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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Abstract |
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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 GSTBPV-1 E6 fusion protein with in vitro-translated cellular proteins were determined with microaffinity column assays as described previously (OConnor et al., 1999 ; Zimmermann et al., 1999
). Briefly, GSTE6 fusion proteins were bound to GSH columns, and the cellular proteins to be studied were translated in vitro and passed over the GSTE6-loaded columns. GSTHPV-11 E6 and GSTHPV-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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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 E6hMCM7 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 17651852), and specifically the TRAM motif (subclone 5, aa 18081826), binds BPV-1 E6. A deletion in the TRAM motif eliminates this interaction (subclone 6, aa 18081819).
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 1438, 4391038, 10391452, 14531882 and 18832378. 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 1438 segment, which contains the C/H1 domain, as well as binding to the 14531882 and 18832378 fragments, the former containing the C/H3 domain and the TRAM motif. These observations suggest that very specific CBP/p300E6 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 structurefunction 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 E6CBP 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 GSTBPV-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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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 p53CBP 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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Acknowledgments |
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Footnotes |
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References |
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Received 25 February 2000;
accepted 22 June 2000.