with the retinoblastoma tumor suppressor gene product - NCBI

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Sep 26, 1989 - complexes with the retinoblastoma tumor suppressor gene product (p105-RB). Similar to the E7 protein of HPV-16, the E7 proteins of HPV-18 ...
The EMBO Journal vol.8 no.13 pp.4099-4105, 1989

Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product

Karl Munger, Bruce A.Werness, Nicholas Dyson', William C.Phelps2, Ed Harlow' and Peter M.Howley Laboratory of Tumor Virus Biology, National Cancer Institute, Bethesda, MD 20892, 'Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724 and 2Burroughs Wellcome Co., Division of Virology, Research Triangle Park, NC 27709, USA

Communicated by M.Yaniv

The E7 proteins encoded by the human papillomaviruses (HPVs) associated with anogenital lesions share siificant amino acid sequence homology. The E7 proteins of these different HPVs were assessed for their ability to form complexes with the retinoblastoma tumor suppressor gene product (p105-RB). Similar to the E7 protein of HPV-16, the E7 proteins of HPV-18, HBV-6b and HPV-11 were found to associate with p105-RB in vitro. The E7 proteins of HPV types associated with a high risk of malignant progression (HPV-16 and HPV-18) formed complexes with p105-RB with equal affinities. The E7 proteins encoded by HPV types 6b and 11, which are associated with clinical lesions with a lower risk for progression, bound to p105-RB with lower affinities. The E7 protein of the bovine papiliomavirus type 1 (BPV-1), which does not share structural similarity in the amino terminal region with the HPV E7 proteins, was unable to form a detectable complex with p105-RB. The amino acid sequences of the HPV-16 E7 protein involved in complex formation with p105-RB in vitro have been mapped. Only a portion of the sequences that are conserved between the HPV E7 proteins and AdElA were necessary for association with p105-RB. Furthermore, the HPV-16 E7-p105-RB complex was detected in an HPV-16-transformed human keratinocyte cell line. Key words: human papillomavirus/retinoblastoma/tumor suppressor

Introduction Of the -60 different types of human papillomaviruses (HPVs) which have been isolated thus far from a variety of squamous epithelial lesions, 18 have been associated with anogenital tract lesions (DeVilliers, 1989). Some of these, such as HPV-6 and HPV- 1, are associated with benign proliferative tumors (e.g. condyloma acuminata) which have a low risk for malignant progression, whereas others, such as HPV-16, HPV-18, HPV-31, HPV-33 and HPV-35, are associated with potentially pre-cancerous genital tract lesions, and with a high percentage of anogenital cancers (zurHausen and Schneider, 1987). The cloned DNAs of those HPV types which are associated with lesions that have a high risk for malignant progression encode cellular transformation properties in established rodent fibroblasts

(Yasumoto et al., 1986), in primary rodent cells (Matlashewski et al., 1987; Phelps et al., 1988; Storey et al., 1988) and in primary human cells (Durst et al., 1987; Pirisi et al., 1987; Schlegel et al., 1988). Genetic analysis of HPV-16 and HPV-18 has revealed that the E7 gene of these viruses encodes an oncoprotein that is sufficient for the induction of focus formation of established rodent fibroblasts (Kanda et al., 1988; Phelps et al., 1988; Vousden et al., 1988; Watanabe and Yoshiike, 1988; Bedell et al., 1989; Tanaka et al., 1989). The HPV-16 E7 protein has been most extensively studied. It is functionally similar to the adenovirus ElA proteins (AdElA) in that it can transactivate the adenovirus E2 promoter and can co-operate with an activated ras oncogene to transform primary baby rat kidney cells (Phelps et al., 1988; Storey et al., 1988). The aminoterminal 38 amino acids of E7 are strikingly similar to portions of conserved region 1 (amino acids 37-49) and conserved region 2 (amino acids 116-137) of the AdElA proteins (Phelps et al., 1988) as well as to portions of the large tumor (T) antigens of papovaviruses. Genetic studies on adenoviruses and SV40 have revealed that these regions of homology in the AdElA and SV40 large T antigen are important in transformation (Kalderon and Smith, 1984; Lillie et al., 1987; Moran and Mathews, 1987; Cherington et al., 1988; Whyte et al., 1988). Like AdElA (Whyte et al., 1988a) and SV40 large T antigen (DeCaprio et al., 1988), the HPV- 16 E7 protein can form a specific complex with the retinoblastoma tumor suppressor gene product (p1O5-RB) (Dyson et al., 1989a). Complex formation between the products of oncogenes and tumor suppressor genes is believed to be important in cellular transformation, providing a mechanism to disrupt the normal physiological functions of the specific tumor suppressor gene products. The exact functions of tumor suppressor proteins are not well understood although they may be involved in the negative regulation of cellular growth and/or differentiation (reviewed in Klein, 1987). Here it is shown that the E7 proteins from genital HPV types, independent of their clinical association of 'low risk' (HPV-6b, HPV-1 1) or 'high risk' (HPV-16, HPV-18) are each capable of in vitro complex formation with p105-RB. The E7 proteins from HPV types 6 and 11 associate with p105-RB with a lower affinity than the E7 proteins from HPV types 16 and 18. The amino acid sequences of the HPV-16 E7 protein necessary for in vitro complex formation with p105-RB have been mapped to a small stretch of amino acids surrounding the cysteine residue at sequence position 24 (C24). Finally evidence is presented for the in vivo association of HPV-16 E7 and p105-RB in a transformed human epithelial cell line.

Results E7 proteins of HPV types 6, 11 and 18 form complexes with p105-RB The capacity of the E7 proteins from various papilloma-

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comparison of the relative amounts of the E7 proteins complexed with p1O5-RB. Quantitation of the E7 protein by densitometric scanning of the X-ray films of several independent experiments confirmed that the HPV-16 and HPV-18 E7 proteins co-precipitated with p1O5-RB were approximately equal. The intensity of the HPV- 11 E7 band was 4- to 6-fold weaker, and the intensity of the HPV-6b E7 protein was 20-fold weaker than that of the HPV- 16 or HPV-18 E7 protein bands. These apparent differences in affinities were further investigated by comparing complex formation of the E7 proteins of HPV-16 ('high risk') and HPV- 11 ('low risk') with p1O5-RB. Initial experiments were carried out to determine the concentration range of p1O5-RB needed to produce a linear relation between the input p1O5-RB in the lysate and E7 binding. The NGP cell extract was serially diluted with phosphate-buffered saline (PBS) containing 3 % BSA, mixed with constant amounts of the HPV- 16 and/or HPV- 11 E7 proteins and immunoprecipitated with the p1O5-RB specific C36 antiserum. The amounts of the E7 proteins coprecipitated in these experiments were directly proportional to the amount of NGP extract when the HPV-16 and HPV-1 1 E7 proteins were added either independently or as a mixture (data not shown). For the competition experiments (Figure 2) the amount of HPV-1 1 E7-specific cRNA was held constant and added to increasing amounts of HPV- 16 E7 cRNA. The cRNA mixtures were co-translated in vitro, incubated with constant amounts of p1O5-RB-containing NGP cell lysate followed by immunoprecipitation with the p1O5-RB-specific antibody C36. The co-precipitated HPV-16 and HPV-1 1 E7 proteins were then separated by SDS -PAGE, visualized by autofluorography and the ratio of the complexed HPV-16 E7 to HPV-1 1 E7 proteins was quantitated for each lane by densitometry (Figure 2). The ratios of the input HPV-16 to HPV-1 1 E7 polypeptides were determined from a densitometric analysis of the co-translated E7 proteins prior to coprecipitation with the C36 antibody. The ratio of the apparent affinity constants for each lane was calculated by dividing the ratio of the plO5-Rb-complexed E7 proteins by the ratio of the input proteins. By this analysis, the relative affinity constant for the HPV-16 E7-105-RB complex was found to be 5- to 10-fold higher than that for the HPV-1 1 E7-p1O5-RB complex. This result is in good agreement with the estimate based on the relative amounts of the E7 proteins in complex with p1O5-Rb shown in Figure 1. -

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Fig. 1. Co-precipitation of E7 proteins from various papillomaviruses with p105-RB. Radioactively labeled E7 proteins of HPV-6b, HPV-l 1, HPV-16, HPV-18 and BPV-1 were synthesized in vitro and incubated with extracts of a p1O5-RB containing neuroblastoma cell line NGP or a p105-RB deficient retinoblastoma cell line Y79. Immunoprecipitations were performed with a p105-RB-specific monoclonal antibody C36 or an SV40 T-specific monoclonal antibody PAb419. Radioactively labeled E7 proteins were resolved by SDS- 14 % PAGE and visualized by autofluorography. The positions of mol. wt markers are given on the left side of the figure. See text for details.

viruses to form complexes with p1O5-RB was tested using an in vitro mixing assay previously described for HPV-16 E7 (Dyson et al., 1989a). The E7 genes of HPV-18, HPV- 1, HPV-6b and BPV-1 were cloned in the sense orientation downstream of the T7 promoter of prokaryotic expression plasmids. Radioactively labeled E7 proteins were synthesized by in vitro translation in a rabbit reticulocyte lysate system in the presence of [35S]cysteine using in vitro transcribed E7 complementary RNAs (cRNAs) as templates. Rabbit reticulocyte lysates containing E7 proteins were incubated with unlabeled NGP neuroblastoma cell extracts that contain normal p1O5-RB, and immunoprecipitations were performed with the p105-RB-specific monoclonal antibody C36. The HPV-16 E7 protein served as a positive control in these experiments and, as shown in Figure 1, the E7 proteins of HPV-1 8, HPV-1 1 and HPV-6b were each co-precipitated with the p1O5-RB-specific antibody C36. In contrast, the BPV-1 E7 protein did not co-precipitate with p1O5-RB. Control immunoprecipitations with the SV40 large T antigen-specific monoclonal antibody PAb419 or the p1O5-RB-deficient retinoblastoma cell lysate Y79 were negative for the HPV E7 proteins. Therefore, each of the E7 proteins of the HPV types tested, regardless of the clinical and biological characteristics of the HPV type, was able to complex with p1O5-RB. Differences in affinity for p105-RB binding of the E7 proteins from HPV types associated with a high or a low risk of malignant progression The co-precipitation experiment shown in Figure 1 suggested that there may be potential differences in the binding affinity of the E7 proteins of different HPV types for p1O5-RB. Since equal amounts of the various HPV E7 proteins of the same specific radioactivity were used in these experiments, the intensities of the co-precipitated E7 bands allowed a direct

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Mapping of the regions on the HPV- 16 E7 protein necessary for association with p105-RB A series of deletion and single amino acid substitution mutants of the HPV-16 E7 protein were tested for their capacity to associate with p1O5-RB in vitro (Figure 3). Deletion of sequences encompassing a major portion of sequence similarity between HPV-16 E7 and conserved region 1 of AdElA (APTLHE) had no effect on p1O5-RB binding. Similarly, a deletion of a stretch of conserved acidic amino acids at the carboxy-terminal end of conserved region 2 (AEDE) did not affect association of HPV-16 E7 with p105-RB. A deletion of four amino acids at the aminoterminal end of conserved region 2 (ADLYC), however, totally abolished p1O5-RB binding. Mutation of the conserved cysteine residue at sequence position 24 to a serine residue (C24-S) markedly decreased p1O5-RB binding. Similarly, mutation of a conserved glutamic acid residue at

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Fig. 2. Competition between the HPV-16 and HPV- 11 E7 proteins for binding to p105-RB. Increasing amounts of HPV-16 E7 cRNA were added to constant amounts of HPV- 11 E7 cRNA, the cRNA mixtures were translated in vitro in a rabbit reticulocyte lysate system and incubated with a constant amount of p105-RB containing NGP cell lysate. Immunoprecipitations were performed with the p105-RB-specific monoclonal antibody C36. Radioactively labeled E7 proteins were analyzed by SDS- 14% PAGE followed by autofluorography and quantitated by densitometric scanning of the X-ray film. The ratio of the input HPV-16 E7 and HPV-l I E7 proteins for each lane (a-h) is given in the first line at the bottom of the figure. On the second line is given the ratio of the HPV-16 E7 to HPV- 11 E7 proteins complexed in p105-RB as estimated by densitometric analysis of each lane. The relative affinity of HPV-16 E7 to HPV-1 1 E7 for the complex with p105-RB was calculated for each lane by dividing the value for the ratios of the p105-RB complexed E7 proteins (line 2) with the value for the E7 input ratios (line 1).

sequence position 26 to a glutamine residue (E26-Q) severely impaired the association of HPV- 16 E7 with

p105-RB.

Evidence that HPV-16 E7 forms a complex with p105-RB in vivo Radioactively labeled extracts of a human keratinocyte cell line that was transformed by the HPV-16 E6/E7 genes were used to study the association of HPV-16 E7 with p1O5-RB in vivo. Co-precipitation experiments (Figure 4) were performed with the p1O5-RB-specific monoclonal antibody C36 (lane 3). Aliquots of the same cell extract were also precipitated with an HPV-16 E7-specific antiserum (lane 2), with a monoclonal antibody to SV40 large T antigen, PAb419 (lane 4) or normal rabbit serum (lane 1). A protein band with the mol. wt of the HPV-16 E7 protein (21 kd, Smotkin and Wettstein, 1986) was detected in the precipitation products obtained with the p105-RB-specific monoclonal antibody C36 (lane 3). This band co-migrated with the HPV-16 E7 protein precipitated with the E7-specific antiserum from the same cell extract (lane 2) and it was

absent in the control lanes using either PAb419 (lane 4) or normal rabbit serum (lane 1). No protein of the size of HPV-16 E7 was detected in immunoprecipitations carried out with an SV40-transformed human keratinocyte line using either the HPV-16-specific antiserum or C36 (data not

shown).

Discussion The transforming proteins encoded by a number of DNA tumor viruses have now been shown to associate with cellular proteins. The polyomavirus middle T antigen forms a stable complex with the product of the cellular proto-oncogene c-src (Courtneidge and Smith, 1983), and the SV40 large T antigen and the 58 kd E1B protein of adenovirus associate with p53 (Lane and Crawford, 1979; Linzer and Levine, 1979; McCormick and Harlow, 1980; Sarnow et al., 1982), a cellular protein which has recently been shown to have a tumor suppressor-like activity (Ben-David et al., 1988; Baker et al., 1989; Finley et al., 1989; Hinds et al., 1989).

The AdElA proteins specifically associate with several host

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Fig. 3. Mapping of amino acid sequences of HPV-16 E7 which are necessary for p1O5-RB binding. Mutant E7 proteins were synthesized by in vitro transcription followed by in vitro translation, mixed with p105-RB-containing neuroblastoma cell (NGP) lysate and precipitated with the p1O5-RBspecific C36 monoclonal antibody. The E7 proteins were resolved by SDS- 14 % PAGE and visualized by autofluorography. The sequence positions of the E7 tested mutants are shown at bottom of the figure.

cell proteins (Yee and Branton, 1985; Harlow et al., 1986) and Whyte et al. (1988a) have demonstrated that one of these associated proteins is the retinoblastoma tumor suppressor gene product, p105-RB. Complex formation with p105-RB was also demonstrated for SV40 T (DeCaprio et al., 1988), the large T antigens from many other polyomaviruses (N.Dyson and E.Harlow, unpublished data), and recently for the HPV-16 E7 oncoprotein, using an in vitro binding assay (Dyson et al., 1989a). We have tested the E7 proteins of other types of HPVs associated with genital tract lesions for their capacity to form specific complexes with p105-RB in vitro. The E7 proteins of HPV-1 8, HPV-1 1 and HPV-6b could all be co-precipitated with the p105-RB-specific monoclonal antibody C36 indicative of their ability to form stable complexes with the retinoblastoma tumor suppressor gene product p1O5-RB in vitro. Interestingly, HPV types 6 and 11 which are associated with lesions which are at 'low risk' of malignant progression contrast with the 'high risk' HPV types 16 and 18 in that they are non-transforming in several in vitro transformation assay systems (Schlegel et al., 1988; Storey et al., 1988; Pecoraro et al., 1989; Woodworth et al., 1988). These intrinsic biological differences may be related at least in part to the proteins encoded by these different viruses. The results obtained in this study indicate that these differences in biological behavior cannot be attributed to the potential of the E7 proteins to associate with p105-RB per

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Fig. 4. Evidence for association of HPV-16 E7 with p105-RB in transformed human keratinocytes. Human keratinocytes transformed by the HPV-16 E6/E7 genes expressed from the human ,B-actin promoter (Munger et al., 1989) were labeled with [35S]cysteine and cellular extracts made as described in Materials and methods. Immunoprecipitations were performed with normal rabbit serum (lane 1), a polyclonal HPV-16 E7-specific antiserum (lane 2), a p1O5-RB-specific monclonal antibody C36 (lane 3) or an SV40 T-specific monoclonal antibody (lane 4) as described in Materials and methods. The positions of mol. wt markers are given on the right side of the figure and the position of the HPV-16 E7 protein is shown (E7).

HPV-E7 - p105-RB association

se. It is notable, however, that the apparent affinity for the association with p1O5-RB was lower for the E7 proteins of the 'low risk' HPV types 6 and 11 compared to the 'high risk' HPV-16 and HPV-18 E7 proteins. It is certainly possible that these differences in the relative affinities may contribute to the differences in biologic characteristics of the E7 proteins, but further experiments will be necessary to examine this hypothesis. These data are consistent with the notion that the ability of the E7 proteins to complex p1O5-RB is necessary but not sufficient for cellular transformation. It seems likely that association of the E7 proteins of the different HPVs with p105-RB reflects a common biological property of the genital associated HPVs, such as the ability to induce proliferation or to alter the differentiation of the infected squamous epithelial cell. All of the lesions caused by the genital papillomaviruses are characterized by hyperplasia of the epithelial cells and an altered differentiation referred to as dysplasia. The ability of the HPV E7 protein to complex p1O5-RB may alter its normal function, resulting in cellular changes which by themselves are not sufficient for carcinogenesis. Tumorigenic progression likely involves additional mutations in the host cell genome. Studies with somatic cell hybrids have implicated the loss of a tumor suppressor gene located on chromosome 11 in human cervical carcinoma cell lines (Saxon et al., 1986; Oshimura et al., 1989). It is also possible that the difference in biological behavior of these two classes of genital tract associated HPVs related to the mechanism by which the E7 proteins are produced. The E7 encoding mRNAs are expressed differently in the high-oncogenic versus the low-oncogenic HPV types (Smotkin et al., 1989). In HPV types 16 and 18, the E7 proteins are expressed from polycistronic mRNAs which also encode either a full-length or internally spliced versions of the E6 protein (Schneider-Gadicke and Schwarz, 1986; Smotkin and Wettstein, 1986; Smotkin et al., 1989). These polycistronic mRNAs are expressed from a promoter (P97 in HPV-16) located just upstream of the E6 ORF in the viral long control region (LCR). In HPV types 6 and 11, the E6and E7-specific mRNAs appear to be transcribed from different promoters. The E7 promoter is located within the E6 ORF (Smotkin et al., 1989). Thus differences in the mechanisms regulating expression of the E7 proteins, rather than differences in the intrinsic biological properties of the E7 proteins, could account for the differences in the biological activities of the different HPVs. In addition to the E7 oncoprotein, the full-length E6 protein is necessary for transformation of primary human cells (Munger et al., 1989; Watanabe et al., 1989). It is therefore possible that the differences in the risk of malignant progression associated with the different HPVs may depend on some properties of the E6 protein. The E6 proteins are structurally related to the carboxy-terminal portions of the E7 proteins (Cole and Danos, 1987). The similarity includes two copies of an amino acid motif Cys-X-X-Cys that has recently been implicated in the binding of Zn2+ ions (Barbosa et al., 1989). This domain, however, does not include the region of sequence similarity between E7, AdElA and the large T antigens of the polyomaviruses. The cellular targets of the E6 proteins are currently under

investigation.

The amino acid residues of the HPV-16 E7 protein which

are necessary for the association with p105-RB in vitro have

been mapped to a small region that is highly conserved in all of the HPVs, in conserved region 2 of AdElA, and in the large T antigens of polyomaviruses (Figure 3). For the AdElA protein, amino acid residues within conserved region 1 as well as conserved region 2 have been implicated in complex formation with p105-RB in vivo (Whyte et al., 1989). The sequences in conserved region 2 are essential for binding, whereas the sequences in conserved region 1 are important for high affinity binding of AdElA to p1O5-RB but are not essential for association (N.Dyson and E.Harlow, unpublished results). Based on our results with the deletion mutant APTLHE, it is clear that the amino acids of HPV-16 E7 homologous to conserved region 1 of AdElA are also not essential for in vitro binding to p1O5-RB. Deletion of a portion of the conserved acidic amino acid sequence motif (AEDE) had no effect on p1O5-RB binding. Mutation of the analogous amino acids in AdElA also had no effect on p1O5-RB binding (Whyte et al., 1989). Deletion of the HPV-16 E7 amino acid residues between D21 and C24 (ADLYC) totally abolished p1O5-RB binding (Figure 3). Two of the four deleted amino acid residues (L22 and C24) are conserved in all HPV E7 sequences as well as in the corresponding parts of the AdElA and SV40 large T antigen. Mutation of C24 to a serine residue markedly decreased P105-RB binding, indicating that this cysteine residue may also be crucial in complex formation. Mutagenesis of glutamic acid residues 26 (E26) to a glutamine residue severely impaired p105-RB binding by the mutated E7 protein. This amino acid residue is conserved in all the HPV E7 proteins studied here, as well as in the homologous sequence positions in AdElA (EI26), SV40 large T antigen (E107) and large T antigens of the other polyomaviruses. In SV40 large T antigen, a mutation of E107 to a lysine residue also abolishes p105-RB binding (DeCaprio et al., 1988). As noted above, mutations in conserved region 2 in AdElA and the homologous sequences in SV40 large T antigen impair transformation. Preliminary analysis of the HPV-16 E7 mutants reveal that the sequence in E7 homologous to conserved region 2 of AdElA is also essential for transformation (our unpublished results). These results are in agreement with the recent report of Edmonds and Vousden (1989) and are consistent with the hypothesis that complex formation with p105-RB may be necessary for transformation. The amino acids necessary for complexing p1O5-RB constitute only a small portion of the conserved amino acid sequences common to AdElA, the papovavirus large T antigens and the HPV E7 proteins, although additional amino acids may be important for high affinity associations. While the data implicate p105-RB association as one common aspect involved in cellular transformation by these different DNA tumor viruses, it is possible that complex formation with other cellular proteins may also be important for the transforming properties of these viral oncoproteins. The AdElA proteins are associated with a series of cellular proteins in transformed cells (Yee and Branton, 1985; Harlow et al., 1986). The binding sites for at least two additional proteins (p1O7 and p300) have been mapped to conserved regions 1 and 2 of AdElA (Whyte et al., 1989) and both of these are required for cellular transformation (Lillie et al., 1987; Whyte et al., 1988b). The identity of these proteins is presently unknown. The binding site on AdElA for p107 maps to amino acid residues 121-127 and

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similar sequences are also present in large T antigens of several polyomaviruses and in the HPV E7 proteins. It was shown recently that p107 also associates with the large T antigens of SV40 and JC virus (Dyson et al., 1989b) and because the analogous domain is in the E7 proteins it is possible that this cellular protein also binds to E7.

Materials and methods Recombinant plasmid DNAs For HPV-18 E7, an NsiI (nt 594) to EcoRI (nt 2440) fragment was cloned in the BamHI and EcoRI sites of pGem2 in the presence of a synthetic oligonucleotide adapter (GATCTCCACCATGCA) to reconstruct the amino terminus of the HPV-18 E7 protein. Similarly, for HPV-I 1, an NsiI (nt 534) to AccI (nt 1376) fragment was cloned in the BamHI and AccI sites of pGEM1 in the presence of the same oligonucleotide adapter as described for the HPV-18 E7 gene. A StyI (nt 466) to EcoRI (nt 2188) fragment of HPV-6b, which contained the entire E7 ORF, was cloned in the Hindll and EcoRI sites of pGem2. The BPV-1 E7 gene was isolated as an XhoI (nt 475) to AvaI (nt 945) fragment from plasmid pXH875 (Schiller et al., 1984) and cloned in the SalI and AvaI sites of pGem2. the HPV-16 E7 expression plasmid has been described earlier (Dyson et al., 1989a). Each of the plasmids was verified by nucleotide sequencing using T7 DNA polymerase (Sequenase, USB). The mutations in the HPV-16 E7 gene were generated by oligonucleotide reconstruction and will be described in more detail elsewhere (W.C.Phelps, K.MMunger, C.L.Yee and P.M.Howley, manuscript in preparation).

Cells The human keratinocyte lines analyzed for the association of HPV-6 E7 with p105-RB in vivo were transformed by a plasmid expressing the HPV-16 E6/E7 genes from the human,B-actin promoter (Munger et al., 1989) or by SV40. Cell cultures were grown to near confluency and pulse-labeled with [35S]cysteine for 4 h followed by lysis in 250 mM NaCl, 50 mM HEPES, pH 7.0, 0.1 % Nonidet P40 at 4°C for 30 min as previously described (Whyte et al., 1988b). The neuroblastoma cell line NGP and the retinoblastoma cell line Y79 were grown and cellular extracts made as described earlier (Whyte et al., 1988b; Dyson et al., 1989a,c).

Immunological techniques The in vitro mixing experiments were performed as described earlier (Dyson et al., 1989a,c). In short, [35S]cysteine-labeled E7 polypeptides were synthesized by transcription and translation in vitro. Synthesis of the E7 proteins was monitored by TCA precipitation and liquid scintillation counting. Equal numbers of TCA-precipitable counts (2 x 106 c.p.m.) of E7 proteins were used in the same experiment. E7 containing rabbit reticulocyte lysate was mixed with unlabeled extracts from neuroblastoma cells (NGP) or retinoblastoma cells (Y79) for 3 h. Immunoprecipitations were performed using C36, a monoclonal antibody raised against human p1O5-RB (Whyte et al., 1988b), PAb419, a monoclonal antibody recognizing SV40 large T antigen (Harlow et al., 1981), a polyclonal rabbit antibody generated against a bacterial fusion protein of HPV-16 E7 (Smotkin and Wettstein, 1986) and normal rabbit serum (Organon Teknika Corp., West Chester, PA). Proteins were analyzed by SDS-14% PAGE followed by autofluorography. Radioactively labeled protein bands were quantitated by densitometric scanning of X-ray films. For competition experiments, the NGP cell lysate was diluted 7:3 with 3 % BSA in PBS, in all other experiments, the lysates were used undiluted.

Acknowledgements We thank Alison McBride for help with the in vitro transcription/in vitro translation techniques, Doug Lowy for the plasmid pHX875, David Smotkin and Felix Wettstein, for the HPV-16 E7 antibody and Janet Byrne for oligonucleotide synthesis. We are grateful to Joe Bolen and Jack Lichy for critically reading this manuscript.

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Received on August 14, 1989; revised on September 26, 1989

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