Journal of General Virology (1990), 71, 165-171.
165
Printed in Great Britain
Co-transformation by human papillomavirus types 6 and 11 Alan Storey, Kit Osborn and Lionel Crawford* Imperial Cancer Research Fund Tumour Virus Group, Department o f Pathology, University o f Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.
Human papillomavirus (HPV) types 6 and 11 are usually found in benign genital lesions and laryngeal papillomas. However, the occasional occurrence of their DNAs in carcinomas of the genital tract and larynx suggests that they have some tumorigenic activity. In this paper, we have examined the cotransforming and transactivation activities of the E7 genes from these virus types and show that they cooperate with ras to transform primary cells, but at a greatly reduced level compared to HPV-16 E7.
Although the efficiencies of transformation in vitro by HPV-6 and HPV-11 are low, it is striking that the cells that are transformed are highly tumorigenic in vivo in immunocompetent animals. Transactivation studies using the adenovirus E2 promoter demonstrated that both HPV-11 E7 and HPV-16 E7 could stimulate transcription to a similar degree. These results separate the transactivation and co-transforming activities of HPV E7 genes.
Introduction
The oncogenic virus types such as HPV-16 appear to generate E7 mRNA by splicing events that cannot occur in the benign types 6 and 11 (Smotkin et al., 1989). Splice donor and acceptor sites found within the E6 open reading frame (ORF) of HPV-16 and other malignant types are not present in either HPV-6 or HPV-11. To test whether the E7 ORFs of types 6 and 11 were functional in a co-transformation assay when isolated from the rest of the genome, we cloned the individual ORFs into the expression vector pJ4D (a gift from J. P. Morgenstern) and co-transfected these plasmids, plus an activated ras oncogene, into primary cultures of BRK ceils. These results show that the HPV-6 and -11 E 7 0 R F s are able to cooperate with ras to transform BRK cells, but with a greatly reduced efficiency, to give rise to cells that are tumorigenic in syngeneic immunocompetent animals. We then examined these genes for their ability to transactivate the adenovirus E2 promoter and show that the observed decrease in the co-transforming potential of these virus types does not correlate with the ability of a particular virus type to transactivate the adenovirus E2 promoter. This is the first demonstration that transfection of HPV-6 or HPV-I 1 DNA into primary epithelial cells can lead to malignant transformation.
The strong association between the DNAs of human papillomavirus (HPV) types 6 and 11 and genital and laryngeal papillomas suggests that they possess a mitogenic activity. Carcinomas associated with HPV-6 and -11 are very rare when compared to other virus types, although HPV-6b and a variant, termed HPV-6vc, have been isolated from vulvar carcinomas (Kasher & Roman, 1988; Rando et al., 1986). The viral early region of the HPV types most commonly associated with cervical carcinomas, types 16, 18, 31 and 33, when placed under the transcriptional control of a strong heterologous promoter, are able to cooperate with activated ras (Storey et al., 1988; Phelps et al., 1988) or fos (Crook et al., 1988) oncogenes to transform primary baby rat kidney (BRK) or baby mouse kidney epithelial cells. This co-transforming activity has been localized to the E7 gene, which was found to be as efficient as the entire early region of the virus in this assay (Storey et al., 1988; Phelps et al., 1988) and is necessary for the maintenance of the transformed phenotype (Crook et al., 1989). However, the early regions of HPV types 6 and 11 were found to be inactive in such experiments (Storey et al., 1988). There are three possible explanations for these findings. First, the E7 genes may have been expressed at a level which was too low to be effective or, second, expression was at normal levels but the proteins themselves were non-functional in these assays or, thirdly, some early protein other than E7 was produced and antagonized the activity of E7.
0000-9083 © 1990SGM
Methods Constructionof E7 expressionplasmids. The HPV typesused in these experimentswere kindlyprovidedfor us by ProfessorH. zur Hausen (HPVs-6b, -11, -16 and -18), Dr A. T. Lorincz(HPV-31)and Dr G.
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A. Storey, K. Osborn and L. Crawford
Orth (HPV-33). The expression vector pJ4fZ was a gift from J. P. Morgenstern (Growth Control and Development Laboratory, Imperial Cancer Research Fund, U.K.) and plasmid constructions containing HPV-16,-18, -31 and -33 DNA were as previously described (Storey et al., 1988). HPV-6 DNA present in the BamHI site of pATI53 was cut with BamHI and MluI, yielding a 5-2 kb fragment, which was endrepaired, BamHl-linked and cloned into pJ4f~, forming the plasmid pJ4f~6. The entire HPV-11 genome present in a pSV2-neo vector was excised with BamHI and cloned into pJ4fL DNA fragments containing HPV-6 and HPV-11 E7 genes alone were as described later. Transfection and selection. Cultures of primary BRK cells were prepared and transfected by the DNA-calcium phosphate coprecipitation method (Wiglet et al., 1979). Samples of DNA-calcium phosphate precipitate (0-8 ml), containing 5 Ixg of each of the indicated plasmids, were added to 90 mm dishes of sub-confluent BRK cells. After glycerol treatment the cells were cultured in Dulbecco's modified Eagle's medium containing 500 p.g/ml G418. Three weeks after transfection colonies were either isolated with cloning rings and propagated as cell lines, or the cells were fixed in formal saline and stained using Giemsa stain. Nucleic acid hybridization. Total genomic DNA was isolated from cell lines transfected with HPV DNA and EJ-ras. DNA samples (10 p.g) were digested with BamHI and electrophoresed on 1 ~ agarose gels and subjected to Southern transfer (Southern, 1975) using 0.4 M-NaOH (Reed & Mann, 1985) and Hybond-N filters (Amersham). Hybridizations were performed using 32p-labelled random primed (Feinberg & Vogelstein, 1983) HPV DNA fragments for 20 h. The filters were washed in 2 x SSC containing 0-1 ~ SDS (1 x SSC is 150 mM-NaCI, 15 mM-trisodium citrate pH 7-0) at room temperature, then in 0-2 x SSC and 0.1 ~ SDS at 55 °C and exposed to Fuji RX X-ray film with screens. Total cellular RNA was isolated using guanidinium isothiocyanate (Chirgwin et al., 1979) and samples of glyoxylated RNA (10 ~tg) were electrophoresed on 1.2~ agarose gels and subjected to Northern blot analysis (Thomas, 1980). Hybridizations were performed as described above. lmmunoprecipitation. Sub-confluent 90 mm dishes of transfected cell lines were starved in medium without cysteine for 1 h and then labelled in medium containing 1 mCi [3sS]cysteine per plate. The cells were lysed in 250111E7 extraction buffer (250 rnM-NaCl, 0. l ~ NP40, 50 mI~HEPES pH 7-0, 1 ~ aprotinin) for 30 min on ice. Debris was removed by centrifugation at 13 000 g for 1 min. The supernatant was removed and E7 protein precipitated with an anti-E7 rabbit polyclonal serum (Smotkin & Wettstein, 1986). Protein A-Sepharose (Pharmacia) was added and incubated at 4 °C for 1 h. Proteins bound to the beads were separated on 15~ PAGE gels, which were subjected to fluorography (Bonner & Laskey, 1974), dried and exposed to Hyperfilm (Amersham). Chloramphenicol acetyltransferase (CAT) assays. CAT assays were performed as previously described (Spalholz et al., 1985) in NIH 3T3 cells. The cells were lysed and the protein concentration was estimated with the Bio-Rad protein assay so that each CAT assay contained an equivalent amount of protein. The extracts were incubated with 0.5 ~tM[~4C]chloramphenicol (53 Ci/mol) and 10 mM-acetyl CoA in 0-25 uTris-HCl pH 7.8. After a 2 h incubation at 37 °C the reaction products were separated by ascending thin-layer chromatography and localized by autoradiography. Acetylated chloramphenicol spots were excised for quantification by liquid scintillation. The CAT activity of an HPV sequence-containing plasmid is expressed as a percentage of the activity of the E l a plasmid pCE. Transfection efficiencies were monitored by including into each transfection experiment 5 Ixg of the plasmid pCH110 (Hall et al., 1983), which contains an Escherichia coil lacZ gene downstream of a simian virus 40 (SV40) promoter, fl-Galactosidase activity in the cell extracts
was determined according to Miller (1972) and the results of each CAT assay are expressed as a function of the fl-galactosidase activity.
Results Experiments using early region D N A derived from HPV types 16, 18, 31 and 33 transfected into primary BRK cells together with an activated ras oncogene showed that the major co-transforming region of these virus types was the E 7 0 R F , which was as active as the entire early region (Storey et al., 1988; Phelps et al., 1988). Repeated attempts to derive transformed colonies using early region DNA from types 6 and 11 failed (Storey et al., 1988). To investigate the co-transforming potential of the isolated E7 ORFs, expression vectors containing E7 D N A derived from HPV types 6 and 11 were generated. HPV-6 DNA present in the BamHI site of pAT153 was cut with NsiI, giving a 1.1 kb fragment (bp 530 to 1640); similarly, a 1.0 kb NsiI fragment of HPV-11 (bp 530 to 1580) was isolated from a genomic clone of HPV-11 present in the BamHI site of pSV2-neo. These NsiI fragments were linker-adapted such that both NsiI sites were regenerated and BamHI sites added at either end. These fragments were cloned into the expression vector pJ4f~, where the E7 ORF is under the transcriptional control of a strong heterologous promoter, the Moloney murine leukaemia virus (MoMLV) long terminal repeat (LTR). These plasmids were designated pJ4~)6. E7 and pJ4fll 1. ET, respectively. Construction of similar expression plasmids containing the E 7 0 R F s of HPV types 16, 18 and 33 have already been described (Storey et al., 1988). The E7 expression plasmids of types 6, 11 and 16 share a common NsiI site at the 5' end of the E 7 0 R F , but have differing endpoints within the E 1 0 R F (Fig. 1).
HPV-16
I E7 I
E1
m
I
~,,e ~ (
HPV-6 I E7 ]
El
| tI ,
e
HPV-11 ~ E7 I %
El
• • N
MoMLVLT
1 ! ! I
CS SD
SA poyCA
pJ4f~(3.7 kb) pBR ori
Amp'
Fig. 1. Structure of HPV E7 DNA-containing plasmids. HPV ORFs are indicated by open boxes. MoMLV LTR, ampicillin resistance marker, the pBR322 origin of replication, multiple cloning sites (MCS), SV40 T poly(A), splice donor (SD) and acceptor (SA) are indicated. The diagram is not to scale.
Co-transformation by HPV-6 and -11
pJ4~l 6. E7
pJ4t)6. E7
pJ4f~l 8. E7
167
pJ4f~33. E7
pJ4fll 1. E7
Fig. 2. Transfection of primary BRK ceils with HPV DNA. All dishes were transfected with pSV2-neo plus pEJ6.6 (containing the Ha-ras oncogene cloned from the E J/T24 bladder carcinoma cell line) and the indicated plasmid in each case. Cultures were grown for 3 weeks after transfection in medium containing 500 p.g/ml G418, before fixing and staining.
These E7-containing plasmids were transfected into BRK cells with an activated ras oncogene present on the plasmid pEJ6.6, which contains the activated Ha-ras oncogene derived from the T24 bladder carcinoma cell line (Shih & Weinberg, 1982). The plasmid pSV2-neo (Southern & Berg, 1982) conferring G418 resistance was included in all transfection experiments. After glycerol shock treatment the cells were placed under G418 selection and colony formation was monitored for 3 to 4 weeks after transfection (Fig. 2). Neither pEJ6.6 nor pSV2-neo gave rise to G418-resistant colonies and no colonies were detected if either plasmid was omitted from the experiment. As shown in Table 1, transfection of pJ4t)6. E7 or pJ4fil 1. E7 plus ras into BRK cells produced about 50 to 100-fold fewer G418-resistant colonies when compared to pJ4~)16. E7 and transfection of the entire early region of HPV-6 or HPV-11 in the plasmids pJ4~6 and pJ4f~l 1 repeatedly yielded no colonies, suggesting that the E7 ORFs of these HPV types possess a markedly lower co-
Table 1. Number of G418-resistant colonies formed 3 weeks after transfection of 5 ~tg each of H P V DNA, pEJ6.6 and pSV2-neo per 90 mm dish of primary BRK cells G418-resistant colonies/90mm dish Experiment Plasmid
1
2
3
pJ4sq 16. E7 pJ4fl6 pJ4f]6. E7 pJ4f~ 11 pJ4f~ 11. E7 pEJ6.6 alone
> 100 0 1 0 2 0
> 100 0 2 0 3 0
42 0 1 0 1 0
transforming potential. It has been shown that high level expression of ras in G418-selected, rat embryo fibroblasts was able to induce tumorigenic conversion (Land et al., 1986). However, no G418-resistant colonies were ever generated by transfection of ras plus pSV2-neo into
A. Storey, K. Osborn and L. Crawford
168
predominantly epithelial cultures of primary B R K cells. Analysis of cells transfected by pJ4~)6. E7 and pJ4~) 11. E7 was performed on individually isolated and expanded colonies. Of eight HPV-6 E7 and ten HPV-11 E7 colonies chosen only one and two, respectively, were able to be propagated as cell lines, which have now been growing in cell culture for more than 6 months and show no sign of senescence. Typical growth curve analysis of these cell lines is shown in Fig. 3. All three cell lines grew more slowly and attained lower saturation densities than cells transfected by H P V - 16 E7 (Fig. 3). Southern blot analysis of cell lines showed that they contained unrearranged copies of HPV-6 and -11 D N A (Fig. 4) and that the pJ4f~l 1.E7 clone 2 contained approximately 10 times more HPV-11 D N A than clone 1. Copy number analysis indicated that the pJ4f26. E7 and the pJ4f~l 1. E7 clone 1 cell lines possess one or two copies of integrated H P V D N A (data not shown). Northern blot analysis showed that the cell lines expressed type-specific transcripts (Fig. 5) and that the p J 4 f ~ l l . E 7 clone 2 cell line expressed more m R N A than clone 1. The pJ4f~l 1.E7 cell lines both produced two species of m R N A , which were both under 18S (about 2 kb), whereas the pJ4~)6. E7 cell line also produced two m R N A species, but of between 18S and 28S (about 5 kb in length) (Fig. 5). We were also interested to see whether the cell lines produced any detectable E7 protein. Immunoprecipitation of [3SS]cysteine-labeUed proteins from these cell lines using a polyclonal serum raised against an HPV-16 E7 trpE fusion protein (Smotkin & Wettstein, 1986) is shown in Fig. 6. N o detectable E7 protein was precipitated from
120
i
I
I
1
I
I
I
I
I
3
4
kb 2.0--
56
1
2
3
4
56
28S--
1-6-18S--
1.0--
0.5 D Fig. 4
Fig. 5
Fig. 4. Southern blot analysis of H P V - 6 a n d H P V - I 1 D N A in transfected cell lines. E a c h lane c o n t a i n s 10 ktg g e n o m i c D N A digested with BamHI. Lanes 1 and 5 contain DNA isolatedfrom pJ4f~l 1. E7/ras
clone 1 cells; lane 2, pJ4~ll.E7/ras clone 2; lanes 3 and 6, pJ4fll 6. E7/ras; lane 4, pJ4f~6.E7/ras. Lanes 1 to 3 were probed with an HPV-11 genomic probe (bp 530 to 1580) and lanes 4 to 6 with an HPV-6 genomic probe (bp 530 to 1640). Fig. 5. Northern blot analysis of total cellular RNA derived from transfected BRK cells. Lanes 1 and 6 contain RNA derived from pJ4f~16.E7/ras cells; lanes 2 and 4, pJ4f~ll.E7/ras clone 2; lane 3, pJ4~6. E7/ras; lane 5, pJ4~l 1. E7/ras clone 1. The probes used were the same as those used for the Southern analysis shown in Fig. 4. Lanes 1 to 3 were probed with an HPV-6 genomic probe and lanes 4 to 6 with an HPV-11 genomic probe.
the p J 4 ~ 6 . E 7 and pJ4O33.E7 cell lines. Proteins of about 15000 Mr immunoprecipitated from the two p J 4 ~ l 1. E7 clones, and a protein of similar size to H P V 16 E7 from a cell line transformed by pJ4~31 plus ras (Fig. 6), may be tentatively identified as HPV-11 E7 and HPV-31 E7 proteins, respectively.
It was important to determine whether the ceils produced by transfection of HPV-6 and -11 D N A s plus ras were tumorigenic in syngeneic immunocompetent animals. When 2 × 105 or 2 × 106 cells containing pJ4f~6.E7 or p J 4 ~ l 1. E7 were injected subcutaneously into the flanks of each of four rats, no differences in the tumorigenic potential of these cells was observed when compared to pJ4O16.E7 cells. Tumours developed at the site of injection within 3 weeks in all cases.
80L 60-
40-
20-
00
2
Tumour production
100 -
,.Q
1
1
2
3
4
5
6
7
8
9
10
T i m e (days) Fig. 3. Typical growth curves of H P V E7 D N A c o n t a i n i n g cell lines.
Cells (5 x 104) were plated on each of ten 60 mm dishes and one plate was counted at each time point. (/X) pJ4Ol6.E7/ras; (A) pJ4~6. E7/ras; (U])pJ4f~l 1. E7/ras clone 1; ( I ) pJ4Q11. E7/ras clone 2.
Transactivational activities of HPV-6 and -11 The HPV-16 E7 protein has sequence homology with conserved E 1a regions of the sequenced adenovirus types and can transactivate the adenovirus E2 promoter (Phelps et al., 1988) and presumably other, as yet unidentified, cellular genes. As H P V s share sequence
Co-transformation by HPV-6 and -11
1
2
3
4
5
6
7
8
9
10
11
169
12
Fig. 6. Immunoprecipitation analysis of [35Slcysteine-labelled proteins using an HPV-16 E7 polyclonal rabbit serum (Smotkin & Wettstein, 1986). Lanes 1 to 6 were precipitated with anti-E7 serum and lanes 7 to 12 with pre-immune rabbit serum. BRK cell lines used were: lane 1, E I a/ras; lane 2, pJ4f216. E7/ras; lanes 3 and 8, pJ4f~6. E7/ras; lanes 4 and 9, pJ4~l 1. E7/ras clone 1; lanes 5 and 10, pJ4f~l 1. E7/ras clone 2; lanes 6 and 11, pJ4f~31/ras; lanes 7 and 12, pJ4D33. E7/ras. The arrow indicates the position of HPV-16 E7 in lane 2. Table 2. Quantification of CA T activity*
pCE pE2: :CAT alone pJ4~6.E7 pJ4f~l l.E7 pJ4Dl6.E7 pJ4f~33. E7
Specific activity of fl-galactosidase (units/mg protein)
Acetylated chloramphenicol (%)
CAT activity in transfected cells (% of pCE value)
190 179 171 177 170 187
80 2 5 78 90 51
(100) 2 7 104 126 64
* Results are expressed relative to the Ela plasmid pCE and are the mean of three separate experiments. h o m o l o g y t h r o u g h o u t t h e i r E7 regions, we were interested to d e t e r m i n e w h e t h e r the d e c r e a s e d c o - t r a n s f o r m ing activities o f H P V - 6 a n d -I 1 E7s could be c o r r e l a t e d w i t h a d e c r e a s e d a b i l i t y to t r a n s a c t i v a t e the a d e n o v i r u s E2 p r o m o t e r p r e s e n t in the p l a s m i d p E 2 : : C A T ( M u r t h y et al., 1985). H P V E7 D N A s in pJ4~) were c o - t r a n s f e c t e d w i t h the p E 2 : : C A T p l a s m i d into dishes o f half-confluent N I H 3T3 cells. F o l l o w i n g glycerol t r e a t m e n t the cells were g r o w n for 48 h, h a r v e s t e d a n d a s s a y e d for C A T activity. T h e r e l a t i v e C A T activities o f t h e different E7 constructs c o m p a r e d to a w i l d - t y p e E l a , p r e s e n t on the p l a s m i d p C E ( S c h n e i d e r et al., 1987), are s h o w n in Fig. 7 a n d T a b l e 2. T h e virus t y p e s 16 a n d 33, w h i c h c o o p e r a t e d efficiently w i t h ras to form G 4 1 8 - r e s i s t a n t colonies, also s t i m u l a t e d t r a n s c r i p t i o n o f the E2 p r o m o t e r . HPV-11 E7 was effective in the t r a n s a c t i v a t i o n assay, d e s p i t e its low c o - t r a n s f o r m i n g activity. It is t h e r e f o r e u n l i k e l y t h a t
G
AC
CM
Fig. 7. Relative expression of the pE2" :CAT plasmid in NIH 3T3 cells transfected pE2: :CAT and HPV E7 DNA. The plasmid pCE contains a wild-type E I a gene. The positions of [14C]chloramphenicol (C M) and its acetylated derivative (AC) are indicated.
170
A. Storey, K. Osborn and L. Crawford
papillomavirus co-transforming activity is simply a consequence of transactivation.
Discussion Sequence homology between the E7 proteins of genital papillomaviruses (Cole & Danos, 1987) suggests that the HPV-6 and HPV-11 E7 proteins might be able to mediate cell transformation with ras, or to transactivate the adenovirus E2 promoter in a similar manner to oncogenic papillomaviruses (types 16, 18, 31 and 33) (Phelps et al., 1988; Storey et al., 1988). The lack of cooperation between the entire early region of HPVs -6 and -11 (Storey et al., 1988) may be due to a functionally inactive E7 protein in these types, or the E7 protein may be active but produced below a 'threshold' level needed for co-transformation. Using a keratinocyte assay, Schlegel et al. (1988) showed that in medium containing serum and calcium macrocolony formation was limited to HPV-16 and -18, but in low calcium HPV-6 and -11 could form micro- and macrocolonies. Recent studies have shown that oncogenic and non-oncogenic papillomaviruses generate functional E7 mRNA by different splicing mechanisms (Smotkin et al., 1989), which could be important in determining the level of E7 protein. To avoid any differences due to splicing efficiency we have cloned the individual E 7 0 R F s from HPV types 6 and 11 directly behind a heterologous promoter, the MoMLV LTR and examined their transactivation and cotransforming activities. In the co-transfection experiments we show that HPV-6 E7 and HPV-11 E7 can cooperate with ras to transform primary BRK cells, but at a level 50- to 100-fold lower than HPV-16 E7 (Table 1). Southern and Northern blot analysis of expanded colonies of pJ4~6.E7/ras and pJ4~ll.E7/ras cells showed the presence of expressed unrearranged HPV DNA. The pJ4f]ll.E7/ras clone 2 cells grew faster, contained more HPV DNA and expressed more E7 mRNA than the clone 1 cells, suggesting that the level of E7 gene expression may be important in determining the in vitro growth rate of these cells. The pJ4~6.E7/ras and pJ4f]ll.E7/ras ceils were clearly transformed, as judged by their ability to form tumours in syngeneic immunocompetent rats within 3 weeks after injection. However, it was surprising that these cells generated tumours at the same rate as cells transformed by HPV-16 E7, as the ability of the HPV-6 and HPV-11 E7 genes to cooperate with ras to generate transformed colonies is massively reduced. The shared E7 protein sequence homology between papillomavirus types suggests that a polyclonal serum raised against HPV-16 E7 may cross-react with other E7 proteins. Immunoprecipitation of [35S]cysteine-labelled proteins
showed that a protein of about 15000 Mr was specifically precipitated from pJ4~l 1.E7/ras cells, suggesting that this may be the HPV-11 E7 protein. A protein of similar Mr to HPV-16 E7 could also be precipitated from pJ4f231. E7/ras cells, but the equivalent protein could not be detected in pJ4~6.E7/ras or pJ4f~33.E7/ras cells (Fig. 6). This may be because some epitopes that are present on HPV-16 E7 are absent from these E7 proteins. Type-specific sera or monoclonal antibodies will be needed to verify the identity of all these E7 proteins. Having established that HPV-6 E7 and HPV-11 E7 could generate malignantly transformed cells we then examined the ability of these DNAs to transactivate the adenovirus 2 promoter, to see whether the transcriptional stimulatory activities of E7 are important for its role in cell transformation. Neither E7 nor E 1a seems to bind to DNA directly (Feldman et al., 1982; Ferguson et al., 1985). Ela proteins contain three distinct regions, which are strongly conserved amongst adenovirus types (van Ormondt et al., 1980; Kimelman et al., 1985). Mutations in conserved regions 1 and 2, which are homologous to papillomavirus E7 N-terminal sequences, interfere with the ability of Ela to immortalize primary cells, to collaborate with ras in transformation and to induce cellular DNA synthesis, but are not required for transactivation (Lillie et al., 1986, 1987; Moran et al., 1986). Ela conserved region 3, which does not have an E7 homologue, is important and on its own can be sufficient for transactivation of early promoters (Glenn & Ricciardi, 1985; Lillie et al., 1986). In similar mutagenesis studies Schneider et al. (1987) suggested that transactivation was unlikely to play an important role in transformation by E 1a, as they were able to isolate mutants that could transform but not transactivate. They further suggested that Ela-mediated transformation occurs by repression of a cellular enhancer. Here we have shown that the ability of a particular viral E7 to transactivate is not always accompanied by an equivalent ability to co-transform, thus separating these two activities of E7 to a large extent. The results further suggest that not only splicing events within the E6 ORF but also the activity of the E7 protein itself are important in determining the oncogenic potential of a particular virus type. These co-transformation assays show both qualitative and quantitative differences in the oncogenic potential of these genital papillomaviruses. The occurrence of HPV-6 and HPV-11 in vulvar and laryngeal carcinomas is rare and this is paralleled in vitro, where greatly diminished co-transforming activities of these types are observed, yet the HPV-6 E7- and HPV-I1 E7-transformed cells are as tumorigenic as cells transformed by HPV-16 E7. Further investigation of the overall structure of these E7 proteins and their interaction with other
Co-transformation by HPV-6 and -11
cellular factors will be necessary to understand the role of E7 in cellular transformation. We would like to thank Dr F. Wettstein for the rabbit anti-E7 polyclonal serum, Carole Pye and Kate Hartley for their technical assistance, Dr ADders Follin for useful discussions during the course of this work and Helen Wilson for preparing the manuscript.
References DONNER, W. M. & LASKEY, R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. European Journal of Biochemistry 46, 83-88. CHIRGWIN, J., PRZYBYLA,A. E., MACDONALD, R. J. & RUTTER, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-5299. COLE, S. T. & DANOS, O. (1987). Nucleotide sequence and comparative analysis of the human papillomavirus 18 genome. Phylogeny of papillomaviruses and repeated structure of the E6 and E7 gene products. Journal of Molecular Biology 193, 599-608. CROOK, T., STOREY, A., ALMOND, N., OSBORN, K. &. CRAWFORD, L. (1988). Human papiltomavirus type 16 cooperates with activated ras and fos oncogenes in the hormone-dependent transformation of primary mouse cells. Proceedingsof the National Academy of Sciences, U.S.A. 85, 8820-8824. CROOK, T., MORGENSTERN, J., CRAWFORD, L. & BANKS, L. (1989). Continued expression of HPV-16 E7 protein is required for maintenance of the transformed phenotype of ceils cotransformed by HPV-16 plus EJ-ras. EMBO Journal 8, 513-519. FEINBERG, A. P. & VOGELSTEtN, B. (1983). A technique for radiolabetling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry 132, 6-13. FELDMAN, L. T., IMPERIALE,M. J. & NEVlNS, J. R. (1982). Activation of early adenovirus transcription by the herpes immediate early gene: evidence for a common cellular control factor. Proceedings of the National Academy of Sciences, U.S.A. 79, 4952-4956. FERGUSON, B., KRIPPLE, B., ANDRISANI, O., JONES, N., WESTPHAL, H. & ROSENBERG, i . (1985). E l a 13S and 12S mRNA products made in Escherichia coil both function as nucleus localized transcription activators but do not directly bind DNA. Molecular and Cellular Biology 5, 2653-2661. GLENN, G. M. & RICCIARDI, R. P. (1985). Adenovirus 5 early region 1A host range mutants hr 3, hr 4 and hr 5 contain point mutations which generate single amino acid substitutions. Journal of Virology 56, 66-74. HALL, C. V., JACOB, P. E., RINGOLD, G. M. & LEE, F. (1983). Expression and regulation of Escherichia coil lacZ gene fusions in mammalian cells. Journal of Molecular and Applied Genetics 2, 101-109. KASHER, M. S. & ROMAN, A. (1988). Characterization of human papillomavirus type 6b DNA isolated from an invasive squamous carcinoma of the vulva. Virology 165, 225-233. KIMELMAN, D., MILLAR, J. S., PORTER, D. & ROBERTS, B. E. (1985). E 1a regions of the human adenoviruses and of the highly oncogenic simian adenovirus 7 are closely related. Journal of Virology 53, 399-409. LAND, H., CHEN, A. C., MORGENSTERN, J. P., PARADA, L. F. 8z WEINBERG, R. A. (1986). Behaviour of myc and ras oncogenes in transformation of rat embryo fibroblasts. Molecular and Cellular Biology 6, 1917-1925. LILLIE, J. W., GREEN, M. & GREEN, M. R. (1986). An adenovirus E l a protein region required for transformation and transcriptional repression. Cell 46, 1043-1051. LILLIE, J. W., LOEWENSTEIN, P. M., GREEN, M. R. & GREEN, M. (1987). Functional domains of adenovirus type 5 E l a proteins. Cell 50, 1091-1100.
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MILLER, J. H. (1972). Experiments in Molecular Genetics. New York: Cold Spring Harbor Laboratory. MORAN, E., ZERLER, B., HARRISON, T. M. & MAI"rHEWS,M. B. (1986). Identification of separate domains in the adenovirus E l a gene for immortalization activity and the activation of virus early genes. Molecular and Cellular Biology 6, 3470-3480. MURTHY, S. C. S., BHAT, G. P. & THIMMAPPAYA,B. (1985). Adenovirus E l a early promoter: transcriptional control elements and induction by the viral pre-eady E l a gene which appears to be sequence independent. Proceedingsof the National Academy of Sciences, U.S.A. 82, 2230-2234. PHELPS, W. C., YEE, C. L., MUNGER, K. & HOWLEY, P. M. (1988). The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus E 1a. Cell 53, 539-547. RANDO, R. F., GROFF, D. E., CH1RIKJIAN, J. G. & LANCASTER,W. D. (1986). Isolation and characterization of a novel human papillomavirus type 6 DNA from an invasive vulval carcinoma. Journal of Virology 57, 353-356. REED, K. C. & MANN, O. A. (1985). Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Research 13, 7207-7221. SCHLEGEL, R., PHELPS, W. C., ZHANG, Y-L. & BARBOSA, M. (1988). Quantitative keratinocyte assay detects two biological activities of human papillomavirus DNA and identifies viral types associated with cervical carcinoma. EMBO Journal 7, 3181-3187. SCHNEIDER, J. F., FISHER, F., GODING, C. P-. 8r~JONES, N. C. (1987). Mutational analysis of the adenovirus E l a gene: the role of transcriptional regulation in transformation. EMBO Journal 6, 2053-2060. SHIH, C. & WEINBERG, R, A. (1982). Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell 29, 161-169. SMOTKIN, n . &. WETTSTEIN, V. O. (1986). Transcription of human papillomavirus type 16 early genes in a cervical cancer and cancerderived cell line and identification of the E7 protein. Proceedings of the National Academy of Sciences, U.S.A. 83, 4681)-4684. SMOTKIN, D., PROKOPH, H. & WE'I'I'STEIN,F. O. (1989). Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms. Journal of Virology63, 1441-1447. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98, 503-517. SOUTHERN, P. J. 8£ BERG, P. (1982). Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. Journal of Molecular and Applied Genetics 1, 327-341. SPALHOLZ, B. A., YANG, Y-C. & HOWLEY, P. M. (1985). Transactivation of a bovine papillomavirus transcriptional regulatory element by the E2 gene product. Cell 42, 183-191. STOREY, A., PIN, D., MURRAY, A., OSBORN, K., BANKS, L. & CRAWFORD, L. (1988). Comparison of the in vitro transforming activities of human papillomavirus types. EMBO Journal 7, 1815-1820. THOMAS, P. S. (1980). Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proceedings of the National Academy of Sciences, U.S.A. 77, 5201-5205. VAN ORMONDT, H., MAAT, J. • DIJKEMA, R. (1980). Comparison of nucleotide sequences of the early E la regions for subgroups A, B and C of human adenoviruses. Gene 12, 63-76. WIGLER, M., PELLICER, A., SILVERSTEIN,S., AXEL, R., URLAUB, G. & CHASIN, L. (1979). DNA-mediated transfer of the adenine phosphoribosyl transferase locus into mammalian cells. Proceedings of the National Academy of Sciences, U.S.A. 76, 1373-1376.
(Received 22 May 1989; Accepted 18 September 1989)
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Printed in Great Britain
Co-transformation by human papillomavirus types 6 and 11 Alan Storey, Kit Osborn and Lionel Crawford* Imperial Cancer Research Fund Tumour Virus Group, Department o f Pathology, University o f Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.
Human papillomavirus (HPV) types 6 and 11 are usually found in benign genital lesions and laryngeal papillomas. However, the occasional occurrence of their DNAs in carcinomas of the genital tract and larynx suggests that they have some tumorigenic activity. In this paper, we have examined the cotransforming and transactivation activities of the E7 genes from these virus types and show that they cooperate with ras to transform primary cells, but at a greatly reduced level compared to HPV-16 E7.
Although the efficiencies of transformation in vitro by HPV-6 and HPV-11 are low, it is striking that the cells that are transformed are highly tumorigenic in vivo in immunocompetent animals. Transactivation studies using the adenovirus E2 promoter demonstrated that both HPV-11 E7 and HPV-16 E7 could stimulate transcription to a similar degree. These results separate the transactivation and co-transforming activities of HPV E7 genes.
Introduction
The oncogenic virus types such as HPV-16 appear to generate E7 mRNA by splicing events that cannot occur in the benign types 6 and 11 (Smotkin et al., 1989). Splice donor and acceptor sites found within the E6 open reading frame (ORF) of HPV-16 and other malignant types are not present in either HPV-6 or HPV-11. To test whether the E7 ORFs of types 6 and 11 were functional in a co-transformation assay when isolated from the rest of the genome, we cloned the individual ORFs into the expression vector pJ4D (a gift from J. P. Morgenstern) and co-transfected these plasmids, plus an activated ras oncogene, into primary cultures of BRK ceils. These results show that the HPV-6 and -11 E 7 0 R F s are able to cooperate with ras to transform BRK cells, but with a greatly reduced efficiency, to give rise to cells that are tumorigenic in syngeneic immunocompetent animals. We then examined these genes for their ability to transactivate the adenovirus E2 promoter and show that the observed decrease in the co-transforming potential of these virus types does not correlate with the ability of a particular virus type to transactivate the adenovirus E2 promoter. This is the first demonstration that transfection of HPV-6 or HPV-I 1 DNA into primary epithelial cells can lead to malignant transformation.
The strong association between the DNAs of human papillomavirus (HPV) types 6 and 11 and genital and laryngeal papillomas suggests that they possess a mitogenic activity. Carcinomas associated with HPV-6 and -11 are very rare when compared to other virus types, although HPV-6b and a variant, termed HPV-6vc, have been isolated from vulvar carcinomas (Kasher & Roman, 1988; Rando et al., 1986). The viral early region of the HPV types most commonly associated with cervical carcinomas, types 16, 18, 31 and 33, when placed under the transcriptional control of a strong heterologous promoter, are able to cooperate with activated ras (Storey et al., 1988; Phelps et al., 1988) or fos (Crook et al., 1988) oncogenes to transform primary baby rat kidney (BRK) or baby mouse kidney epithelial cells. This co-transforming activity has been localized to the E7 gene, which was found to be as efficient as the entire early region of the virus in this assay (Storey et al., 1988; Phelps et al., 1988) and is necessary for the maintenance of the transformed phenotype (Crook et al., 1989). However, the early regions of HPV types 6 and 11 were found to be inactive in such experiments (Storey et al., 1988). There are three possible explanations for these findings. First, the E7 genes may have been expressed at a level which was too low to be effective or, second, expression was at normal levels but the proteins themselves were non-functional in these assays or, thirdly, some early protein other than E7 was produced and antagonized the activity of E7.
0000-9083 © 1990SGM
Methods Constructionof E7 expressionplasmids. The HPV typesused in these experimentswere kindlyprovidedfor us by ProfessorH. zur Hausen (HPVs-6b, -11, -16 and -18), Dr A. T. Lorincz(HPV-31)and Dr G.
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A. Storey, K. Osborn and L. Crawford
Orth (HPV-33). The expression vector pJ4fZ was a gift from J. P. Morgenstern (Growth Control and Development Laboratory, Imperial Cancer Research Fund, U.K.) and plasmid constructions containing HPV-16,-18, -31 and -33 DNA were as previously described (Storey et al., 1988). HPV-6 DNA present in the BamHI site of pATI53 was cut with BamHI and MluI, yielding a 5-2 kb fragment, which was endrepaired, BamHl-linked and cloned into pJ4f~, forming the plasmid pJ4f~6. The entire HPV-11 genome present in a pSV2-neo vector was excised with BamHI and cloned into pJ4fL DNA fragments containing HPV-6 and HPV-11 E7 genes alone were as described later. Transfection and selection. Cultures of primary BRK cells were prepared and transfected by the DNA-calcium phosphate coprecipitation method (Wiglet et al., 1979). Samples of DNA-calcium phosphate precipitate (0-8 ml), containing 5 Ixg of each of the indicated plasmids, were added to 90 mm dishes of sub-confluent BRK cells. After glycerol treatment the cells were cultured in Dulbecco's modified Eagle's medium containing 500 p.g/ml G418. Three weeks after transfection colonies were either isolated with cloning rings and propagated as cell lines, or the cells were fixed in formal saline and stained using Giemsa stain. Nucleic acid hybridization. Total genomic DNA was isolated from cell lines transfected with HPV DNA and EJ-ras. DNA samples (10 p.g) were digested with BamHI and electrophoresed on 1 ~ agarose gels and subjected to Southern transfer (Southern, 1975) using 0.4 M-NaOH (Reed & Mann, 1985) and Hybond-N filters (Amersham). Hybridizations were performed using 32p-labelled random primed (Feinberg & Vogelstein, 1983) HPV DNA fragments for 20 h. The filters were washed in 2 x SSC containing 0-1 ~ SDS (1 x SSC is 150 mM-NaCI, 15 mM-trisodium citrate pH 7-0) at room temperature, then in 0-2 x SSC and 0.1 ~ SDS at 55 °C and exposed to Fuji RX X-ray film with screens. Total cellular RNA was isolated using guanidinium isothiocyanate (Chirgwin et al., 1979) and samples of glyoxylated RNA (10 ~tg) were electrophoresed on 1.2~ agarose gels and subjected to Northern blot analysis (Thomas, 1980). Hybridizations were performed as described above. lmmunoprecipitation. Sub-confluent 90 mm dishes of transfected cell lines were starved in medium without cysteine for 1 h and then labelled in medium containing 1 mCi [3sS]cysteine per plate. The cells were lysed in 250111E7 extraction buffer (250 rnM-NaCl, 0. l ~ NP40, 50 mI~HEPES pH 7-0, 1 ~ aprotinin) for 30 min on ice. Debris was removed by centrifugation at 13 000 g for 1 min. The supernatant was removed and E7 protein precipitated with an anti-E7 rabbit polyclonal serum (Smotkin & Wettstein, 1986). Protein A-Sepharose (Pharmacia) was added and incubated at 4 °C for 1 h. Proteins bound to the beads were separated on 15~ PAGE gels, which were subjected to fluorography (Bonner & Laskey, 1974), dried and exposed to Hyperfilm (Amersham). Chloramphenicol acetyltransferase (CAT) assays. CAT assays were performed as previously described (Spalholz et al., 1985) in NIH 3T3 cells. The cells were lysed and the protein concentration was estimated with the Bio-Rad protein assay so that each CAT assay contained an equivalent amount of protein. The extracts were incubated with 0.5 ~tM[~4C]chloramphenicol (53 Ci/mol) and 10 mM-acetyl CoA in 0-25 uTris-HCl pH 7.8. After a 2 h incubation at 37 °C the reaction products were separated by ascending thin-layer chromatography and localized by autoradiography. Acetylated chloramphenicol spots were excised for quantification by liquid scintillation. The CAT activity of an HPV sequence-containing plasmid is expressed as a percentage of the activity of the E l a plasmid pCE. Transfection efficiencies were monitored by including into each transfection experiment 5 Ixg of the plasmid pCH110 (Hall et al., 1983), which contains an Escherichia coil lacZ gene downstream of a simian virus 40 (SV40) promoter, fl-Galactosidase activity in the cell extracts
was determined according to Miller (1972) and the results of each CAT assay are expressed as a function of the fl-galactosidase activity.
Results Experiments using early region D N A derived from HPV types 16, 18, 31 and 33 transfected into primary BRK cells together with an activated ras oncogene showed that the major co-transforming region of these virus types was the E 7 0 R F , which was as active as the entire early region (Storey et al., 1988; Phelps et al., 1988). Repeated attempts to derive transformed colonies using early region DNA from types 6 and 11 failed (Storey et al., 1988). To investigate the co-transforming potential of the isolated E7 ORFs, expression vectors containing E7 D N A derived from HPV types 6 and 11 were generated. HPV-6 DNA present in the BamHI site of pAT153 was cut with NsiI, giving a 1.1 kb fragment (bp 530 to 1640); similarly, a 1.0 kb NsiI fragment of HPV-11 (bp 530 to 1580) was isolated from a genomic clone of HPV-11 present in the BamHI site of pSV2-neo. These NsiI fragments were linker-adapted such that both NsiI sites were regenerated and BamHI sites added at either end. These fragments were cloned into the expression vector pJ4f~, where the E7 ORF is under the transcriptional control of a strong heterologous promoter, the Moloney murine leukaemia virus (MoMLV) long terminal repeat (LTR). These plasmids were designated pJ4~)6. E7 and pJ4fll 1. ET, respectively. Construction of similar expression plasmids containing the E 7 0 R F s of HPV types 16, 18 and 33 have already been described (Storey et al., 1988). The E7 expression plasmids of types 6, 11 and 16 share a common NsiI site at the 5' end of the E 7 0 R F , but have differing endpoints within the E 1 0 R F (Fig. 1).
HPV-16
I E7 I
E1
m
I
~,,e ~ (
HPV-6 I E7 ]
El
| tI ,
e
HPV-11 ~ E7 I %
El
• • N
MoMLVLT
1 ! ! I
CS SD
SA poyCA
pJ4f~(3.7 kb) pBR ori
Amp'
Fig. 1. Structure of HPV E7 DNA-containing plasmids. HPV ORFs are indicated by open boxes. MoMLV LTR, ampicillin resistance marker, the pBR322 origin of replication, multiple cloning sites (MCS), SV40 T poly(A), splice donor (SD) and acceptor (SA) are indicated. The diagram is not to scale.
Co-transformation by HPV-6 and -11
pJ4~l 6. E7
pJ4t)6. E7
pJ4f~l 8. E7
167
pJ4f~33. E7
pJ4fll 1. E7
Fig. 2. Transfection of primary BRK ceils with HPV DNA. All dishes were transfected with pSV2-neo plus pEJ6.6 (containing the Ha-ras oncogene cloned from the E J/T24 bladder carcinoma cell line) and the indicated plasmid in each case. Cultures were grown for 3 weeks after transfection in medium containing 500 p.g/ml G418, before fixing and staining.
These E7-containing plasmids were transfected into BRK cells with an activated ras oncogene present on the plasmid pEJ6.6, which contains the activated Ha-ras oncogene derived from the T24 bladder carcinoma cell line (Shih & Weinberg, 1982). The plasmid pSV2-neo (Southern & Berg, 1982) conferring G418 resistance was included in all transfection experiments. After glycerol shock treatment the cells were placed under G418 selection and colony formation was monitored for 3 to 4 weeks after transfection (Fig. 2). Neither pEJ6.6 nor pSV2-neo gave rise to G418-resistant colonies and no colonies were detected if either plasmid was omitted from the experiment. As shown in Table 1, transfection of pJ4t)6. E7 or pJ4fil 1. E7 plus ras into BRK cells produced about 50 to 100-fold fewer G418-resistant colonies when compared to pJ4~)16. E7 and transfection of the entire early region of HPV-6 or HPV-11 in the plasmids pJ4~6 and pJ4f~l 1 repeatedly yielded no colonies, suggesting that the E7 ORFs of these HPV types possess a markedly lower co-
Table 1. Number of G418-resistant colonies formed 3 weeks after transfection of 5 ~tg each of H P V DNA, pEJ6.6 and pSV2-neo per 90 mm dish of primary BRK cells G418-resistant colonies/90mm dish Experiment Plasmid
1
2
3
pJ4sq 16. E7 pJ4fl6 pJ4f]6. E7 pJ4f~ 11 pJ4f~ 11. E7 pEJ6.6 alone
> 100 0 1 0 2 0
> 100 0 2 0 3 0
42 0 1 0 1 0
transforming potential. It has been shown that high level expression of ras in G418-selected, rat embryo fibroblasts was able to induce tumorigenic conversion (Land et al., 1986). However, no G418-resistant colonies were ever generated by transfection of ras plus pSV2-neo into
A. Storey, K. Osborn and L. Crawford
168
predominantly epithelial cultures of primary B R K cells. Analysis of cells transfected by pJ4~)6. E7 and pJ4~) 11. E7 was performed on individually isolated and expanded colonies. Of eight HPV-6 E7 and ten HPV-11 E7 colonies chosen only one and two, respectively, were able to be propagated as cell lines, which have now been growing in cell culture for more than 6 months and show no sign of senescence. Typical growth curve analysis of these cell lines is shown in Fig. 3. All three cell lines grew more slowly and attained lower saturation densities than cells transfected by H P V - 16 E7 (Fig. 3). Southern blot analysis of cell lines showed that they contained unrearranged copies of HPV-6 and -11 D N A (Fig. 4) and that the pJ4f~l 1.E7 clone 2 contained approximately 10 times more HPV-11 D N A than clone 1. Copy number analysis indicated that the pJ4f26. E7 and the pJ4f~l 1. E7 clone 1 cell lines possess one or two copies of integrated H P V D N A (data not shown). Northern blot analysis showed that the cell lines expressed type-specific transcripts (Fig. 5) and that the p J 4 f ~ l l . E 7 clone 2 cell line expressed more m R N A than clone 1. The pJ4f~l 1.E7 cell lines both produced two species of m R N A , which were both under 18S (about 2 kb), whereas the pJ4~)6. E7 cell line also produced two m R N A species, but of between 18S and 28S (about 5 kb in length) (Fig. 5). We were also interested to see whether the cell lines produced any detectable E7 protein. Immunoprecipitation of [3SS]cysteine-labeUed proteins from these cell lines using a polyclonal serum raised against an HPV-16 E7 trpE fusion protein (Smotkin & Wettstein, 1986) is shown in Fig. 6. N o detectable E7 protein was precipitated from
120
i
I
I
1
I
I
I
I
I
3
4
kb 2.0--
56
1
2
3
4
56
28S--
1-6-18S--
1.0--
0.5 D Fig. 4
Fig. 5
Fig. 4. Southern blot analysis of H P V - 6 a n d H P V - I 1 D N A in transfected cell lines. E a c h lane c o n t a i n s 10 ktg g e n o m i c D N A digested with BamHI. Lanes 1 and 5 contain DNA isolatedfrom pJ4f~l 1. E7/ras
clone 1 cells; lane 2, pJ4~ll.E7/ras clone 2; lanes 3 and 6, pJ4fll 6. E7/ras; lane 4, pJ4f~6.E7/ras. Lanes 1 to 3 were probed with an HPV-11 genomic probe (bp 530 to 1580) and lanes 4 to 6 with an HPV-6 genomic probe (bp 530 to 1640). Fig. 5. Northern blot analysis of total cellular RNA derived from transfected BRK cells. Lanes 1 and 6 contain RNA derived from pJ4f~16.E7/ras cells; lanes 2 and 4, pJ4f~ll.E7/ras clone 2; lane 3, pJ4~6. E7/ras; lane 5, pJ4~l 1. E7/ras clone 1. The probes used were the same as those used for the Southern analysis shown in Fig. 4. Lanes 1 to 3 were probed with an HPV-6 genomic probe and lanes 4 to 6 with an HPV-11 genomic probe.
the p J 4 ~ 6 . E 7 and pJ4O33.E7 cell lines. Proteins of about 15000 Mr immunoprecipitated from the two p J 4 ~ l 1. E7 clones, and a protein of similar size to H P V 16 E7 from a cell line transformed by pJ4~31 plus ras (Fig. 6), may be tentatively identified as HPV-11 E7 and HPV-31 E7 proteins, respectively.
It was important to determine whether the ceils produced by transfection of HPV-6 and -11 D N A s plus ras were tumorigenic in syngeneic immunocompetent animals. When 2 × 105 or 2 × 106 cells containing pJ4f~6.E7 or p J 4 ~ l 1. E7 were injected subcutaneously into the flanks of each of four rats, no differences in the tumorigenic potential of these cells was observed when compared to pJ4O16.E7 cells. Tumours developed at the site of injection within 3 weeks in all cases.
80L 60-
40-
20-
00
2
Tumour production
100 -
,.Q
1
1
2
3
4
5
6
7
8
9
10
T i m e (days) Fig. 3. Typical growth curves of H P V E7 D N A c o n t a i n i n g cell lines.
Cells (5 x 104) were plated on each of ten 60 mm dishes and one plate was counted at each time point. (/X) pJ4Ol6.E7/ras; (A) pJ4~6. E7/ras; (U])pJ4f~l 1. E7/ras clone 1; ( I ) pJ4Q11. E7/ras clone 2.
Transactivational activities of HPV-6 and -11 The HPV-16 E7 protein has sequence homology with conserved E 1a regions of the sequenced adenovirus types and can transactivate the adenovirus E2 promoter (Phelps et al., 1988) and presumably other, as yet unidentified, cellular genes. As H P V s share sequence
Co-transformation by HPV-6 and -11
1
2
3
4
5
6
7
8
9
10
11
169
12
Fig. 6. Immunoprecipitation analysis of [35Slcysteine-labelled proteins using an HPV-16 E7 polyclonal rabbit serum (Smotkin & Wettstein, 1986). Lanes 1 to 6 were precipitated with anti-E7 serum and lanes 7 to 12 with pre-immune rabbit serum. BRK cell lines used were: lane 1, E I a/ras; lane 2, pJ4f216. E7/ras; lanes 3 and 8, pJ4f~6. E7/ras; lanes 4 and 9, pJ4~l 1. E7/ras clone 1; lanes 5 and 10, pJ4f~l 1. E7/ras clone 2; lanes 6 and 11, pJ4f~31/ras; lanes 7 and 12, pJ4D33. E7/ras. The arrow indicates the position of HPV-16 E7 in lane 2. Table 2. Quantification of CA T activity*
pCE pE2: :CAT alone pJ4~6.E7 pJ4f~l l.E7 pJ4Dl6.E7 pJ4f~33. E7
Specific activity of fl-galactosidase (units/mg protein)
Acetylated chloramphenicol (%)
CAT activity in transfected cells (% of pCE value)
190 179 171 177 170 187
80 2 5 78 90 51
(100) 2 7 104 126 64
* Results are expressed relative to the Ela plasmid pCE and are the mean of three separate experiments. h o m o l o g y t h r o u g h o u t t h e i r E7 regions, we were interested to d e t e r m i n e w h e t h e r the d e c r e a s e d c o - t r a n s f o r m ing activities o f H P V - 6 a n d -I 1 E7s could be c o r r e l a t e d w i t h a d e c r e a s e d a b i l i t y to t r a n s a c t i v a t e the a d e n o v i r u s E2 p r o m o t e r p r e s e n t in the p l a s m i d p E 2 : : C A T ( M u r t h y et al., 1985). H P V E7 D N A s in pJ4~) were c o - t r a n s f e c t e d w i t h the p E 2 : : C A T p l a s m i d into dishes o f half-confluent N I H 3T3 cells. F o l l o w i n g glycerol t r e a t m e n t the cells were g r o w n for 48 h, h a r v e s t e d a n d a s s a y e d for C A T activity. T h e r e l a t i v e C A T activities o f t h e different E7 constructs c o m p a r e d to a w i l d - t y p e E l a , p r e s e n t on the p l a s m i d p C E ( S c h n e i d e r et al., 1987), are s h o w n in Fig. 7 a n d T a b l e 2. T h e virus t y p e s 16 a n d 33, w h i c h c o o p e r a t e d efficiently w i t h ras to form G 4 1 8 - r e s i s t a n t colonies, also s t i m u l a t e d t r a n s c r i p t i o n o f the E2 p r o m o t e r . HPV-11 E7 was effective in the t r a n s a c t i v a t i o n assay, d e s p i t e its low c o - t r a n s f o r m i n g activity. It is t h e r e f o r e u n l i k e l y t h a t
G
AC
CM
Fig. 7. Relative expression of the pE2" :CAT plasmid in NIH 3T3 cells transfected pE2: :CAT and HPV E7 DNA. The plasmid pCE contains a wild-type E I a gene. The positions of [14C]chloramphenicol (C M) and its acetylated derivative (AC) are indicated.
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A. Storey, K. Osborn and L. Crawford
papillomavirus co-transforming activity is simply a consequence of transactivation.
Discussion Sequence homology between the E7 proteins of genital papillomaviruses (Cole & Danos, 1987) suggests that the HPV-6 and HPV-11 E7 proteins might be able to mediate cell transformation with ras, or to transactivate the adenovirus E2 promoter in a similar manner to oncogenic papillomaviruses (types 16, 18, 31 and 33) (Phelps et al., 1988; Storey et al., 1988). The lack of cooperation between the entire early region of HPVs -6 and -11 (Storey et al., 1988) may be due to a functionally inactive E7 protein in these types, or the E7 protein may be active but produced below a 'threshold' level needed for co-transformation. Using a keratinocyte assay, Schlegel et al. (1988) showed that in medium containing serum and calcium macrocolony formation was limited to HPV-16 and -18, but in low calcium HPV-6 and -11 could form micro- and macrocolonies. Recent studies have shown that oncogenic and non-oncogenic papillomaviruses generate functional E7 mRNA by different splicing mechanisms (Smotkin et al., 1989), which could be important in determining the level of E7 protein. To avoid any differences due to splicing efficiency we have cloned the individual E 7 0 R F s from HPV types 6 and 11 directly behind a heterologous promoter, the MoMLV LTR and examined their transactivation and cotransforming activities. In the co-transfection experiments we show that HPV-6 E7 and HPV-11 E7 can cooperate with ras to transform primary BRK cells, but at a level 50- to 100-fold lower than HPV-16 E7 (Table 1). Southern and Northern blot analysis of expanded colonies of pJ4~6.E7/ras and pJ4~ll.E7/ras cells showed the presence of expressed unrearranged HPV DNA. The pJ4f]ll.E7/ras clone 2 cells grew faster, contained more HPV DNA and expressed more E7 mRNA than the clone 1 cells, suggesting that the level of E7 gene expression may be important in determining the in vitro growth rate of these cells. The pJ4~6.E7/ras and pJ4f]ll.E7/ras ceils were clearly transformed, as judged by their ability to form tumours in syngeneic immunocompetent rats within 3 weeks after injection. However, it was surprising that these cells generated tumours at the same rate as cells transformed by HPV-16 E7, as the ability of the HPV-6 and HPV-11 E7 genes to cooperate with ras to generate transformed colonies is massively reduced. The shared E7 protein sequence homology between papillomavirus types suggests that a polyclonal serum raised against HPV-16 E7 may cross-react with other E7 proteins. Immunoprecipitation of [35S]cysteine-labelled proteins
showed that a protein of about 15000 Mr was specifically precipitated from pJ4~l 1.E7/ras cells, suggesting that this may be the HPV-11 E7 protein. A protein of similar Mr to HPV-16 E7 could also be precipitated from pJ4f231. E7/ras cells, but the equivalent protein could not be detected in pJ4~6.E7/ras or pJ4f~33.E7/ras cells (Fig. 6). This may be because some epitopes that are present on HPV-16 E7 are absent from these E7 proteins. Type-specific sera or monoclonal antibodies will be needed to verify the identity of all these E7 proteins. Having established that HPV-6 E7 and HPV-11 E7 could generate malignantly transformed cells we then examined the ability of these DNAs to transactivate the adenovirus 2 promoter, to see whether the transcriptional stimulatory activities of E7 are important for its role in cell transformation. Neither E7 nor E 1a seems to bind to DNA directly (Feldman et al., 1982; Ferguson et al., 1985). Ela proteins contain three distinct regions, which are strongly conserved amongst adenovirus types (van Ormondt et al., 1980; Kimelman et al., 1985). Mutations in conserved regions 1 and 2, which are homologous to papillomavirus E7 N-terminal sequences, interfere with the ability of Ela to immortalize primary cells, to collaborate with ras in transformation and to induce cellular DNA synthesis, but are not required for transactivation (Lillie et al., 1986, 1987; Moran et al., 1986). Ela conserved region 3, which does not have an E7 homologue, is important and on its own can be sufficient for transactivation of early promoters (Glenn & Ricciardi, 1985; Lillie et al., 1986). In similar mutagenesis studies Schneider et al. (1987) suggested that transactivation was unlikely to play an important role in transformation by E 1a, as they were able to isolate mutants that could transform but not transactivate. They further suggested that Ela-mediated transformation occurs by repression of a cellular enhancer. Here we have shown that the ability of a particular viral E7 to transactivate is not always accompanied by an equivalent ability to co-transform, thus separating these two activities of E7 to a large extent. The results further suggest that not only splicing events within the E6 ORF but also the activity of the E7 protein itself are important in determining the oncogenic potential of a particular virus type. These co-transformation assays show both qualitative and quantitative differences in the oncogenic potential of these genital papillomaviruses. The occurrence of HPV-6 and HPV-11 in vulvar and laryngeal carcinomas is rare and this is paralleled in vitro, where greatly diminished co-transforming activities of these types are observed, yet the HPV-6 E7- and HPV-I1 E7-transformed cells are as tumorigenic as cells transformed by HPV-16 E7. Further investigation of the overall structure of these E7 proteins and their interaction with other
Co-transformation by HPV-6 and -11
cellular factors will be necessary to understand the role of E7 in cellular transformation. We would like to thank Dr F. Wettstein for the rabbit anti-E7 polyclonal serum, Carole Pye and Kate Hartley for their technical assistance, Dr ADders Follin for useful discussions during the course of this work and Helen Wilson for preparing the manuscript.
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(Received 22 May 1989; Accepted 18 September 1989)
