International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy1
Author for correspondence: Lawrence Banks. Fax +39 040 226555. e-mail banks{at}icgeb.trieste.it
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Abstract |
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Many studies have been performed to investigate the mechanisms of action of HPV E6. High-risk E6 proteins, derived from HPV-16 and HPV-18, interact with a large number of cellular proteins whose functions are associated with the regulation of cell growth and survival. One of the most important of these interactions is with the p53 tumour suppressor protein (Werness et al., 1990 ). In this case, E6 targets p53 for ubiquitin-mediated degradation (Scheffner et al., 1990
; Huibregtse et al., 1991
) and thereby overcomes a vital checkpoint in the host cells defence against incorporation of DNA damage and subsequent progression to malignancy (Lane, 1992
; Wu & Levine, 1994
; Foster et al., 1994
; Kessis et al., 1993
).
Recent studies have demonstrated that the HPV-16 E6 and bovine papillomavirus type 1 (BPV-1) E6 can also interact with the p300/CBP co-activator protein (Patel et al., 1999 ; Zimmermann et al., 1999
, 2000
). As a result of this interaction, the transcriptional activity of p300 is greatly reduced, suggesting that E6 can interfere with the normal functioning of p300 (Patel et al., 1999
; Zimmermann et al., 1999
). This was a particularly important observation, since a number of other DNA tumour virus oncoproteins, including simian virus 40 LT and adenovirus E1a, have also been shown to require interaction with p300 in order to efficiently transform cells (Stein et al., 1990
; Smith & Ziff, 1988
; Eckner et al., 1996
). Thus, the demonstration that E6 could likewise inhibit p300 activity suggested that this interaction may be important for the ability of HPV E6 to contribute toward cell transformation. At present, however, there is little biological evidence to support this hypothesis. In addition, conflicting results have been published as to whether the interaction between E6 and p300 is restricted only to the oncogenic HPV types (Patel et al., 1999
; Zimmerman et al., 1999
).
We therefore proceeded to investigate the ability of high-and low-risk HPV E6 proteins to interact with p300 in a series of in vitro GST pull-down assays. Different regions of p300 were expressed as GST fusion proteins (Fig. 1A). These proteins were induced and purified as described previously (Massimi et al., 1996
), and then incubated with in vitro translated, radiolabelled HPV-11 E6 and HPV-18 E6. The results from representative binding assays are shown in Fig. 1(B)
. As can be seen, both E6 proteins show significant levels of interaction with p300. Interestingly, the fragments of p300 bound by both E6 proteins are the same, and overlap with the CH1, CH2 and CH3 domains of p300. No significant binding is seen with fragments of p300 spanning residues 6721193 and 18932414. This is largely in agreement with earlier observations where the CH1 and CH3 domains were bound by HPV-16 E6, although binding to CH2 was not assessed in those studies (Patel et al., 1999
; Zimmermann et al., 1999
). To further verify the specificity of these interaction assays, we then compared the relative ability of the HPV-11 and HPV-18 E6 proteins to bind p300 in comparison with their abilities to interact with p53 and the amino-terminal region of the Discs Large (Dlg) protein (NTdlg), as positive and negative control respectively (Werness et al., 1990
; Gardiol et al., 1999
). As can be seen, HPV-18 E6 binds strongly to p300 and, interestingly, this is similar to the level of interaction seen between p53 and p300 (Fig. 1C
). HPV-11 E6 also shows a significant degree of interaction with p300 when compared with the NTdlg negative control and p53 (Fig. 1D
), although the levels of interaction between HPV-11 E6 and p300 are considerably weaker than that observed with HPV-18 E6 (see Fig. 1C
and 1D
). Taken together, these results demonstrate that both HPV-18 and HPV-11 E6 also interact with p300, with stronger levels of association being observed with the high-risk E6 protein compared with the low-risk E6 protein.
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In this study the interaction between E6 and p300 has been further analysed. We found that the E6 proteins derived from both high- and low-risk HPV types were capable of interacting with p300. Taken together with the recent observation that BPV-1 E6 also interacts with p300 (Zimmermann et al., 2000 ), it indicates that this activity is conserved across many PV types, suggesting that this interaction may be a common requirement for virus replication. In addition, the HPV-18 and HPV-11 E6 binding sites on p300 overlap with the CH1, CH2 and CH3 domains of the protein, although at present we do not know whether these three regions of p300 are bound directly by E6, or whether any of these interactions are mediated via an unknown intermediate protein present within the reticulocyte lysate. However, previous studies would indicate that HPV-16 E6 binding to CH1 and CH3 was indeed direct (Patel et al., 1999
). Interestingly, the CH3 region of p300 is also the contact point for other viral transforming proteins such as adenovirus E1a, simian virus 40 LT and human immunodeficiency virus Tat (Eckner et al., 1994
, 1996
; Hottiger & Nabel, 1998
). In addition, as we have seen with HPV E6, adenovirus E1a also interacts with multiple sites on p300/CBP (Kurokawa et al., 1998
; Korzus et al., 1998
), implying that diverse viral transforming proteins may have evolved common strategies for interfering with the normal function of p300.
We then performed a series of studies to investigate directly the potential biological significance of the E6p300 interaction. Using a mutant of adenovirus E1a unable to bind p300 and, as a consequence, defective in transformation of primary BRK cells, we were able to efficiently rescue this defect by co-transfecting HPV E6. In contrast, a mutant of E6 that was unable to bind p300 failed to complement the defect of the adenovirus E1a mutant. This activity of E6 appears to be unrelated to its ability to degrade p53 since a mutant of E6 unable to degrade p53 nonetheless retained the ability to complement the E1a mutant. Interestingly, low-risk E6 was not as effective as high-risk E6 protein in these assays which is consistent with the weaker interaction with p300 demonstrated here, and supports previous studies where HPV-6 E6 had only marginal effects on p300 transcriptional activity (Patel et al., 1999 ).
Taken together, these results demonstrate that the interaction between E6 and p300 is biologically relevant and suggests that this may contribute towards E6-induced cellular transformation.
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References |
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Received 24 September 2001;
accepted 5 December 2001.