Cell. Vol. 67, 547-556,

November 1, 1991, Copyright 0 1991 by Cell Press

Degradation of ~53 Can Be Targeted by HPV E6 Sequences Distinct from Those Required for ~53 Binding and Trans-Activation Tim Crook, John A. Tidy,’ and Karen H. Vousden Ludwig Institute for Cancer Research St. Mary’s Hospital Medical School Norfolk Place, London W2 1PG England ‘Department of Gynaecological Oncology Samaritan Hospital for Women Marylebone Road London NW1 5YE England

Summary Human papillomavirus (HPV) types 16 and 18 appear to play a role in the development of ano-genital malignancies, whereas HPV 6 and 11 are usually associated wlth benign lesions. One HPV16 oncoprotein, E6, complexes with and promotes degradation of the cellular tumor suppressor ~53. Here we show that E6 proteins of both oncogenlc and benign HPV types associate in vitro with ~53, but binding by E6 proteins of benign HPV types cannot target p53 for degradation. A C-terminal region of E6 conserved among all HPV types is important for ~53 binding. However, N-terminal sequences of E6 conserved only between oncogenie HPV types are necessary to direct ~53 degradation. ~53 binding by E6 appears necessary but not sufficient for this activity. All E6 proteins tested showed comparable transcriptional trans-activating activity, a property that does not require the ability to bind or direct degradation of ~53. Introduction Over 60 different types of lluman papillomavirus (HPV) have been identified, many of which infect ano-genital epithelium (De Villiers, 1989). These genital HPV types can be placed into one of two broad groups: those that are predominantly associated with benign hyperproliferations and appear to carry only a low risk for malignant progression and those frequently found associated with anogenital carcinomas, which are considered high risk, or oncogenic, HPV types (Vousden, 1989). The most common oncogenic genital HPV types are HPV16 and HPV18, and a large body of experimental evidence clearly indicates that theseviral DNAs encode transforming and immortalizing activities in rodent and human cells (Vousden, 1990). These activities have been localized to two viral open reading frames, E6 and E7, and although E7 shows the predominant transforming and immortalizing activities in rodent cells (Kandaet al., 1988; Phelps et al., 1988; Vousden et al., 1988; Bedell et al., 1989). cooperation between E6 and E7 is necessary for efficient immortalization of primary human genital epithelial cells (Hawley-Nelson et al., 1989; Munger et al., 1989). Many studies of primary ano-genital carcinomas have revealed that only the E6 and E7 region

of these HPV types is consistently retained and expressed in these tumors (Schneider-Gadicke and Schwarz, 1986; Smotkin and Wettstein, 1986) supporting the hypothesis that these viral proteins are contributing to the development and possibly also the maintenance of the malignant phenotype. Interestingly, the E6 and E7 proteins encoded by the low risk genital HPV types such as HPV6 or HPVl 1 function only very poorly or not at all in the in vitro transformation/immortalization assays (Storey et al., 1988; Woodworth et al., 1989), and it is clear that this difference is at least in part due to intrinsic differences in protein functions rather than differences in levels of expression (Barbosa et al., 1991). HPV E6 and E7 are small proteins that show some similarity to each other and are proposed to have arisen following amplification and divergence of a 33 amino acid peptide (Danos and Yaniv, 1987). The main feature that the two proteins share is a series of Cys-x-x-Cys motifs, which occur four times in E6 and twice in E7, and are thought to play a role in zinc binding by both proteins (Barbosa et al., 1989; Rawls et al., 1990). Recent studies have shown that HPV E7 proteins can form a complex with the product of the cellular retinoblastoma gene (RB) (Dyson et al., 1989, Mijnger et al., 1989; Barbosa et al., 1990) and that HPV16 and HPV18 E6 proteins can bind to the cell-encoded ~53 protein (Werness et al., 1990). The oncogenic genital HPV types therefore show some similarity to other small DNA tumor viruses such as SV40 and adenovirus, which also encode RB- and p53-binding proteins (Lane and Crawford, 1979; Sarnow et al., 1982; DeCaprio et al., 1988; Whyte et al., 1988). Since both RB and ~53 are able to negatively control cell growth and suppress transformation (Huang et al., 1988; Finlay et al., 1989; Baker et al., 1990; Diller et al., 1990; Mercer et al., 1990), it seems likely that one of the mechanisms by which the viruses contribute to cell transformation is by binding and inactivating the wild-type function of these cellular proteins. Although binding by the SV40 large T antigen results in the stabilization of p53 (Yewdell et al., 1986), E6 binding leads to an increased rate of ~53 degradation by a ubiquitin-directed system (Scheffner et al., 1990) and the enhancement of ~53 degradation has been shown to be mediated only by E6 proteins encoded by the oncogenic HPV types. Although there is no direct evidence that p53 binding and degradation by E6 is important for the immortalizing activity of the viral protein, studies of ano-genital carcinomas have provided evidence for the importance of this association. Analysis of ~53 sequences in tumors and tumor cell lines has shown that while HPV-negative tumors express mutant ~53 sequences, only wild-type ~53 is detected in HPV-positive cancers (Crook et al., 1991a, 1991 b; Scheffner et al., 1991). This suggests that loss of wild-type ~53 function is important in the genesis of these tumors and that this can be achieved either by somatic mutation or by the expression of HPV E6 protein. However, other activities of E6 may also contribute to cellular transformation. The E6 protein encoded by bovine papillomavirus type-l

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Position of amino acids conserved throughout all genital HPV types are boxed, with the conserved Cys-x-x-Cys in bold boxes. Amino acids conserved only among the oncogenically associated HPV types are circled. The positions of the various mutations discussed in this study are also Indicated.

(BPV-1) shows strong transforming activity in mouse cells, and mutational analyses have shown that this activity correlates with transcriptional trans-activation by the E6 sequences (Lamberti et al., 1990). Although BPV-1 E6 and the E6 encoded by the oncogenic genital HPV types appear to have somewhat different transforming activities, transcriptional activation may also contribute to the transforming function of the HPV E6 proteins. There is some evidence that wild-type p53 also has the ability to control transcription (Fields and Jang, 1990; Raycroft et al., 1990) and it is possible that transcriptional activation by E6 is mediated via p53 binding. We have undertaken a mutational analysis of HPV16 E6 to identify important functional regions of the protein. Using a series of small deletions, we have mapped a region of E6 required for p53 binding. This portion of E6 is well conserved between all the genital HPV E6 proteins, and we were able to show that E6 encoded by the low risk HPV types 6 and 11 also bound ~53, albeit with a lower avidity than HPV16 E6. However, HPV6 E6 was clearly unable to direct the rapid degradation of p53 seen following HPV16 E6 binding. Using chimeric HPV6/16 E6 proteins, we have shown that the putative p53-binding region in the C-terminus of the E6 proteins could be functionally separated from a region in the N-terminal half of the protein that participates in directed rapid degradation of ~53. Mutations within N-terminal amino acids conserved in E6 proteins from oncogenic HPV types indicated two important regions for this activity. Analysis of the ability of E6 mutants to trans-activate a heterologous promoter demonstrated

that this activity is not dependent on the ability of the E6 protein to direct degradation of ~53. Results Construction

of HPV16

E6 Mutants

A series of mutations within the HPV16 E6 gene were constructed by in vitro mutagenesis (Figure 1). The majority of the mutations comprised in-frame deletions of 5 codons, which were named according to the numbers of the deleted amino acids. Plasmids encoding point mutations resulting in single or triple amino acid substitutions were also constructed and named according to the number of the altered codon and name of the substituted amino acid(s). Point mutations were targeted to the conserved Cys-x-x-Cys motifs and amino acids conserved among E6 proteins encoded by the oncogenic HPV types. The latter mutations were designed to result in substitution by the residue found at this position in benign HPV types. Two premature termination mutations were introduced into the E6 sequence, which resulted in the expression of truncated E6 proteins consisting of the N-terminal 59 (16E6A59) or 110 (16E6AllO) amino acids. Localization

of p53-Binding

Domain

Each of the mutant E6 proteins was translated in wheat germ extract or rabbit reticulocyte lysate and [3sS]cysteine-labeled proteins made in these systems were tested for the ability to bind unlabeled in vitro translated human ~53. All of the E6 mutants expressed stable protein in the

p53 Binding by HPV E6 Proteins 549

Table 1. Association of Wild-Type and Mutant 16E6 Proteins wrth p53 following lmmunoprecrpitation of p53

HPVl6

E6 Protein

Wild type 16E6

Binding (Percent of 16E6)

100

Premature terminations 16E6A59 16E6AllO