Alternatively spliced HPV-18 E6* protein inhibits E6 mediated degradation of p53 and suppresses transformed cell growth David Pim, Paola Massimi and Lawrence Banks International Centre for Genetic Engineering and Biotechnology. Padriciano 99, I-34012, Trieste, Italy 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. How- ever, 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 transcrip- tional 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. Keywords: HPV; E6; p53; transformation Introduction Human Papillomaviruses (HPVs) are a group of strongly epitheliotropic small DNA viruses, a subset of which infect the anogenital mucosa. Of this subset some HPV types, such as 6 and 11, are found associated with benign lesions whereas other types, such as 16 and 18, are commonly associated with lesions that can progress to high-grade cervical intraepithelial neoplasia and ultimately to cervical cancer (zur Hausen and Schneider, 1987). The early viral proteins E6 and E7 are continually expressed in cell lines derived from cervical tumours (Smotkin and Wettstein, 1986; Androphy et al., 1987; Banks et al., 1987). They interact with key cellular tumour suppressors; E7 with pRb (Dyson et al., 1989) and E6 with p53 (Werness et al., 1990), and can cooperate to immortalise primary human keratinocytes (Barbosa and Schlegel, 1989; Hawley-Nelson et al., 1989; Munger et al., 1989). These ®ndings are considered to be central to the role of these HPVs as initiators of tumourigenesis, and infection of cervical keratinocytes by these `high-risk' HPVs can therefore be said to predispose them to immortalisation and subsequent progression to malignancy. In contrast to the simple monocistronic mRNAs which encode E6 and E7 from the non oncogenic HPVs (Chow et al., 1987; Smotkin et al., 1989), the splicing pattern for the early viral transcripts of the oncogenic types, such as HPV-16 and HPV-18, is complex. These HPVs produce not only bi-cistronic mRNAs for E6 and E7, but also alternatively spliced mRNAs from which a large portion of E6 has been removed, with the mRNA splicing back into a dierent reading frame before reaching an adjacent termination codon. These alternatively spliced species are termed E6* and form the majority of early viral transcripts both in cervical tumours and in tumour-derived cell- lines (Schneider-GaÈdicke and Schwarz, 1986; Smotkin and Wettstein, 1986). Indeed in many tumours, transcripts for unspliced, full-length E6 are in extremely low abundance (Doorbar et al., 1990; Bohm et al., 1993; Grassmann et al., 1996). It was originally proposed that these splicing events increased the translation eciency for the E7 open reading frame by increasing the inter-cistronic spacing between E6 and E7 (Sedman et al., 1991). However, recent studies have demonstrated that E7 is, in fact, as eciently translated from E6-E7 bicistronic mRNA as it is from spliced transcripts (Stacey et al., 1995) and that mutation of the splice donor site in E6 has no eect upon the levels of PCNA induced by E7 in dierentiating keratinocytes (Cheng et al., 1995). These results imply that the splicing of E6 mRNA may have a dierent purpose. In addition, monoclonal antibodies made against a peptide derived from a putative HPV-18 E6* protein identi®ed a protein of the predicted molecular weight in nuclear extracts of HPV- 18 containing human tumour cells which had been grown in nude mice (Schneider-Gadicke et al., 1988). Interestingly, this same protein was not found in extracts from HPV-containing cell lines originally derived from cervical tumours, even though E6* mRNA is the majority species found in these lines (Schneider-GaÈ dicke et al., 1988). Since the potential to encode E6* proteins is a property of only the tumour-associated HPV types, considerable eort has been expended in attempting to demonstrate that E6* proteins possess a transforming activity. These studies in fact demonstrate the reverse, in that coexpression of E6* with E6 and E7 in primary human keratinocytes produces substantially fewer immortalised colonies than those obtained in the absence of E6* (Sedman et al., 1991). Previous studies have demonstrated some potential transcriptional transactivating activity for a form of E6* (E6*I) derived from HPV-16 (Shirasawa et al., 1994). In addition, a recent study demonstrated that the E6*IV form of E6* from HPV-16 was able to inhibit E6 directed degradation of p53 in vitro (Shally et al., Correspondence: D Pim Received 6 December 1996; revised 14 April 1997; accepted 16 April 1997 Oncogene (1997) 15, 257 ± 264  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00