Alternatively spliced HPV-18 E6* protein inhibits E6 mediated degradation of p53 and suppresses transformed cell growth

Abstract

The E6 proteins originating from the tumour-associated Human Papillomavirus (HPV) types 16 and 18 have been shown to bind to and target the tumour suppressor protein, p53, for ubiquitin-mediated degradation. However, in cell lines derived from cervical neoplasias, the predominant early region transcripts are spliced and encode truncated forms of E6, termed E6*. We report here that HPV-18 E6* protein will interact both with the full-length E6 proteins from HPV-16 and HPV-18 and also with E6-AP, and subsequently blocks the association of full length E6 protein with p53. We also show that, as a result of this block, E6* can inhibit E6-mediated degradation of p53 both in vitro and in vivo. The biological consequences of this are increased transcriptional activity on p53-responsive promoters and an inhibition of cell growth in cells transfected with E6*. This is the ®rst report of a potential biological function for this polypeptide and may represent a means by which HPV is able to modulate the activity of the full-length E6 protein with respect to p53 during viral infection.

HPV-18 E6*I protein modulates the E6-directed degradation of p53 by binding to full-length HPV-18 E6

“…We have previously shown that overexpression of HPV-18 E6*I in CaSKi cells has an antiproliferative e ect, which was in marked contrast to the lack of e ect in cells which were negative for p53 and/or E6 (Pim et al, 1997). We demonstrated that this antiproliferative activity of E6*I correlated with its ability to inhibit E6 mediated degradation of p53 both in vitro and in vivo and with the subsequent increase in p53 transcriptional transactivation.…”

“…We had previously shown that overexpression of HPV-18 E6*I protein has a growth suppressive e ect in CaSKi cells, but not in cells lacking p53 and/or oncogenic E6 protein (Pim et al, 1997). It has been shown previously that p53 is not Figure 4 Binding of wild-type E6*I and deletion mutants D1, D2 and D3 expressed as GST-fusions, to: (a) in vitro transcribed/ translated E6-AP; (b) in vitro transcribed/translated full-length HPV-18 E6.…”

“…Tracks 3 ± 6: 4 ml of in vitro translated, radiolabelled p53 incubated with 3 ml of in vitro translated HPV-18E6 which had been pre-incubated with 20 ml of in vitro translated wild-type (WT) E6*I or mutants D1, D2 or D3 as indicated. Residual p53 was visualized by immunoprecipitation and polyacrylamide gel electrophoresis readily activated in HeLa cells but that it can be activated in CaSKi cells (Butz et al, 1995;Pim et al, 1997;Mantovani and Banks, 1999). In addition, since the mutant E6*I proteins bound HPV-16 E6 in a similar manner to HPV-18 E6 (data not shown), we proceeded to investigate the antiproliferative capacities of the three mutants in a CaSKi cell colony-forming assay.…”

“…We have previously demonstrated that E6*I expression leads to the transcriptional upregulation of p53 target genes in cells derived from cervical tumours (Pim et al, 1997). Since the induction of p53 leads to either cellcycle arrest (El-Deiry et al, 1993) or to the initiation of apoptosis (Clarke et al, 1993;Lowe et al, 1993), we were interested in whether either of these pathways was responsible for the antiproliferative e ects of E6*I.…”

“…The construction of the open reading frame for HPV-18 E6*I by PCR has been described previously (Pim et al, 1997). The cloning of this into vector pCDNA3 for in vivo expression, and into pSP64 for in vitro translation has also been previously described, as has the 95 kDa E6-AP clone and the full-length HPV-18 E6 clone used for in vitro translation (Pim et al, 1997).…”