Interaction with CBP/p300 enables the bovine papillomavirus type 1 E6 oncoprotein to downregulate CBP/p300-mediated transactivation by p53

Holger Zimmermann1, Choon-Heng Koh1, Roland Degenkolbe1, Mark J. O’Connorb,1, Andreas Müller2, Gertrud Steger2, Jason J. Chen3, Yun Lui3, Elliot Androphy3,4 and Hans-Ulrich Bernard1

Laboratory for Papillomavirus Biology, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore1
Institut für Virologie der Universität zu Köln, Cologne, Germany2
Department of Dermatology, New England Medical Center, Boston, MA, USA3
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111, USA4

Author for correspondence: Hans-Ulrich Bernard. Fax +65 779 1117. e-mail mcbhub{at}imcb.nus.edu.sg


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The E6 oncoprotein of bovine papillomavirus type 1 (BPV-1) can transform cells independently of p53 degradation. The precise mechanisms underlying this transformation are not yet completely understood. Here it is shown that BPV-1 E6 interacts with CBP/p300 in the same way as described for the E6 proteins of oncogenic human papillomaviruses. This interaction results in an inhibition of the transcriptional coactivator function of CBP/p300 required by p53 and probably by other transcription factors. The comparison of the CBP/p300-binding properties of BPV-1 E6 mutants previously characterized in transcription and transformation studies suggests (i) that the E6–CBP/p300 interaction may be necessary, but not sufficient, for cell transformation, and (ii) that the transcriptional activator function, inherent to the E6 protein, is not derived from forming a complex with CBP/p300.


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Bovine papillomavirus type 1 (BPV-1) causes fibropapillomas in its natural host (cattle), sarcoids in horses, non-productive fibromas in rodents after experimental infection and foci in cell cultures like mouse c127 fibroblasts (Howley, 1996 ). As there is great interest in human papillomaviruses (HPVs) as a cause of genital cancer (International Agency for Research on Cancer, 1995 ; zur Hausen, 1996 ), BPV-1 has served as a paradigm for various aspects of papillomavirus biology, making it one of the best-studied papillomaviruses. Initial subcloning studies mapped sequences responsible for cellular transformation to a fragment containing 69% of the BPV-1 genome (Lowy et al., 1980 ). Further dissection of this fragment revealed that genes E5 and E6 are the most important for the BPV-1-induced transformation process (Neary & DiMaio, 1989 ).

The 137 amino acid BPV-1 E6 protein contains two hypothetical zinc finger structures (Ullman et al., 1996 ; Rapp & Chen, 1998 ) that are conserved among HPVs. It is thought that the E6 oncoprotein brings about transformation by acting as an adaptor protein that interacts with important regulatory cellular proteins. An increasing number of cellular E6-binding proteins have been identified in the case of the E6 proteins of oncogenic HPVs, such as HPV-16, but only some of these have been studied in BPV-1. As a consequence of this, the mechanism of BPV-1-induced cell transformation is much less understood than that of HPV-16. Most importantly, BPV-1 E6 does not lead to degradation of the cell cycle regulator and transcriptional activator p53, as does the HPV-16 E6 protein, although it binds cofactor E6AP, which is involved in this reaction (Scheffner et al., 1990 ; Werness et al., 1990 ; Mietz et al., 1992 ; Das et al., 2000 ).

Our group and others have recently shown that HPV-16 E6 can abrogate p53 transcriptional function by another mechanism, namely by interfering with the formation of a complex between p53 and coactivator CBP/p300 (Zimmermann et al., 1999 ; Patel et al., 1999 ), which is required for p53-dependent transactivation. Here, we investigated whether BPV-1 E6 can interfere with p53 function in a similar manner, by capturing CBP/p300. These experiments were complemented with studies of the binding of BPV-1 E6 to several of the cellular proteins known to interact with HPV-16 E6. Furthermore, by exploiting BPV-1 E6 mutants characterized previously, we investigated the effect of such an interaction on the transforming phenotype of BPV-1 E6.

Interactions of a GST–BPV-1 E6 fusion protein with in vitro-translated cellular proteins were determined with microaffinity column assays as described previously (O’Connor et al., 1999 ; Zimmermann et al., 1999 ). Briefly, GST–E6 fusion proteins were bound to GSH columns, and the cellular proteins to be studied were translated in vitro and passed over the GST–E6-loaded columns. GST–HPV-11 E6 and GST–HPV-16 E6 served as negative and positive controls, respectively. As an initial control for the native conformation of all E6 proteins, we confirmed their ability to form homodimers (Fig. 1A; Daniels et al., 1997 ). As positive controls for E6-bound cellular proteins, we checked E6 binding to the E6BP and paxillin full-length proteins, and confirmed the binding of both cellular proteins to HPV-16 E6 and BPV-1 E6 (Fig. 1B; Chen et al., 1995 , 1997 ; Tong & Howley, 1997 ; Tong et al., 1997 ; Vande Pol et al., 1998 ). We also confirmed that paxillin binds BPV-1 E6 with greater strength than does HPV-16 E6 (Vande Pol et al., 1998 ).



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Fig. 1. Interactions of the E6 oncoproteins of BPV-1, HPV-11 and HPV-16 with the cellular proteins CBP/p300, E6BP, paxillin, hDLG and hMCM7. (A) A control experiment for the native confirmation of all three GST–E6 fusion proteins shows dimer formation between the GSH column-bound GST–E6 proteins and the respective in vitro-translated (IVT) E6 protein. (B) GST microaffinity columns were used to analyse interactions of the in vitro-translated and radiolabelled cellular proteins E6BP, paxillin, hDLG, hMCM7 and CBP with column-bound GST–E6 fusion proteins. The data show that BPV-1 E6 can bind CBP and hMCM7 and, as previously known, E6BP and paxillin. As controls, HPV-16 binds all five proteins, while HPV-11 fails to bind all except hMCM7. The lowest panel shows a representative Coomassie blue-stained SDS gel of GST and GST–E6 fusion proteins eluted from the microaffinity columns. (C) Schematic representation of CBP (Zimmermann et al., 1999 ). The C/H3 domain (aa 1765–1852) and two subclones, including one previously termed the TRAM domain (aa 1808–1826), are shown. GST–CBP fusion clones of these domains that bound BPV-1 E6, subclones 2,4 and 5, are shown as black bars. Those failing to bind, subclones 1, 3 and 6, are shown as grey bars. The outcome of the GST pulldown experiments is shown on the right (upper panel). The panel below shows a Coomassie blue-stained SDS gel of GST and GST–CBP fusion proteins. (D) Schematic representation of p300. Five GST–p300 subclones spanning the whole p300 protein are shown. The two panels to the right show that only subclones 1, 4 and 5 of p300 bind BPV-1 E6 and HPV-16 E6. The lowest panel shows the Coomassie blue-stained SDS gel of GST and GST–p300 fusion proteins.

 
Next, we examined the binding of BPV-1 E6 to the cellular proteins hDLG, hMCM7 and CBP (Fig. 1B). HPV-16 E6 bound hDLG as expected (Kiyono et al., 1997 ), but we could not detect an interaction with BPV-1 E6. This can be taken as support for the requirement of the XS/TXV/L motif, located at the C terminus of HPV-16 E6, for the interaction between the two proteins (Lee et al., 1997 ; Kiyono et al., 1997 ). Since this sequence is missing in the BPV-1 E6 protein it suggests that hDLG binding is not involved in BPV-1 E6-dependent transformation. In light of the observation that BPV-1 E6 binds paxillin with greater strength than HPV-16 E6 does, one can speculate that this strong interaction may compensate for the lack of a BPV-1 E6–hDLG interaction, since both cellular proteins function through membrane contacts that seem to influence cell shape.

The human minichromosome maintenance protein hMCM7, which is involved in the control of the cellular replication process, binds to E6 proteins of different HPV types. The hMCM7-binding domain was mapped to the N terminus of the HPV-16 E6 protein. Yeast two-hybrid results indicated a stronger binding of hMCM7 to the E6 proteins of oncogenic HPV types than to HPV-6 and -11 (Kukimoto et al., 1998 ), whereas our data and a previous publication (Kühne & Banks, 1998 ) show similar in vitro binding activities. Our experiments show that the BPV-1 E6 protein is also able to interact with hMCM7, although more weakly than HPV-16 E6. Although E6–hMCM7 binding does not seem to be a prerequisite for the carcinogenicity of HPV-16, it may be involved in transformation mechanisms.

Interestingly, Fig. 1(B) shows that CBP binds BPV-1 E6 in a manner similar to the previously reported binding to HPV-16 E6. This opens the possibility that BPV-1 interferes with p53 function without degradation of p53 by blocking the binding of CBP to p53 as in the case of HPV-16 E6.

We recently identified a transcriptional adaptor motif (TRAM) within a C-terminal segment of CBP (CBP II) as a binding site for HPV-16 E6 (Zimmermann et al., 1999 ). The TRAM motif is bracketed by the C/H3 domain, one of three segments of CBP that bind HPV-16 E6, as defined by another research group (Patel et al., 1999 ). Here, we examined the binding of this region of CBP to BPV-1 E6, and we observed, as shown in Fig. 1(C), that the whole C/H3 domain (subclone 2, aa 1765–1852), and specifically the TRAM motif (subclone 5, aa 1808–1826), binds BPV-1 E6. A deletion in the TRAM motif eliminates this interaction (subclone 6, aa 1808–1819).

Patel et al. (1999 ) observed that HPV-16 E6 binds two segments of CBP/p300 in addition to the C/H3 domain; the C/H1 domain within the N-terminal third and a C-terminal segment of the protein. In order to study whether BPV-1 E6 shows a conserved pattern of CBP/p300 interaction sites or deviates from this pattern, we divided the p300 protein into five fragments spanning amino acid residues 1–438, 439–1038, 1039–1452, 1453–1882 and 1883–2378. These fragments were expressed in the form of GST fusion proteins and their binding to in vitro-translated BPV-1 E6 and HPV-16 E6 proteins was studied. Fig. 1(D) shows binding of both papillomavirus E6 proteins to the 1–438 segment, which contains the C/H1 domain, as well as binding to the 1453–1882 and 1883–2378 fragments, the former containing the C/H3 domain and the TRAM motif. These observations suggest that very specific CBP/p300–E6 interaction mechanisms have been conserved between two viruses with a biology as different and remotely related to one another as the carcinogenic HPV-16 and the fibropapillomavirus BPV-1 (Chan et al., 1995 ).

To investigate binding of CBP, E6AP and paxillin to BPV-1 E6 mutants in vitro, several research groups have constructed BPV-1 E6 mutants to study structure–function relationships. Here, we used published mutants 212 (I41T), 228 (R46S; Y47H), 359 (C90S), 471 (N-terminal deletion of 11 aa), 491 (N-terminal deletion of 4 aa; Vousden et al., 1989 ) and GST fusion derivatives (J. J. Chen & E. J. Androphy, unpublished data). Mutant 212 has been reported to transform c127 cells as efficiently as wild-type BPV-1 E6, whereas mutant 228 shows a reduced transformation activity. Mutants 359, 471 and 491 show no transformation activity at all. We also examined mutant R42W, which has been instrumental in showing that transformation by BPV-1 E6 is independent of the transcription activation function inherent to this protein (Lamberti et al., 1990 ; Ned et al., 1997 ).

To evaluate the importance of the BPV-1 E6–CBP interaction for the transformation biology of BPV-1, we measured the affinities of these E6 mutants to CBP, E6AP and paxillin. Fig. 2(A) shows impairment of E6AP and paxillin binding to some in vitro-translated E6 mutant proteins, corresponding to published results (Chen et al., 1995 ; Tong & Howley, 1997 ; Vande Pol et al., 1998 ). Surprisingly, only one out of six mutants, mutant 359, was unable to bind CBP. This result could be confirmed by a reciprocal experiment with GST–BPV-1 E6 mutants and in vitro-translated cellular proteins (Fig. 2B). This shows that BPV-1 E6 can lose the ability to transform, while maintaining the competence to bind CBP.



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Fig. 2. In vitro interaction of BPV-1 E6 and BPV-1 E6 mutants with E6AP, paxillin and CBP. (A) BPV-1 E6 and BPV-1 E6 mutant proteins were in vitro-translated and assessed against GST–E6AP, GST–paxillin and GST–CBP fusion proteins immobilized on GSH beads. These experiments were done with the E6 interaction domains E6AP (aa 391–408) and CBP (aa 1765–1852), which is reflected in the small size increase of the two GST fusion proteins, relative to GST alone (lower panel). (B) Microaffinity column experiment using GST–BPV-1 E6 and GST–BPV1 E6 mutants, tested in a reciprocal experiment against IVT-CBP, in order to confirm some of the CBP binding results shown in (A). (C) Overexpression of full-length CBP leads to superactivation of p53-dependent transcription, which can be overcome by E6 proteins that are able to bind CBP (HPV-16 E6 mutant L50G, BPV-1 E6 and BPV-1 E6 mutant 228), but not by E6 proteins that do not bind CBP (HPV-11 E6 and BPV-1 E6 mutant 359). U2-OS cells were transfected with the reported p53-responsive PG13CAT or the control vector MG15 with mutated p53 binding sites.

 
To examine whether these in vitro data reflect the conditions encountered by the BPV-1 E6 and CBP proteins in vivo, we performed a yeast two-hybrid experiment. The full-size BPV-1 E6 protein in its wild-type form, the BPV-1 E6 mutant 359, and the HPV-11 E6 protein were cloned into the bait-vector pAS2-1. The CBP interaction domain (aa 1715–1870 and alternatively aa 1808–1852) was cloned into the activation domain vector pGAD10 (Clontech Laboratories) and sets of constructs were transformed into the yeast strain Y190. When tested for {beta}-galactosidase activity, both CBP constructs gave a colour reaction with X-Gal in the case of the BPV-1 E6 wild-type protein, while there was no detectable activity with the BPV-1 mutant or the HPV-11 E6 construct. We conclude that, in the eukaryotic nucleus, BPV-1 E6 interacts with CBP in a manner similar to the observations made in vitro.

As the interaction between HPV-16 E6 and CBP represses the transcriptional coactivator function of CBP targeted to the DNA-binding factor p53 (Zimmermann et al., 1999 ; Patel et al., 1999 ), one may hypothesize that the corresponding interaction of BPV-1 E6 in vitro may have a similar effect in vivo. To examine this possibility, we transfected U2-OS cells with either a reported p53-responsive construct (PG13CAT) or a control vector with mutated p53 binding sites. These recipient cells were cotransfected with a CBP expression vector and E6 expression vector coding for HPV-11 E6 (negative control); HPV-16 E6 mutant L50G, which is unable to degrade p53 but which interfers with the p53–CBP interaction (positive control; Nagakawa et al., 1995 ; Zimmermann et al., 1999 ); BPV-1 E6; or the BPV-1 E6 mutant 359, which is unable to bind CBP in vitro. We observed that the over-expression of CBP stimulates p53-dependent transcription (Zimmermann et al., 1999 ; data not shown), and that this effect can be overcome by the HPV-16 E6 L50G protein as well as by the BPV-1 E6 wild-type protein and mutant 228, which still bind CBP. However, CBP-dependent transcriptional stimulation of PG13CAT is unaffected by HPV-11 E6 or the BPV-1 E6 mutant protein 359, both of which are unable to bind CBP (Table 1). These data are evidence that the BPV-1 E6 oncoprotein can downregulate p53 transcriptional activity (and probably the activity of other transcription factors dependent on CBP/p300) in the same manner as the HPV-16 E6 protein, although it does not trigger the degradation of p53, as HPV-16 E6 does.


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Table 1. Comparative studies of E6 proteins and six cellular proteins

 
All binding experiments are summarized in Table 1 and are compared with the properties of E6 proteins to act as transcriptional activators, to transform c127 cells, and to repress CBP–p53 transcription. These comparisons show that the transcriptional activator function inherent to BPV-1 E6 does not stem from its binding of the CBP coactivator, since mutant R42W is able to bind CBP but unable to transactivate. Since there is at present no structural information available we cannot exclude that certain mutations disrupt the whole E6 structure. In summary, these data do not show a strict correlation between the transformation of c127 cells and the binding of E6AP, E6BP or at best, paxillin. However, the correlation is best in the case of the latter protein, as suggested by Das et al. (2000) . We take the correlation between transformation, CBP/p300 binding and CBP/p300-dependent transcription downregulation in the case of BPV-1 E6 wild-type and mutant 228, and the lack of all three functions in the case of mutant 359, as a suggestion that CBP/p300–E6 interaction is necessary, but not sufficient, to trigger full cellular transformation.


   Acknowledgments
 
We thank Edward Manser for the paxillin gene constructs, Tadahito Kanda for a GST–hMCM7 expression vector, and M. Ishibashi and T. Kiyono for plasmids encoding the hDLG gene.


   Footnotes
 
b Present address: KuDOS Pharmaceuticals Ltd, Cambridge CB4 4GW, UK.


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Received 25 February 2000; accepted 22 June 2000.