Department of Medical Microbiology and Immunology, Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA1
Author for correspondence: Van Wilson. Fax +1 409 845 3479. e-mail v-wilson{at}tamu.edu
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Abstract |
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Introduction |
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Bovine papillomavirus type 1 (BPV-1) serves as a prototype for the study of papillomavirus mechanisms of transcriptional regulation, transformation and DNA replication. BPV-1 is a member of a class of papillomaviruses that are able to induce proliferation in dermal fibroblasts as well as latent infection of basal epithelium and productive infection of differentiated epithelium. Wild-type BPV-1 replicates as an episome and is capable of inducing transformation when transfected into certain rodent cell types (Law et al., 1981 ). These characteristics of BPV-1 have been exploited for the identification of the viral gene products and mechanisms involved in the processes of BPV-1 DNA replication and transformation. While much information exists on the structure and function of the viral gene products, much less is known about hostvirus interactions, how viral proteins are modified by host systems and how modification may regulate the activities of viral products.
The major viral DNA replication protein of BPV-1 is the 605 amino acid phosphoprotein product of the entire E1 ORF (Sun et al., 1990 ). The 68 kDa E1 protein is an ATP-dependent helicase that specifically binds and unwinds the virus replication origin (Seo et al., 1993a
; Wilson & Ludes-Meyers, 1991
; Yang et al., 1993
). The binding site for the E1 protein lies within the replication origin in the BPV-1 long control region and consists of an 18 bp imperfect inverted repeat centred around the HpaI site at nt 1 (Holt & Wilson, 1995
; Holt et al., 1994
; Ustav et al., 1991
). Mapped functional domains of the E1 protein include a DNA-binding domain, an ATP-binding domain, a nuclear localization sequence and regions for interaction with the viral transcriptional transactivator protein E2 (Leng & Wilson, 1994
; Lentz et al., 1993
; MacPherson et al., 1994
; Sarafi & McBride, 1995
; Thorner et al., 1993
). Both the E1 and full-length E2 proteins are required in vivo for replication of BPV-1 origin-containing DNA (Spalholz et al., 1993
; Ustav & Stenlund, 1991
). Complex formation of the E1 protein with the 48 kDa transcriptional transactivator product of the E2 ORF enhances E1 binding to the origin in the presence of binding sites for the E1 and E2 proteins (Blitz & Laimins, 1991
; Mohr et al., 1990
; Sedman & Stenlund, 1995
; Seo et al., 1993b
). Apart from the E1 and E2 proteins, the DNA replication machinery is supplied by the host cell. E1 interacts with cellular polymerase pol-
(Park et al., 1994
) and replication factor A (Han et al., 1999
) and may serve to recruit these and other cellular replication factors to the virus replication origin. Recently, E1 has been shown to interact with and be phosphorylated by cyclin E/cyclin-dependent kinase (CDK) complexes, and this phosphorylation appears to be required for replication function (Cueille et al., 1998
; Ma et al., 1999
). Other phosphorylation sites have also been identified, although their functional significance remains less clear (Lentz et al., 1993
; Santucci et al., 1990
; Zanardi et al., 1997
).
From the above reports, it is apparent that the E1 protein is multifunctional and has complex regulation mediated through both post-translational modification and proteinprotein interactions. Mutational studies map most E1 functions to the central and C-terminal portions of the protein and it has been reported that deletion of the first 131 amino acids yields an E1 protein that retains replication capacity, though at a reduced level (Ferran & McBride, 1998 ). In order to evaluate possible functional contributions of the N-terminal region, we previously compared the available E1 sequences for conserved features within the first 100 amino acids (McShan & Wilson, 1997a
). Our examination revealed an N-terminal motif consisting of a serine followed by a stretch of acidic residues that was consistently present between amino acids 25 and 60 in human and animal papillomavirus E1 proteins. This conserved motif resembled the recognition sequence for casein kinase II (CKII) and we showed that the corresponding serine in BPV-1 E1, serine-48, was an efficient substrate for CKII in vitro. In the current study, we performed a mutational analysis of this serine to evaluate its potential functional contribution to viral DNA replication. The mutational results are consistent with a requirement for a negative charge at this residue, such as would be supplied by phosphorylation, and support a positive phosphoregulatory role for this residue in activating E1 replication function.
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Methods |
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Cell culture.
C127 cells used in focus formation and transient DNA replication assays were grown in an atmosphere of 5% CO2 in Dulbeccos modified Eagles medium (JRH BioSciences) supplemented with 10% foetal bovine serum (HyClone), 0·1% penicillinstreptomycin and 0·25% fungizone (both from JRH BioSciences). Chinese hamster ovary (CHO) cells used in transient DNA replication assays were grown in HAMs F12 medium supplemented as above and were also maintained in 5% CO2.
Focus formation assay.
C127 cells were 80% confluent when harvested for electroporation. Electroporations for transformation assays were done with 50 ng or 1 µg supercoiled pdBPV.1 and pdBPV.1(S48G) DNA, harvested by large-scale preparation (Qiagen) from E. coliTB1, which had been cut with BamHI to liberate plasmid sequences and religated. Carrier DNA was added to a total DNA content of 50 µg per electroporation. Each electroporation used 2x106 cells suspended in 250 µl growth medium as described above with 5 mM BES buffer, pH 7·2. Electroporation conditions were 960 µF and 230 V. Following electroporation, the contents of each cuvette were resuspended in 10 ml DMEM growth medium with 5 mM BES and 1 ml of the final cell suspension was applied to each of three 100 mm2 plates containing 10 ml DMEM growth medium with 5 mM BES. Transfected cells were maintained for 21 days (50 ng electroporations) or 14 days (1 µg electroporations), at which time they were fixed with 70% isopropanol and stained with 1% methylene blue in 70% isopropanol. Foci were then counted.
Transient DNA replication assays.
Transient in vivo replication assays were performed as described previously (McShan & Wilson, 1997b ). Wild-type and mutant pdBPV.1 DNAs were assayed in C127 cells and the BPV-1 sequences were separated from the vector by BamHI digestion prior to electroporation. Five µg wild-type or mutant BPV DNA was used per electroporation. The wild-type or mutant pCH-E1 expression vectors were tested for ability to support replication of an origin-positive plasmid, pBOR, in CHO cells. Electroporations were done with 5 µg supercoiled pBOR (Holt & Wilson, 1995
), 5 µg wild-type or mutant pCH-E1 and 3 µg of the E2 protein-expression vector pCEAG-E2 (provided by Arne Stenlund, Cold Spring Harbor Laboratory), with carrier DNA to a total DNA content of 50 µg.
Western blotting of pCH-E1 and pCH-E1(S48G) expressed proteins.
Ten µg pCH-E1, pCH-E1(S48G) and control pCH110 was transfected individually into COS-1 cells by electroporation as described previously (Leng & Wilson, 1994 ). At 72 h post-transfection, cells were lysed on ice in culture dishes by using SDS gel sample buffer (50 mM TrisHCl, pH 6·8, 10% glycerol, 2% SDS, 2% 2-mercaptoethanol, 0·01% bromophenol blue). Fifteen µl of each sample was loaded on 10% SDSpolyacrylamide gels and electrophoresed (Laemmli, 1970
). Proteins were transferred electrophoretically from the SDS gels to Hybond-ECL Western nitrocellulose membrane (Amersham) at 2·5 mA/cm2 of gel in a Polyblot unit (American Bionetics). Immunological detection of the expressed wild-type and S48G E1 proteins was performed by using E1-specific polyclonal antisera (5996 and 5997; Sandler et al., 1993
), according to the manufacturers recommendations for the enhanced chemiluminescence detection procedure (Amersham). Western blots of bacterially expressed E1 protein were performed as described previously (Wilson & Ludes-Meyers, 1991
).
Yeast one-hybrid assay.
The one-hybrid system is as described previously and consisted of two reporter strains, p53BS-LACZ and E1BST-LACZ, containing minimal promoters with triplet copies of either the p53-binding site or E1-binding site, respectively (Gonzalez et al., 2000 ). Introduction of pGAD-E1(F), which expresses a GAL4 activation domain (AD)E1 fusion protein, induces
-galactosidase in the E1BST-LACZ strain with minimal induction in the p53BS-LACZ strain. For the current study, the S48G and S48D mutations were introduced into pGAD-E1(F) by using the QuikChange kit (Stratagene) as above. Each mutant plasmid DNA was transformed into both the E1BST-LACZ and p53BS-LACZ strains and five independent clones were tested for
-galactosidase activity by a colony filter assay as described previously (Gonzalez et al., 2000
). All five clones for each mutant exhibited similar phenotypes and a representative clone for each was chosen for quantitative
-galactosidase measurement by liquid culture assay with CRPG (chlorophenol red
-D-galactopyranoside) as the substrate (Gonzalez et al., 2000
).
Yeast two-hybrid system.
The ability of wild-type and mutant E1 proteins to interact with themselves and with E2 protein was tested in vivo in yeast SFY526 cells. For the E1E2 studies, the wild-type and mutant (S48G or S48D) E1 genes were cloned into pGBT9 to produce vectors (pGBT9E1) that expressed GAL4 DNA-binding domain (DBD)E1 fusion proteins. Likewise, the full-length E2 gene was cloned into pGAD424 to generate a vector (pGADE2) that expressed a GAL4 ADE2 fusion protein. Each purified pGBT9E1 DNA was co-transfected into SFY526 cells along with pGADE2 DNA and double transformants were selected on minimal medium lacking leucine and tryptophan (SD/-L/-T). Corresponding control clones were generated by co-transforming the wild-type and mutant pGBT9E1 DNAs with the parental pGAD424 vector that expresses only the unfused AD. Five independent clones were chosen from each transformation and fusion protein expression was verified by Western blotting. For each set of five clones, the -galactosidase expression phenotypes were similar by the colony filter assay, so a representative clone was chosen for quantitative liquid culture
-galactosidase determination as above.
The E1E1 interaction was examined in a similar way to the E1E2 interaction. To complement the pGBT9E1 vectors (DBDE1 fusions), wild-type and mutant E1 DNAs were cloned into pGAD424 to generate the corresponding ADE1 fusion proteins. Homologous pairs of constructs (DBDE1 plus ADE1) were co-transfected into SFY526 cells and selected on SD/-L/-T medium and five clones for each pair were characterized as for the E1E2 pairs.
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Results |
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E1 protein with the S48G mutation fails to support transient DNA replication in vivo
Since the stable replication ability of S48G BPV-1 could not be assessed, the replication function was examined in transient assays. The ability to replicate transiently as an episome when transfected into C127 cells is a well-established property of the wild-type BPV-1 genome and requires a functional E1 protein (Spalholz et al., 1993 ; Ustav & Stenlund, 1991
; Ustav et al., 1991
). BPV-1 genomic DNA containing the S48G mutation was compared with the wild-type genome in a standard transient replication assay. The wild-type genomic BPV-1 DNA replicated as expected, producing a DpnI-resistant product (Fig. 2
, lane 3). In contrast, four independent preparations of the S48G mutant genomic DNA gave no detectable replication (Fig. 2
, lanes 47).
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Recently, we developed a yeast one-hybrid system for assaying binding of E1 to its recognition sequence in vivo (Gonzalez et al., 2000 ). In this system, wild-type E1 is expressed as a fusion with the GAL4 AD and is tested in reporter strains that have an integrated lacZ gene downstream of a minimal promoter containing triple copies of either the E1-binding site (E1BS) or the control p53-binding site (p53BS). The wild-type ADE1 fusion protein produced substantially higher
-galactosidase expression in the E1BS reporter strain compared with the p53BS strain, while the AD protein alone could not activate either promoter (Fig. 5A
; data not shown). These results confirmed that E1 could bind its recognition sequence specifically in vivo in the context of the yeast genome. To examine the binding properties of the serine-48 mutants in vivo, the S48G and S48D mutations were each engineered into the ADE1-expression vector (pGBT9E1) and were tested for promoter activation (Fig. 5A
). Both mutants activated the E1BS promoter specifically compared with the control p53BS promoter and the levels of
-galactosidase expression were comparable to those observed with the wild-type ADE1 fusion. These combined in vitro and in vivo results demonstrate that the structural integrity of the DNA-binding domain was not disrupted significantly by the S48G substitution and, consequently, lack of origin recognition cannot account for the observed total replication defect. Furthermore, introduction of the constitutive negative charge at residue 48 did not increase the origin-binding capacity of E1, so it is unlikely that the phosphorylation of this residue is necessary to activate DNA binding by E1.
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In order to evaluate E1E1 interaction, wild-type and mutant E1 proteins were tested as homologous pairs in the two-hybrid system (Fig. 5C). When wild-type E1 was expressed as a GAL4 DBDE1 fusion in conjunction with the unfused AD, there was no detectable
-galactosidase expression, while the converse pair (ADE1 plus unfused DBD) gave a low but consistent background of
-galactosidase activity. Co-expressing the ADE1 plus DBDE1 fusions resulted in a 10-fold stimulation of
-galactosidase activity, consistent with an E1E1 interaction in vivo. Both of the ADE1 and DBDE1 fusions have also been tested against other partners not known to bind E1, and no stimulation of
-galactosidase activity above background was observed (data not shown), confirming that E1 is not highly promiscuous for protein binding. We conclude from these studies that E1 exhibits intrinsic self-association in vivo that is consistent with the observed capacity of E1 to oligomerize in vitro (Fouts et al., 1999
; Sedman & Stenlund, 1996
, 1998
). Like wild-type E1, both the S48D and S48G mutants exhibited in vivo self-association, although their fold stimulations over background were less than for wild-type (approximately 3- and 4-fold, respectively, compared with 10-fold for wild-type). Nonetheless, both mutants clearly retained self-interaction capacity and, again, the similarity between the S48D and S48G mutants suggests that any absolute difference from wild-type is not sufficient to explain replication differences.
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Discussion |
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While a cellular kinase that modifies serine-48 in vivo has not been defined, the most likely candidate is CKII. CKII is a ubiquitous enzyme that is an essential serine/threonine kinase in Saccharomyces and Dictyostelium (Litchfield et al., 1994 ). CKII phosphorylates numerous substrates, including many transcription factors (Allende & Allende, 1995
; Issinger, 1993
) and the simian virus 40 origin recognition protein, large T antigen (Rihs & Peters, 1989
; Rihs et al., 1991
). Phosphorylation by CKII has been shown to modulate both DNA binding and proteinprotein interactions and these activities were examined as possible explanations for the replication defect of the S48G mutant E1 protein. However, the S48G mutant showed no gross defect in origin binding, E2 interaction or E1 interaction in vivo compared to the wild-type E1 or the pseudophosphorylated S48D E1 mutant. It should be noted, however, that subtle defects in any of these interactions might be sufficient to prevent formation of a functional replication complex, yet not be detected in the yeast one- and two-hybrid systems. In addition, there are now several known interactions between E1 and host cell proteins (Bonne-Andrea et al., 1995
; Han et al., 1999
; Liu et al., 1998
; Park et al., 1994
; Swindle & Engler, 1998
; Yasugi et al., 1997
), any one of which could be affected by phosphorylation of serine-48. Consequently, the mechanistic basis for the severe replication defect of the S48G mutant remains to be determined.
The global phosphoregulation of papillomavirus E1 proteins is still largely unexplored. The full-length BPV-1 E1 protein exhibits multiple phosphorylations on serine residues and at least one threonine phosphorylation at residue 102, although mutation of threonine-102 had no effect on viral DNA replication (Lentz et al., 1993 ). More recently, both serine-90 (Lambert et al., 1990
; Zanardi, 1997
) and serine-109 (Zanardi et al., 1997
) of BPV-1 E1 were shown to be phosphorylated in vivo. Mutation of either serine-90 or -109 to an alanine resulted in increased transient DNA replication in vivo. Consistent with the increase in replication, mutant E1 protein expressed in and purified from Sf9 cells exhibited increased origin binding (S90A) and increased helicase activity (S109A) in vitro, suggesting that phosphorylation at either site had a negative regulatory effect. Conversely, positive phosphoregulation of E1 replication activity has been demonstrated for HPV-11 E1 via CDKs (Ma et al., 1999
), although the mechanistic basis for the enhanced replication was not explored. As both the cyclin/CDK-binding motif (RXL motif) and candidate phosphorylation sites for CDKs are present in other E1 proteins, it is likely that E1 activation by CDK phosphorylation will be a general regulatory mechanism. Our results with the serine-48 mutations are consistent with phosphorylation of this residue also being required for activation of some E1 replication function. Interestingly, while our S48G mutant was totally unable to support detectable replication, Ferran & McBride (1998)
demonstrated that an E1 protein deleted for the first 131 amino acids retained at least minimal replication function. A possible explanation consistent with the available phosphorylation data is that the N terminus of E1 protein contains phosphorylation sites that regulate E1 activity positively (serine-48 and possibly other residues) and negatively (serine-90, serine-109 and possibly other residues) in a combinatorial fashion. Mutation of serine-48 to a non-phosphorylatable residue would eliminate an activation event without affecting phosphorylation of the negative regulatory sites, the net result being a constitutively inactive E1 protein. In contrast, removal of the entire N-terminal region might eliminate both the positive and negative regulatory control, resulting in an E1 protein with the observed basal activity. The existence of multiple positive and negative modification sites suggests a complex regulatory network that could modulate E1 activity incrementally as a function of both cell cycle and cell differentiation state. This combinatorial system may provide the precise control of virus replication required for the long-term persistence of papillomavirus infections in the epidermis.
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Acknowledgments |
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Footnotes |
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References |
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Belyavskyi, M., Westerman, M., Dimichele, L. & Wilson, V. G. (1996). Perturbation of the host cell cycle and DNA replication by the bovine papillomavirus replication protein E1.Virology 219, 206-219.[Medline]
Berg, L. J., Singh, K. & Botchan, M. (1986). Complementation of a bovine papilloma virus low-copy-number mutant: evidence for a temporal requirement of the complementing gene.Molecular and Cellular Biology 6, 859-869.[Medline]
Bergman, P., Ustav, M., Sedman, J., Moreno-Lopez, J., Vennstrom, B. & Pettersson, U. (1988). The E5 gene of bovine papillomavirus type 1 is sufficient for complete oncogenic transformation of mouse fibroblasts.Oncogene 2, 453-459.[Medline]
Blitz, I. L. & Laimins, L. A. (1991). The 68-kilodalton E1 protein of bovine papillomavirus is a DNA binding phosphoprotein which associates with the E2 transcriptional activator in vitro. Journal of Virology 65, 649-656.[Medline]
Bonne-Andrea, C., Santucci, S., Clertant, P. & Tillier, F. (1995). Bovine papillomavirus E1 protein binds specifically DNA polymerase but not replication protein A.Journal of Virology 69, 2341-2350.[Abstract]
Chen, G. & Stenlund, A. (1998). Characterization of the DNA-binding domain of the bovine papillomavirus replication initiator E1. Journal of Virology 72, 2567-2576.
Chiang, C. M., Broker, T. R. & Chow, L. T. (1992a). Properties of bovine papillomavirus E1 mutants.Virology 191, 964-967.[Medline]
Chiang, C. M., Ustav, M., Stenlund, A., Ho, T. F., Broker, T. R. & Chow, L. T. (1992b). Viral E1 and E2 proteins support replication of homologous and heterologous papillomaviral origins.Proceedings of the National Academy of Sciences, USA 89, 5799-5803.[Abstract]
Cueille, N., Nougarede, R., Mechali, F., Philippe, M. & Bonne-Andrea, C. (1998). Functional interaction between the bovine papillomavirus virus type 1 replicative helicase E1 and cyclin ECDK2.Journal of Virology 72, 7255-7262.
Ferran, M. C. & McBride, A. A. (1998). Transient viral DNA replication and repression of viral transcription are supported by the C-terminal domain of the bovine papillomavirus type 1 E1 protein.Journal of Virology 72, 796-801.
Fouts, E. T., Yu, X., Egelman, E. H. & Botchan, M. R. (1999). Biochemical and electron microscopic image analysis of the hexameric E1 helicase.Journal of Biological Chemistry 274, 4447-4458.
Gonzalez, A., Bazaldua-Hernandez, C., West, M., Woytek, K. & Wilson, V. G. (2000). Identification of a short, hydrophilic amino acid sequence critical for origin recognition by the bovine papillomavirus E1 protein.Journal of Virology 74, 245-253.
Han, Y., Loo, Y.-M., Militello, K. T. & Melendy, T. (1999). Interactions of the papovavirus DNA replication initiator proteins, bovine papillomavirus type 1 E1 and simian virus 40 large T antigen, with human replication factor A.Journal of Virology 73, 4899-4907.
Holt, S. E. & Wilson, V. G. (1995). Mutational analysis of the 18-base-pair inverted repeat element at the bovine papillomavirus origin of replication: identification of critical sequences for E1 binding and in vivo replication.Journal of Virology 69, 6525-6532.[Abstract]
Holt, S. E., Schuller, G. & Wilson, V. G. (1994). DNA binding specificity of the bovine papillomavirus E1 protein is determined by sequences contained within an 18-base-pair inverted repeat element at the origin of replication.Journal of Virology 68, 1094-1102.[Abstract]
Issinger, O. G. (1993). Casein kinases: pleiotropic mediators of cellular regulation. Pharmacology & Therapeutics59, 130.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 277, 680-685.
Lambert, P. F., Monk, B. C. & Howley, P. M. (1990). Phenotypic analysis of bovine papillomavirus type 1 E2 repressor mutants.Journal of Virology 64, 950-956.[Medline]
Law, M. F., Lowy, D. R., Dvoretzky, I. & Howley, P. M. (1981). Mouse cells transformed by bovine papillomavirus contain only extrachromosomal viral DNA sequences.Proceedings of the National Academy of Sciences, USA 78, 2727-2731.[Abstract]
Le Moal, M. A., Yaniv, M. & Thierry, F. (1994). The bovine papillomavirus type 1 (BPV1) replication protein E1 modulates transcription activation by interacting with BPV1 E2.Journal of Virology 68, 1085-1093.[Abstract]
Leng, X. & Wilson, V. G. (1994). Genetically defined nuclear localization signal sequence of bovine papillomavirus E1 protein is necessary and sufficient for the nuclear localization of E1-galactosidase fusion proteins.Journal of General Virology 75, 2463-2467.[Abstract]
Leng, X., Ludesmeyers, J. H. & Wilson, V. G. (1997). Isolation of an amino-terminal region of bovine papillomavirus type 1 E1 protein that retains origin binding and E2 interaction capacity.Journal of Virology 71, 848-852.[Abstract]
Lentz, M. R., Pak, D., Mohr, I. & Botchan, M. R. (1993). The E1 replication protein of bovine papillomavirus type 1 contains an extended nuclear localization signal that includes a p34cdc2 phosphorylation site.Journal of Virology 67, 1414-1423.[Abstract]
Litchfield, D. W., Dobrowolska, G. & Krebs, E. G. (1994). Regulation of casein kinase II by growth factors: a re-evaluation. Cellular & Molecular Biology Research40, 373381.[Medline]
Liu, J. S., Kuo, S. R., Makhov, A. M., Cyr, D. M., Griffith, J. D., Broker, T. R. & Chow, L. T. (1998). Human HSP70 and HSP40 chaperone proteins facilitate human papillomavirus-11 E1 protein binding to the origin and stimulate cell-free DNA replication. Journal of Biological Chemistry 273, 30704-30712.
Lusky, M. & Fontane, E. (1991). Formation of the complex of bovine papillomavirus E1 and E2 proteins is modulated by E2 phosphorylation and depends upon sequences within the carboxyl terminus of E1. Proceedings of the National Academy of Sciences, USA 88, 6363-6367.[Abstract]
Ma, T. L., Zou, N. X., Lin, B. Y., Chow, L. T. & Harper, J. W. (1999). Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication.Proceedings of the National Academy of Sciences, USA 96, 382-387.
MacPherson, P., Thorner, L., Parker, L. M. & Botchan, M. (1994). The bovine papilloma virus E1 protein has ATPase activity essential to viral DNA replication and efficient transformation in cells.Virology 204, 403-408.[Medline]
McShan, G. D. & Wilson, V. G. (1997a). Casein kinase II phosphorylates bovine papillomavirus type 1 E1 in vitro at a conserved motif.Journal of General Virology 78, 171-177.[Abstract]
McShan, G. D. & Wilson, V. G. (1997b). Reconstitution of a functional bovine papillomavirus type 1 origin of replication reveals a modular tripartite replicon with an essential AT-rich element. Virology 237, 198-208.[Medline]
Mansky, K. C., Batiza, A. & Lambert, P. F. (1997). Bovine papillomavirus type 1 E1 and simian virus 40 large T antigen share regions of sequence similarity required for multiple functions.Journal of Virology 71, 7600-7608.[Abstract]
Mohr, I. J., Clark, R., Sun, S., Androphy, E. J., MacPherson, P. & Botchan, M. R. (1990). Targeting the E1 replication protein to the papillomavirus origin of replication by complex formation with the E2 transactivator.Science 250, 1694-1699.[Medline]
Moscufo, N., Sverdrup, F., Breiding, D. E. & Androphy, E. J. (1999). Two distinct regions of the BPV1 E1 replication protein interact with the activation domain of E2.Virus Research 65, 141-154.[Medline]
Park, P., Copeland, W., Yang, L., Wang, T., Botchan, M. R. & Mohr, I. J. (1994). The cellular DNA polymerase alpha-primase is required for papillomavirus DNA replication and associates with the viral E1 helicase.Proceedings of the National Academy of Sciences, USA 91, 8700-8704.[Abstract]
Ravnan, J. B., Gilbert, D. M., Ten-Hagen, K. G. & Cohen, S. N. (1992). Random-choice replication of extrachromosomal bovine papillomavirus (BPV) molecules in heterogeneous, clonally derived BPV-infected cell lines. Journal of Virology 66, 6946-6952.[Abstract]
Rihs, H.-P. & Peters, R. (1989). Nuclear transport kinetics depend on phosphorylation-site-containing sequences flanking the karyophilic signal of the simian virus 40 T-antigen.EMBO Journal 8, 1479-1484.[Abstract]
Rihs, H. P., Jans, D. A., Fan, H. & Peters, R. (1991). The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen. EMBO Journal 10, 633-639.[Abstract]
Sandler, A. B., Vande-Pol, S. B. & Spalholz, B. A. (1993). Repression of bovine papillomavirus type 1 transcription by the E1 replication protein.Journal of Virology 67, 5079-5087.[Abstract]
Santucci, S., Androphy, E. J., Bonne-Andrea, C. & Clertant, P. (1990). Proteins encoded by the bovine papillomavirus E1 open reading frame: expression in heterologous systems and in virally transformed cells.Journal of Virology 64, 6027-6039.[Medline]
Sarafi, T. R. & McBride, A. A. (1995). Domains of the BPV-1 E1 replication protein required for origin-specific DNA binding and interaction with the E2 transactivator.Virology 211, 385-396.[Medline]
Sedman, J. & Stenlund, A. (1995). Co-operative interaction between the initiator E1 and the transcriptional activator E2 is required for replicator specific DNA replication of bovine papillomavirus in vivo and in vitro.EMBO Journal 14, 6218-6228.[Abstract]
Sedman, J. & Stenlund, A. (1996). The initiator protein E1 binds to the bovine papillomavirus origin of replication as a trimeric ring-like structure.EMBO Journal 15, 5085-5092.[Abstract]
Sedman, J. & Stenlund, A. (1998). The papillomavirus E1 protein forms a DNA-dependent hexameric complex with ATPase and DNA helicase activities.Journal of Virology 72, 6893-6897.
Sedman, T., Sedman, J. & Stenlund, A. (1997). Binding of the E1 and E2 proteins to the origin of replication of bovine papillomavirus.Journal of Virology 71, 2887-2896.[Abstract]
Seo, Y. S., Müller, F., Lusky, M. & Hurwitz, J. (1993a). Bovine papilloma virus (BPV)-encoded E1 protein contains multiple activities required for BPV DNA replication.Proceedings of the National Academy of Sciences, USA 90, 702-706.[Abstract]
Seo, Y.-S., Müller, F., Lusky, M., Gibbs, E., Kim, H.-Y., Phillips, B. & Hurwitz, J. (1993b). Bovine papilloma virus (BPV)-encoded E2 protein enhances binding of E1 protein to the BPV replication origin.Proceedings of the National Academy of Sciences, USA 90, 2865-2869.[Abstract]
Spalholz, B. A., McBride, A. A., Sarafi, T. & Quintero, J. (1993). Binding of bovine papillomavirus E1 to the origin is not sufficient for DNA replication. Virology 193, 201-212.[Medline]
Sun, S., Thorner, L., Lentz, M., MacPherson, P. & Botchan, M. (1990). Identification of a 68-kilodalton nuclear ATP-binding phosphoprotein encoded by bovine papillomavirus type 1.Journal of Virology 64, 5093-5105.[Medline]
Swindle, C. S. & Engler, J. A. (1998). Association of the human papillomavirus type 11 E1 protein with histone H1. Journal of Virology 72, 1994-2001.
Syrjanen, K. J. (1989). Epidemiology of human papillomavirus (HPV) infections and their associations with genital squamous cell cancer.APMIS 97, 957-970.[Medline]
Thorner, L. K., Lim, D. A. & Botchan, M. R. (1993). DNA-binding domain of bovine papillomavirus type 1 E1 helicase: structural and functional aspects.Journal of Virology 67, 6000-6014.[Abstract]
Ustav, M. & Stenlund, A. (1991). Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.EMBO Journal 10, 449-457.[Abstract]
Ustav, M., Ustav, E., Szymanski, P. & Stenlund, A. (1991). Identification of the origin of replication of bovine papillomavirus and characterization of the viral origin recognition factor E1.EMBO Journal 10, 4321-4329.[Abstract]
Wilson, V. G. & Ludes-Meyers, J. (1991). A bovine papillomavirus E1-related protein binds specifically to bovine papillomavirus DNA.Journal of Virology 65, 5314-5322.[Medline]
Yang, L., Mohr, I., Fouts, E., Lim, D. A., Nohaile, M. & Botchan, M. (1993). The E1 protein of bovine papilloma virus 1 is an ATP-dependent DNA helicase.Proceedings of the National Academy of Sciences, USA 90, 5086-5090.[Abstract]
Yasugi, T., Vidal, M., Sakai, H., Howley, P. M. & Benson, J. D. (1997). Two classes of human papillomavirus type 16 E1 mutants suggest pleiotropic conformational constraints affecting E1 multimerization, E2 interaction, and interaction with cellular proteins.Journal of Virology 71, 5942-5951.[Abstract]
Zanardi, T. (1997). Regulation of bovine papillomavirus DNA replication by phosphorylation of the viral E1 protein. PhD dissertation, Texas A&M University, College Station, TX, USA.
Zanardi, T. A., Stanley, C. M., Saville, B. M., Spacek, S. M. & Lentz, M. R. (1997). Modulation of bovine papillomavirus DNA replication by phosphorylation of the viral E1 protein.Virology 228, 1-10.[Medline]
Received 25 February 2000;
accepted 27 April 2000.