Therapeutics, Preclinical Pharmacology |
Peptides Inhibitory for the Transcriptional Regulatory Function of Human Papillomavirus E2
Takuma Fujii,
David Austin,
David Guo,
Srinivasan Srimatkandada,
Tao Wang,
Kaneyuki Kubushiro,
Nobuo Masumoto,
Katsumi Tsukazaki,
Shiro Nozawa and
Albert B. Deisseroth1
Yale University, New Haven, Connecticut 06520 [T. F., D. A., D. G., S. S., T. W.]; Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, 160-8582 Japan [T. F., K. K., N. M., K. T., S. N.]; and the Sidney Kimmel Cancer Center, San Diego, California 92121 [A. B. D.]
 |
ABSTRACT
|
|---|
Purpose: Human papillomavirus (HPV) infections are associated with cervical neoplasia. Cellular and viral proteins are known to interact with the papillomavirus E2 protein to initiate transcription and DNA replication in the HPV life cycle. Our aim was to identify peptides that bind to the HPV16 E2 protein and thereby inhibit its ability to alter the transcriptional activity of other genes.
Experimental Design: The HPV16 E2 protein was expressed and purified to near homogeneity in bacteria. We screened a phage display library of random peptides for ones that bound to HPV16 E2 protein. Among the isolated phage clones, we found that tryptophan-rich peptide sequences appeared repetitively in successive cycles of phage library panning. Replacement of the tryptophan amino acids in these dodecapeptides reduced the degree to which these peptides bound to the E2 protein. These E2-binding peptides were tested for their ability to inhibit the transcriptional regulatory function of E2 in a test cell line, which contained an E2 gene and a luciferase reporter gene driven by an E2-dependent transcriptional promoter.
Results: Delivery of four of the E2 binding peptides into the intracellular compartment of the test cell line resulted in suppression of the E2-dependent luciferase expression. Deletion of the tryptophan residues from these peptides reduced their E2 binding and their ability to suppress E2-dependent luciferase expression in the test cell line.
Conclusions: These results suggest a strategy for the development of chemical inhibitors of E2-dependent transcription of viral genes in HPV-infected cells as an approach to the therapy of chronic HPV infections.
|
INTRODUCTION
|
|---|
Chronic infections with the HPVs2
are associated with the development of several benign epithelial proliferative lesions, including genital warts and respiratory laryngeal papillomatosis (1
, 2) . Furthermore, epidemiological data suggest that chronic HPV infection is one of main risk factors for the development of cervical cancer, which is the second most common cause of cancer death in women (3)
.
In the viral life cycle, the HPV protein E2 can function as both a transcriptional activator and repressor, probably by interaction with cellular transcriptional factors such as SP1 and TATA-binding protein (4, 5, 6)
. The E2 protein also plays an auxiliary role in promoting the formation of the HPV DNA replication initiation complex by recruiting the E1 protein onto the HPV origin of DNA replication (ori), which leads to the assembly of the replication initiation complex, including recruitment of the host cell DNA polymerase (7
, 8)
. E2 protein is composed of three domains: the NH2-terminal domain, which is involved in transcriptional regulation and direct association with the HPV E1 protein; a COOH-terminal region involved in DNA binding and dimerization; and a hinge region involved in tethering the HPV episomal circular DNA to the chromosomal DNA (9, 10, 11, 12)
. Because both cellular and viral proteins are known to interact with the HPV E2 protein, it may be useful to identify peptide motifs that contribute to E2 binding to host proteins as well as other viral proteins.
On the basis of such information, low molecular weight chemical inhibitors of E2 function can be designed to disrupt the interaction of E2 with its binding proteins and thereby inhibit E2-dependent DNA replication and transcription. Sakai et al. (13)
reported a mutant E2 protein, which disrupts the interaction of the wild-type E2 with the E1 protein, and thereby suppresses its replication. Kasukawa et al. (14)
used 15 amino acid peptide to inhibit viral replication in vitro. However, there is currently insufficient information available for design of low molecular weight chemical inhibitors of such protein-protein interactions. Screening of phage display libraries of random amino acid peptides has been used successfully in the past to identify amino acid motifs that contribute to binding to the E2 protein to host and viral proteins (15, 16, 17, 18)
. Structural information on these peptides and their target domains could lead to predictions as to which host cell proteins may be interacting with E2 as well as to the design of low molecular weight inhibitors of such interactions.
Here we report that certain peptide sequences isolated by screening a random peptide phage display library for HPV E2 binding depend on tryptophan for the E2 binding. We were able to show in a cell-based assay that introduction of these peptides into the intracellular environment of a cell that contains an E2 transcription unit as well as a luciferase gene that is governed by an E2-dependent transcriptional promoter inhibits the transcription of the luciferase reporter gene. These studies suggest that the chemical peptidomimetics of these peptides might form the basis of drugs that could be used to treat chronic HPV infections for prevention of cervical cancer.
|
MATERIALS AND METHODS
|
|---|
Phage Display Screening.
The HPV-16 E2 gene was amplified by PCR from a viral DNA HPV-16 114/K clone, provided by Dr. Matthias Dürst (University of Jena, Jena, Germany) and cloned into XhoI sites of a pRSET A vector (Invitrogen, San Diego, CA). The primers for PCR amplification were 5'-cccgctcgagatggagactctttgccaacgttta-3' and 5'-cccgctcgagtcatatagacataaatccagtaga-3'. In the 114/K clone, the nucleotide sequence differed from that of the accepted sequence of the HPV16 E2 gene. This single difference was identical to that found in a previous report (5)
. The pRSET-HPV16E2 clone was transformed into Escheria coli BL21(DE3; plysS; Novagen, Madison, WI). Purification of the HPV E2 protein was carried out as described previously (19)
.
E2 protein (2.5 µg) in 20 x 10-3 M Tris-HCl (pH 9.1) was immobilized to the bottoms of wells in 96-well-microtiter plates (Falcon 3915) by overnight incubation at 4°C. The following day, the wells were blocked by incubation overnight at 4°C with blocking buffer containing 50 mM Tris-HCl (pH.7.4), 150 x 10-3 M NaCl, and 1% nonfat milk (Carnation, Los Angeles, CA). The wells were rinsed with washing buffer containing 50 x 10-3 M Tris-HCl (pH.7.4), 150 x 10-3 M NaCl, and 0.1% Tween 20. We used a phage display library that has a random hepta- or dodecapeptide sequence fused to a minor coat protein (pIII) of the M-13 phage (New England Biolabs, Beverly, MA). The plate was panned for 1 h at room temperature with the phage particles. The plates were washed six times for 1 h, then the bound phage was eluted with 0.2 M Glycine-HCl (pH 2.2) followed by neutralization with 1.0 M Tris-HCl (pH 9.1). The recovered phage were amplified in Luria-Bertani plates containing stationary phase ER2537 cells. Amplified phage were screened in additional rounds and then plated to obtain isolated clones.
To characterize the binding of individual clones to E2, plaques were picked at random, amplified, and tested for E2 binding by ELISA using horseradish peroxidase coupled to an anti-M13 antibody (Amersham Biosciences, Piscataway, NJ). To exclude phage clones that bind to the plastic surface, the E2 protein and BSA was immobilized to the plates individually. The absorbance value (A) was determined by subtraction of the raw A of the BSA plates from that of the E2 plates.
Analysis of Amino Acid Motifs in Phage Clones That Are Found in E2 Binding Peptides.
The nucleotide sequences coding for random peptides displayed on the phage coat proteins were determined in the Yale Howard Hughes Medical Institute/Keck laboratory. To confirm whether the peptide sequences obtained from our phage library screen were binding to the E2 protein in a sequence-dependent manner, the corresponding biotinylated peptides were synthesized (Yale Howard Hughes Medical Institute/Keck Protein Synthesis Laboratory) and tested for binding to E2. The E2 protein was immobilized to the plate followed by blocking with blocking buffer. The synthetic peptides were incubated in the wells for 2 h. After washing, 1–2000 diluted horseradish peroxidase conjugated streptavidin (Pierce, Rockford, IL) was incubated before addition of substrate.
Peptide Internalization and Visualization.
We arranged for the synthetic coupling of fluorescent-labeled E2 binding peptides to the SV40 nuclear localizing signal (PKKKRKV) at Peptide Laboratories (Osaka, Japan). C33a cells derived from human cervical cancer were obtained from the American Type Culture Collection. C33a cells were grown in DMEM containing 10% heat-inactivated FCS. For each assay, 1 x 105 cells were seeded on a Lab-Tek-II chamber slide (Nalge Nunc International, Rochester, NY) and cultured for 8 h. The cells were incubated at 37°C for 8 h with the fresh medium, which was supplemented with sufficient fluorescein-labeled peptide to produce a concentration of 10 µM. The peptide-supplemented medium was changed three times. After 24 h, cells were washed three times with PBS, fixed with ethanol for 30 min at 4°C, and washed three times again with PBS. Cells were then treated with PBS containing 15 µM propidium iodide at room temperature for 5 min, and then washed three times again with PBS. Data were obtained using confocal scanning laser microscope MRC 1024 (Bio-Rad, Hercules, CA) equipped with a x10 or x60 oil immersion lens.
Inhibitory Effect of the Peptide Sequences on the Transcriptional Activity.
The HPV 16 E2 expression vector driven by the cytomegalovirus promoter and the luciferase reporter vector that contains six cognate E2 binding sites, Sp1 binding sites and thymidine kinase core promoter followed by the luciferase gene was constructed as reported previously (19)
. C33a cells were seeded in triplicate onto six-well plates, and transfected with the luciferase reporter vector and the HPV16 E2 expression vector using Lipofectamine (Life Technologies, Inc., Rockville, MD). The amount of input DNA was equal to that of the backbone vector. The synthetic peptides were added at 0, 3, and 12 h after transfection in the culture medium. Cells were lysed at 24 h with a reporter lysis buffer (Promega, Madison, WI) and measured with a luminometer (Turner Designs, Sunnyvale, CA). All of the transfection experiments were performed at least three times to assess reproducibility.
|
RESULTS
|
|---|
Isolation of the Peptides Bound to the E2 Protein.
We used the full-length HPV16 E2 protein as a target for the panning of the phage display library of random peptides (see Fig. 1
). The first screen involved a random heptapeptide phage display library, which was assumed to contain all of the possible seven amino acid sequences. Even after the fourth round of biopanning, the frequency of each individual phage was not high, suggesting that the candidate phage clones isolated in this screen were low affinity in their binding to E2 and were, therefore, not being amplified such that they would become dominant in the population.
View larger version (41K):
[in this window]
[in a new window]
[Download PPT slide]
|
Fig. 1. Purification of the HPV16 E2 protein produced in bacteria. Lane 1, 1 µg of BSA; Lane 2, prestained broad range protein markers (Bio-Rad); Lanes 3 and 4, 1 and 3.3 µg of the HPV16 E2 protein, respectively. These proteins were loaded on a 15% SDS-PAGE with Coomassie blue staining. Molecular weights are indicated in kDa on the left of the gel.
|
|
Therefore, we screened a dodecapeptide phage library to isolate candidate peptides that would bind to E2 at higher affinity. The dodecapeptide phage library we used consists of 1.9 x 109 independent clones in which only 1 in 106 of the sequences can be assumed to have been displayed in the screen. Despite these limitations, we expected that a higher affinity binding peptide could be isolated because of the increased length of the amino acid residues bound to the HPV16 E2 protein. Of importance, we noticed that tryptophan rich peptides were preferentially isolated in this screen (see Table 1
). We first used the ELISA to test whether the affinity of isolated phage clones bound to the HPV16 E2 protein was high or not. Among isolated clones, the 5 tryptophan-rich clones tended to give high absorbance values in the ELISA assay whereas the 6 tryptophan-negative peptides gave low absorbance values (see Table 1
, and Figs. 2
and 3
).
View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
|
Fig. 3. Analysis of biotinylated synthetic peptide bound to the HPV16 E2 protein by ELISA. The HPV16 E2 protein was immobilized on the 96-well plate before incubation with 10-6 M of biotinylated synthetic peptide (0.5% DMSO). Streptavidin-conjugated horseradish peroxidase and the substrate were added to each well, and the A value was then measured as outlined in "Materials and Methods." Statistical analysis was performed by Student’s t test.
|
|
Site-Directed Mutagenesis of Clones Identified as High Affinity in the ELISA.
We next decided to formally test the role of tryptophan residues in the binding of the peptides to the HPV E2, because tryptophan was encountered so commonly in the third round of biopanning. Therefore, we selected and synthesized two of the peptide sequences from the isolated phage clones that contained tryptophan and that exhibited high binding affinity to the E2 protein in the ELISA assay results. As shown in Fig. 3
, synthetic peptides from clone TWFNPFGYYSWA and clone TLWPWAWRHNWQ were tested for binding to the HPV16 E2 protein. Increasing our interest in tryptophan containing peptides was the report, albeit in bovine papillomavirus E2 that tryptophan residues in the dimerization of E2, which is important for E2 function (20, 21, 22)
. We hypothesized that tryptophan containing residues could contribute to the interaction of the E2 protein with either DNA or other proteins. To test this hypothesis, we introduced alanine for every tryptophan in these peptides to determine whether tryptophan is important for the affinity of the synthetic peptides for E2. As shown in Fig. 3
, the tryptophan containing peptide sequences selected from the phage library bound to the E2 protein, whereas the mutated peptides with the tryptophan substituted by alanine (TLAPAAARHNAQ) appeared to bind to E2 to a much lower degree than does the wild-type peptide.
Peptide Internalization and Visualization.
Peptides were conjugated with FITC for monitoring the intracellular location of the peptides and then linked synthetically to the SV40 nuclear localizing signal. Uptake into the cultured cells exposed to the FITC-conjugated peptides linked to the SV40 localizing signal was detectable by 3 h (data not shown). After incubation of the cells with the peptides for 24 h, >90% of the cells were positive for FITC-mediated fluorescence in both the nucleus and the cytoplasm (see Fig. 4
). The peptide internalization was examined microscopically. Internalization was not found to be such a substantial variable among peptides. These data showed that the FITC-conjugated peptides linked to the SV40 nuclear localizing sequence internalized very efficiently.
View larger version (44K):
[in this window]
[in a new window]
[Download PPT slide]
|
Fig. 4. Delivery of the peptides into C33a cells. Confocal microscopic observation of the C33a cells first incubated in propidium iodide, then incubated with the peptide conjugated with FITC and synthetically linked to the SV40 nuclear localizing sequence peptide. Accumulation of the peptide into both the cytosol and nucleus was observed. The peptide internalization was not significantly different among peptides.
|
|
Inhibitory Effect by the Peptide on the Transcriptional Activity.
Because the HPV16 E2 protein is known to be a transcriptional regulatory protein as well as promoter of HPV DNA replication initiation (7
, 8)
, we had shown that the FITC-labeled peptides that were linked to the SV40 nuclear localizing sequence entered the C33a cells, and we then tested whether the isolated peptides could inhibit the E2-dependent expression of a luciferase transcription unit driven by an E2-dependent promoter. As outlined in a previous publication from our laboratory (19)
, we had constructed an artificial transcriptional promoter composed of 6 E2 binding sites, 2 Sp1 sites, and a truncated thymidine kinase promoter. This promoter had been shown to be dependent on the binding of E2 protein. Therefore, we constructed a plasmid containing a luciferase transcription unit driven by this E2-dependent promoter. We also constructed a plasmid that contained a cytomegalovirus driven E2 transcription unit. We first plated C33a cells in triplicate in six-well plates. Then we cotransfected using Lipofectamine both of these plasmids into the C33a cervical cancer cell line. The FITC-conjugated E2 binding peptides, which were linked to the SV40 nuclear localizing sequence, were added to the medium of the wells containing the transfected C33a cells at 0, 3, and 12 h after the transfection.
After lysis of the cells, they were analyzed in the luminometer for luciferase activity. As shown in Fig. 5
, addition of peptide 2 to the C33a cells reduced the luciferase activity to 78% of the control. Addition of the mutant peptide 2 in which the tryptophan residues had been substituted by alanine residues no longer suppressed the luciferase activity in the C33a cells. This suggested that peptide 2 could suppress the transcriptional activation function of E2 with the E2-dependent promoter. It was clear that there is a statistically significant difference between the suppression of luciferase by tryptophan-positive peptides 3–4 and the tryptophan-negative peptide 6.
View larger version (25K):
[in this window]
[in a new window]
[Download PPT slide]
|
Fig. 5. Transcriptional repression by peptides, which bound to the HPV 16 E2 protein. C33a cells in the six-well plates were transfected with the HPV 16 E2 expression vector along with the luciferase reporter vector that is driven by the E2-dependent promoter. The Luciferase activity was indicated as 100% in the absence of added peptide (Y-axis). Statistics analysis was performed by the Dunnett multiple comparison post-test. The tryptophan containing peptides 3–4 were shown to bind to a statistically significantly greater degree than the typtophan-negative peptide 6.
|
|
We next tested the effect of adding various concentrations of the FITC-conjugated tryptophan containing E2 binding peptide 2, which had been linked to the SV40 nuclear localizing sequence. In the absence of added E2 binding peptide to the C33a cell line (transiently cotransfected with the E2 and luciferase/E2-dependent promoter expression vectors), the expression of luciferase was 5.5 times that seen in the C33a cells transfected with the luciferase/E2-dependent promoter expression vector without the E2 expression vector (see Fig. 6
). As the concentration of the tryptophan containing peptide 2 was increased from 5 to 20 µM in the medium bathing the C33a cells transfected with both the E2 and luciferase/E2 promoter expression vector, the level of the luciferase activity decreased 2-fold. The basal level of luciferase expression in the C33a cells transfected with the luciferase/E2-dependent promoter expression vector in the absence of the E2 expression vector did not change in the presence of increasing concentrations of peptide 2. These experiments suggested that the effect of the FITC-conjugated E2 binding peptide 2, which was linked to the SV40 nuclear localizing sequence, was specific for E2-dependent expression of the luciferase transcription unit.
View larger version (39K):
[in this window]
[in a new window]
[Download PPT slide]
|
Fig. 6. Transcriptional repression by the E2 binding peptide Pep-2 in a dose-dependent manner. Relative Luciferase activity was arbitrarily assigned the value 1 in the absence added peptide (Y-axis). The concentration of Pep-2 in the culture medium is indicated on the abcissa (X-axis). Statistical analysis was performed by Student’s t test.
|
|
|
DISCUSSION
|
|---|
Because HPV is not easily propagated in mammalian cells, it is difficult to investigate the precise nature of interactions between the E2 protein with other viral and host cell proteins as they relate to the papillomavirus life cycle. To circumvent this problem, we decided to use a phage display screen to identify peptides that bound to E2 and then to look for peptides within that E2 binding group that would disrupt the transcriptional regulatory function of E2 in a transient transfection assay of E2-dependent reporter gene expression. The goal of this exercise was to define amino acid structures that were key to the E2 binding and disruption of E2 transcriptional regulatory function.
The results of our experiments demonstrated that the E2 protein was able to bind peptides displayed on a phage surface proteins, which are rich in tryptophan. Site-directed mutagenesis experiments show that the tryptophan rich sequences are important for the binding of peptides to the HPV 16 E2 protein. Search of protein structure databases showed that these tryptophan-rich motifs are found in many viral and host cell proteins, which are known to bind to the E2 protein, or to play an important role in DNA replication or transcription (data not shown).
Protein-protein interactions are known to be crucial for many biological processes, especially regulation of transcription. The discovery of potential interactions between E2 and otherwise uncharacterized proteins may provide insight into their role with respect to the transcriptional regulatory function of E2.
In our experiments, dodecapeptides were found through the phage display screening, which bound at high affinity to the HPV E2 protein. Many of these were rich in tryptophan containing motifs. These regions may be crucial to the stabilization of the protein-protein interactions necessary for the functions of the E2 protein. Alternatively, tryptophan may be favored in a phage display library, because it provides a hydrophobic scaffold and it improves the binding activity of a displayed peptide (23)
. To exclude this trivial explanation, we used site-directed mutagenesis to show that the tryptophan residues were important for the high affinity binding of peptides to the E2 protein. We assume that the change of the tryptophan to alanine and the resultant decrement in the bulkiness, aromaticity, and polarity of the amino acids resulted in the observed decrease in the binding of the peptides to E2. These data suggest that tryptophan-rich peptides may play a role in the interactions between the E2 protein and other proteins.
We additionally examined the inhibitory effect on the E2-binding peptides on the transcriptional regulatory function of E2. One problem in carrying out this analysis was the poor solubility of the peptides and the internalization of the test peptides into test cells, which contained transiently transfected plasmid transcription units for the E2 and a luciferase/E2-dependent promoter. At first we decided to use penetratin-1, the DNA-binding region of the Drosophila antennapedia homeoprotein to promote the internalization of the test peptides into the test cells. However, peptides conjugate to the antennapedia homoprotein would still pose a problem of the solubility in aqueous solvents. In view of this, we decided to conjugate the SV40 nuclear localizing sequence to the test peptides to overcome both the solubility and internalization problems.
The test peptides that were conjugated to FITC and synthetically linked to the SV40 nuclear localization sequence were found to successfully internalize not only into the cytoplasm but also into the nucleus as shown in Fig. 4
. We found that the E2-binding peptides identified in the phage display screen (Pep-1, Pep-2, Pep-3, and Pep-4) were all capable suppressing E2-dependent transcription of the luciferase reporter gene when it was under the control of an E2-dependent transcriptional promoter. In contrast, a mutant version of the E2-binding peptide, Pep-2, in which tryptophan was substituted by alanine, showed reduced binding to E2 and was unable to suppress the E2-dependent transcription of the luciferase transcription unit, suggesting that tryptophan-rich sequences were associated with the transcriptional regulatory function of E2.
Interestingly, the E2-binding peptide Pep-1 was homologous to the HPV 16 capsid protein L2 protein (amino acid: 434–442). The bovine papillomavirus L2 protein has also been reported to interact with the E2 protein (24)
. Therefore, our data support the hypothesis that the L2 protein in some way modulates the transcriptional activity of the E2 protein.
A sequence homology search showed that the E2-binding peptide Pep-2 was homologous to DNA polymerase 
(amino acid: 169–175). The E2-binding peptide Pep-3 was found to be homologous to TAF-II 100 (amino acid: 591–599). Therefore, these proteins may also be involved in the modulation of the E2 transcriptional regulatory or DNA replication functions as well.
Our data are the first demonstration showing inhibition by synthetic peptides of the E2 transcriptional regulatory function in a cell-based assay. Furthermore, the data we have generated for amino acid motifs that are necessary for E2 binding and inhibition of the transcriptional regulatory function of E2 may help in the future in the design of chemical mimics for suppression of HPV replication.
|
ACKNOWLEDGMENTS
|
|---|
We thank Matthias Dürst, University of Jena (Jena, Germany), for his gift of HPV-16 114/K clone. We thank Eiji Ohta and Miyuki Saito for technical assistance. We also acknowledge help from Dr. Alan Garen (Yale University), Yoshinori Hara (Yamanouchi Pharmaceutical Co., Ltd.), and Atsushi Takayanagi and Nobuyoshi Shimizu (Genetics Division, Keio University School of Medicine).
|
FOOTNOTES
|
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by Yale Cancer Center Development Fund, the Hull Development Fund, the George and Barbara Bush Leukemia Research Fund, the Ensign Professorship of Medicine, Support from Sidney Kimmel, Grant-in-Aid for Scientific Research (C), Japan Society for the Promotion of Science, and Research Grants for Life Sciences and Medicine, The Keio University Medical Science Fund.
1 To whom requests for reprints should be addressed, at Office of the President and CEO, Sidney Kimmel Cancer Center, 10835 Altman Row, San Diego, CA 92121. Phone: (858) 410-4205; Fax: (858) 587-8873; E-mail: adeisseroth{at}skcc.org 
2 The abbreviations used are: HPV, human papillomavirus.
Received 12/16/02;
revised 4/14/03;
accepted 4/14/03.
|
REFERENCES
|
|---|
- Shah K. V., Buscema J. Genital warts, papillomaviruses, and genital malignancies. Ann. Rev. Med., 39: 271-379, 1988.
- zur Hausen H. Viruses in human cancers. Science (Wash. DC), 254: 1167-1173, 1991.[Abstract/Free Full Text]
- Parkin D. M., Pisani P., Ferlay J. Estimates of the worldwide incidence of 25 major cancers in l990. IJC, 80: 827-841, 1999.
- Tanj S. H., Leong L. E., Walker P. A., Bernard H. U. The HPV type 16 E2 transcription factor binds with low cooperativity to two flanking sites and represses the E6 promoter through displacement of Sp1 and TFIID. J. Virol., 68: 6411-6420, 1994.[Abstract/Free Full Text]
- Kovelman R., Bilter G. K., Glezer E., Tsou A. Y., Barbosa M. S. Enhanced transcriptional activation by E2 proteins from the oncogenic HPV. J. Virol., 70: 7549-7560, 1996.[Abstract]
- Bouvard V., Storey A., Pim D., Banks L. Characterization of the HPV E21 protein: evidence of trans-activation and trans-repression in cervical keratinocytes. EMBO J., 13: 5451-5459, 1994.[Medline]
- Melendy T., Sedman J., Stenlund A. Cellular factors required for papillomavirus DNA replication. J. Virol., 69: 7857-7867, 1995.[Abstract]
- McBride A. A., Bolen J. B., Howley P. M. Phosphorylation sites of the E2 transcriptional regulatory proteins of bovine papillomavirus type 1. J. Virol., 63: 5076-5085, 1989.[Abstract/Free Full Text]
- Lehman C. W., Botchan M. R. Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation. Proc. Natl. Acad. Sci. USA, 95: 4338-4343, 1998.[Abstract/Free Full Text]
- Ilves I., Kivi S., Ustav M. Long-term episomal maintenance of bovine papillomavirus type 1 plasmids is determined by attachment to host chromosomes, which is mediated by the viral E2 protein and its binding sites. J. Virol., 73: 44040-4412, 1999.
- Spalholz B. A., Yang Y. C., Howley P. M. Transactivation of a bovine papilloma virus transcriptional regulatory element by the E2 gene product. Cell, 42: 183-191, 1985.[CrossRef][Medline]
- Stundel W., Huang Z., Tan S. H., O’Connor M. J., Bernard H. U. Nuclear matrix attachment regions of HPV type 16 repress or activate the E6 promoter, depending on the physical state of the viral DNA. J. Virol., 74: 2489-2501, 2000.[Abstract/Free Full Text]
- Sakai H., Yasugi T., Benson J. D., Dowhanick J. J., Howley P. M. Targeted mutagenesis of the HPV 16 E2 transactivation domain reveals separable transcriptional activation and DNA replication functions. J. Virol., 70: 1602-1611, 1996.[Abstract]
- Kasukawa H., Howley P. M., Benson J. D. A fifteen-amino-acid peptide inhibits HPV E1–E2 interaction and HPV DNA replication in vitro. J. Virol., 72: 8166-8173, 1998.[Abstract/Free Full Text]
- Devlin J. J., Panganiban L. C., Devlin P. E. Random peptide libraries: a source of specific protein binding molecules. Science (Wash. DC), 249: 404-406, 1990.[Abstract/Free Full Text]
- Oldenburg K. R., Loganathan D., Goldstein I. J., Schultz P. G., Gallop M. A. Peptide ligands for a sugar-binding protein isolated from a random peptide library. Proc. Natl. Acad. Sci. USA, 89: 5393-5397, 1992.[Abstract/Free Full Text]
- Scott J. K., Loganathan D., Easley R. B., Gong X., Goldstein I. J. A family of concanavalin A-binding peptides from a hexapeptide epitope library. Proc. Natl. Acad. Sci. USA, 89: 5398-5402, 1992.[Abstract/Free Full Text]
- Gardsvoll H., van Zonneveld A. J., Holm A., Eldering E., van Meijer M., Dano K., Pannekoek H. Selection of peptides that bind to plasminogen activator inhibitor 1 (:AI-1) using random peptide phage-display libraries. FEBS Lett., 431: 170-174, 1998.[CrossRef][Medline]
- Fujii T., Brandsma J. L., Peng X., Srimatkandada S., Li L., Canaan A., Deisseroth A. B. High and low levels of cottontail rabbit papillomavirus E2 protein generate opposite effects on gene expression. J. Biol. Chem., 276: 867-874, 2001.[Abstract/Free Full Text]
- Liang H., Petros A. M., Meadows R. P., Yoon H. S., Egan D. A., Walter K., Holzman T. F., Robins T., Fesik S. W. Solution structure of the DNA-biding domain of a HPV E2 protein: evidenced for flexible DNJA-binding regions. Biochemistry, 35: 2095-2103, 1996.[CrossRef][Medline]
- Corina K., Grossman S. R., Barsoum J., Prakash S. S., Androphy E. F., Pepinsky R. B. The tryptophan bridge is a critical feature of the papillomavirus E2 binding domain. Virology, 197: 391-396, 1993.[CrossRef][Medline]
- Hegde R. X., Androphy E. F. Crystal structure of the E2 DNA-binding domain from HPV type 16: implications for its DNA binding-site selection mechanism. J. Mol. Biol., 284: 1479-1489, 1998.[CrossRef][Medline]
- Koivunen E., Restel B. H., Rajotte D., Lahdenranta J., Hagedorn M., Arap W., Pasqualini R. Integrin-binding peptides derived from phage display libraries. Methods Mol. Biol., 129: 3-17, 1999.[Medline]
- Heino P., Zhou J., Lambert P. F. Interaction of the papillomavirus transcription/replication factor #2 and the viral capsid protein L2. Virology, 276: 3040-314, 2000.
Top
ABSTRACT
INTRODUCTION
0.05 or 0.1. One of replicate experiments was shown in Fig. 2