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The E7 functions of human papillomaviruses in rat 3Y1 cells
VIROLOGY
187, 107-l 14 (1992)
The E7 Functions SUMIE WATANABE,’ Department
of Human Papillomaviruses
in Rat 3Yl Cells
HIRONORI SATO,’ NAOKI KOMIYAMA,‘TADAHITO of Enteroviruses,
National institute of Health, Kamiosaki,
Received July 8. 199 1; accepted
November
KANDA, AND KUNITO YOSHIIKE
Shinagawa-ku,
Tokyo 14 1, Japan
5, 199 1
Among more than 60 human papillomavirus (HPV) genotypes, several HPVs are believed to be high risk because they are found in close association with cervical carcinoma. We compared the E7 genes from HPVs 1,6b, 16, 18, and 33 for their transactivating, transforming, and mitogenic functions in a single cell line rat 3Yl. Whereas both the low-risk (1 and 6b) and the high-risk (16.18, and 33) HPVs were transactivating for the adenovirus E2 promoter, only the high-risk HPVs were capable of focal transformation as assayed by an efficient method using the SRLu-promoter and in conjunction with the HPV 16 E6 gene. The putative oncogenicity of HPVs appears to be reflected in vitro by the focal transformation, but not by the transactivation. Transient expression of the E7 genes controlled by the dexamethasone-responsive MMTV-LTR showed that the HPV 16 mutant E7s only with residual transforming activity were not mitogenic, but that, although the low-risk HPV E7s were less efficient, both the low-risk and high-risk HPV E7s were capable of inducing cellular DNA synthesis. Probably, the capability to induce cell DNA synthesis is necessary but not sufficient o 1992 Academic PDZSS, inc. for the E7-mediated focal transformation.
by mitosis (Sato et al., 1989a). Correlation of the E7 functions and transformation has been studied with HPV 16 E7 mutants (Edmonds and Vousden, 1989; Watanabe et al., 1990; Storey et a/., 1990a) and the synthetic polypeptides of E7 (Rawls et al., 1990), but has not been fully elucidated as yet. More than 60 different HPV genotypes have been isolated and identified to date (de Villiers, 1989) and a group of HPVs (including 16, 18, and 33), which have been frequently found in cervical carcinomas, are considered high-risk types for these cancers. The variety of HPV types has facilitated comparative studies of the E7 functions of the HPVs with and without putative oncogenic potential (Storey et al., 1989, 1990b; Smotkin eta/., 1989; Gage eta/., 1990; Mungeretal., 1991). In this study we examined transactivating and transforming capabilities and the capacity to induce cell DNAsynthesis of HPVs 1, 6b, 16, 18, and 33 in a single cell line, rat 3Y1, in an attempt to correlate the in vitro E7 functions to the oncogenicity of HPVs.
INTRODUCTION Human papillomavirus type 16 (HPV 16) (Dilrst eta/., 1983) a probable causative agent for carcinoma of the cervix in humans (zur Hausen and Schneider, 1987) encodes two viral oncoproteins E6 and E7 capable of binding to tumor suppressor gene products (Dyson et a/., 1989; Werness et a/., 1990). Their ability to immortalize or transform rodent and human cells is believed to reflect in vitro the putative oncogenicity of HPV 16. Whereas expression of the two genes is required for extension of the life span of human fibroblasts (Watanabe et al., 1989) and for immortalization (Mllnger et a/., 1989a; Hawley-Nelson et al., 1989) or efficient immortalization (Halbet-t eta/., 1991) of human keratinocytes, expression of the E7 alone is sufficient for focal transformation of rat cells (Kanda eta/., 1988a,b) or morphological transformation of mouse cells (Phelps et a/., 1988). Like adenovirus ElA and SV40 large-T, the nuclear (Sato eta/., 1989b) HPV 16 E7 oncoprotein of 98 amino acid residues long (Seedot-f et al., 1985) is multifunctional. Expression of HPV 16 E7 is capable of activating chloramphenicol acetyltransferase (CAT) expression controlled by the adenovirus E2 promoter (Phelps et a/., 1988) and of inducing cell DNA synthesis followed
MATERIALS Expression
AND METHODS
plasmids
Appropriate HPV DNA fragments with appropriate linkers were inserted into expression vectors, pSV2-0 (at HindIll site), pSRa-0 (at HindIll site), and pMSN (at Xbal site), in a sense orientation. The entire genomic DNA sequences have been determined for HPV 1 (Danos et al., 1983) HPV 6b (Schwarz et a/., 1983), HPV 16 (Seedot-f et al., 1985), HPV 18 (Cole and Danos, 1987) and HPV 33 (Cole and Streeck, 1986).
’ To whom reprint requests should be addressed. ’ Present address: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892. ’ Present address: Meiji Institute of Health Science, 540 Naruda, Odawara 250. Japan. 107
0042-6822192
$3.00
Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.
108
WATANABE
The HPV DNA fragments that contained only the E7 open reading frame (ORF) intact were HP1 E7 (HPV 1 fragment nucleotides (nt) 487 to 971) HP6bE7 (HPV 6b nt 474 to 1074), E7P or HP1 6E7 (HPV 16 nt 554 to 875) HP18E7 (HPV 18 nt 512 to 923) and HP33E7 (HPV 33 nt 531 to 1077). Fragment E6 (HPV 16 nt 25 to 657) contained E6 ORF alone. Fragment E6E7 (HPV 16 nt 25 to 875) contained the E6 and E7 ORFs intact. E6(f)E7 and E6(/V)E7 have a frameshift mutation at ,Fnu4HI and lvcfel sites, respectively, within E6 ORF in the E6E7 (Watanabe et al., 1989). Fragments El (HPV 16 nt 686 to 321 1) and E2 (nt 2713 to 4470) contained El and E2 ORFs intact, respectively. Construction of HPV 16 E7 mutants 7H02D (His-2 replaced with Asp), 7L13S (Leu-13 replaced with Ser), 7C24G (Cys-24 replaced with Gly), and 7SS (Ser-31 -Ser-32 replaced with Arg) had been described previously (Watanabe et al., 1990). Expression vector pSV2-0 (Kanda et a/., 1988a) has the SV40 promoter and transcriptional regulatory elements, pSRar-0 (Kanda et al., 1991) has the SRorpromoter (Takebe et a/., 1988) consisting of SV40 ori and the HTLVl -LTR R-U5 sequence and the SV40 regulatory elements, and pMSN (Sato et al., 1989a), derived from pMDSG, has a dexamethasone-inducible promoter (MMTV-LTR) and the SV40-derived transcriptional elements and an independent transcriptional unit for the neomycin-resistance gene for dominant selection. Construction and preparation of plasmids and DNA were according to standard methods (Maniatis et a/., 1982). Detection
of HPV 16 E7 protein
For detection of steady-state E7 expression, monkey COS-1 (Gluzman, 1981) cells on coverslips were transfected by the DEAE-dextran method (McCutchan and Pagano, 1968) with pSV2-E7P or pSRor-E7 (1 pg of DNA per coverslip). At 48 hr after transfection, the transfected cells were processed as described previously with the anti-lac-E7 serum (Sato et a/., 198913) and fluorescent cells were scored. For detection of pulse-labeled E7, the COS-1 cells transfected with pSV2-E7P or pSRcu-E7or the E-/-transformed rat cells were labeled with [35S]methionine-cysteine mixture (trans35S-label; ICN Radiochemical, Inc., Irvine, CA). The labeled cells were processed as described previously (Watanabe et a/., 1990). Detection
of focal transformation
Subconfluent rat 3Yl (Kimura et al., 1975) cells (30mm dish) were transfected by the calcium coprecipitation method (Graham and van der Eb, 1973) with plasmid DNA (0.3 to 10 pg per culture). Transfected cells were replated at a split ratio of 1:30 and maintained for
ET AL
4 to 5 weeks as described previously (Kanda et al., 1987). Foci were scored with the cultures stained with 5% Giemsa. Before staining some foci were isolated for characterization. Anchorage-independent growth was tested as described (Kanda et a/., 1987). Detection
of transactivation
Rat 3Yl cells (60-mm dish) were transfected with pE2CAT (5 pg) and pSRa-plasmids with HPV E7 (5 pg) under the conditions used previously (Watanabe et a/., 1990). CAT activities were assayed for [14C]chloramphenicol (Amersham International plc., Buckinghamshire, England) with the cell extracts prepared 48 hr after transfection, as described by Gorman et al. (1982). Reporter plasmid pSRa-CAT (pSRa-0 containing CAT gene) was used for the test of transactivating function of pSRa-E6. Analyses
of RNA
Total RNA extracted from 3Y 1 cells was analyzed by the Northern (RNA) blot method as described previously (Watanabe et a/., 1990). RNA on membrane filters was probed with HPV 16 E6 or HPV 6 E7 fragments isolated from pSV2-expression plasmids and labeled with 32Pby nick translation. Induction
of ceil DNA synthesis
Rat 3Y 1 cells were transfected with pMSN plasmids containing HPV DNA fragments and were grown in the presence of G418 (400 pg/ml), as described previously (Sato et al,, 1989a). Randomly selected G418 resistant clones were maintained in the medium without serum for 2 days and, then, dexamethasone (lop6 M) was added to the medium. Twenty-two hours after the addition of the hormone, at which time the induced cell DNA synthesis reaches the highest level, cells were labeled with [3H]thymidine (Amersham) (2 &i/ml) for 2 hr and acid-insoluble fractions were measured for radioactivity. The ratio of radioactivity of dexamethasone-treated to nontreated cultures was used as an index for stimulation of DNA synthesis. RESULTS Expression of HPV 16 E7 ORF controlled SRa-promoter
by the
Use of the SRa-promoter in place of the SV40 promoter markedly enhanced expression of the HPV 16 E7 ORF placed under its control in monkey COS-1 cells and rat 3Yl cells (Table 1). The number of immunofluorescent cells (cells transiently producing E7 protein at a level detectable by immunofluorescence staining) at 48 hr after transfection increased by 20-fold. Increase
HPV E7 FUNCTIONS
109
IN RAT CELLS
TABLE 1
TABLE 2
COMPARISONOF pSV2 AND PSRL~EXPRESSIONPLASMIDSFOR HPV 16 E7
E7 TRANSACTIVATINGAND TRANSFORMINGFUNCTIONSOF HPVs
Plasmid
Steady-state E7 protein in monkey COS-1 cell9 No. of nuclei/fig DNA
Transforming activity for rat 3Yl cell9 No. of foci/l 0 pg DNA
pSV2-E7P pSRa-HP16E7
3.5 x lo3 7.9 x lo4
39 630
a Transfected COS-1 cells were stained with anti&c-E7 48 hr after transfection. ’ Number of foci of transformed cells scored 4 weeks after transfection.
in 35S-labeled E7 protein was also confirmed by the immunoprecipitation of the pulse-labeled proteins in the transfected cultures (data not shown). The efficiency of focus formation by the HPV 16 E7 gene in rat 3Yl cells was increased by 15-fold. Within a range of 0.3 to 10 gg DNA per culture, the number of foci increased proportionally with the increasing amount of transfecting DNA (data not shown). Several clones of pSRa-E7-transformed cells were isolated and examined for the presence of E7 mRNA (data not shown) and protein (Fig. 1A). The levels of expression in these clones were, on the average, higher than those of the cell clones obtained by transfection with pSV2-E7P. The results show that pSRor-0 is an efficient vehicle for expressing HPV 16 E7 transiently or permanently.
A
B 12
34
5
12
Plasmid
1
2
1
2
pSRor-0 pSRol-HP1 E7 pSRa-HPGbE7 pSRa-HP16E7 pSRa-HP18E7 pSRa-HP33E7
1.0 7.6 4.0 4.6 NT” 6.3
1 .o 8.4 4.4 5.2 3.4 6.0
0 0 0 590 330 140
0 0 0 340 180 50
a CAT activities (percentage conversion to acetylated form per protein content) of pE2CAT with each plasmid were normalized to that with pSRa-0. which is a nontransactivating backbone vector. ’ Number of foci scored 4 weeks after transfection. ’ Not tested.
E7 transactivating
and transforming
functions
Using the pSRa-expression vector, we compared HPVs 1, 6b, 16, 18, and 33 for their E7 transactivating and transforming functions in rat 3Yl cells (Table 2). The E7s from all the HPV types tested enhanced expression of CAT placed under the control of adenovirus E2 promoter in rat cells, but only HPVs 16, 18, and 33, which are from cervical carcinoma and thus considered to be high risk, were capable of focal transformation of rat 3Yl cells. Apparently, the two E7 functions in rat cells can be differentiated among HPV types. Synergetic
FIG. 1. Expression of HPV 16 E7 or E6 gene in transformed rat cells. (A) Expression of E7 protein under the control of the SV40 promoter and SRol promoter. Protein was immunoprecipitated with anti-lac-E7 from cell clones containing pSV2-E7P (lanes 1 and 2) and pSRti-E-I (lanes 3, 4, and 5). E7 protein is denoted by arrow. Bars on the left indicate the positions for molecular markers of 15, 20,28, 47, and 74 kDa (from the bottom). (B) Expression of E6 mRNA in rat cells isolated from foci of morphologically transformed cells in transfection with pSRa-E6. Total RNAs from cell clone E6-236 (lane 1) and clone E6-224 (lane 2) were examined by the Northern (RNA) blot method. E6 mRNA is denoted by arrow. Bars on the left indicate the positions for molecular size markers of 0.3, 1.4. and 2.4 kb (from the bottom).
Transforming activity for rat 3Yl cell@ No. of foci/l 0 pg DNA in Expt
Transactivation for Ad E2 promoter in rat 3Yl cell9 in Expt
transforming
function
of E7 and E6
Use of the SRa-promoter revealed the E6 transforming function, which is undetectable when placed under the control of the SV40 promoter (Kanda et al., 1988a), for rat 3Yl cells (Table 3). First, foci of the morphologically transformed cells were found at a very low frequency in the cultures transfected with the E6 alone (pSRa-E6). Second, the HPV 16 DNA fragment containing both E6 and E7 ORFs intact (pSRa-E6E7) generated significantly more foci than the DNA fragment containing E7 ORF alone (pSRa-E-l). Two HPV 16 DNA fragments, E6(F)E7 and E6(/V)E7, containing a frameshift mutation within the E6 ORF (at fnu4HI site and at lvo’el site, respectively) were tested for their transforming activities. The two fragments with mutated E6 failed to show the enhanced transforming activity (Table 3). Furthermore, the enhanced focal transformation was also noted when the mixture of ~SRCX-E6and pSRor-E7 was used, in place of pSRa-
110
WATANABE TABLE 3 COTRANSFORMATIONOF RAT 3Yl CELLS BY HPV 16 E6 AND E7 Transforming activity for rat 3Yl cells No. of foci/ 10 fig DNA in Expt Plasmid
1
2
pSRol-E6 pSRol-E7 pSRcu-E6E7’ pSRa-EG(F)E7* PSR~-EG(N)E~~
1 840 1980 500 210
1 660 2100 450 450
aTwo ORFs E6 and E7 are placed under a single set of SRapromoter. b E6E7 with a frameshift mutation at Fnu4HI site in E6 ORF. ’ E6E7 with a frameshift mutation at Mel site in E6 ORF.
E6E7, for transfection (data not shown). The results indicate that expression of the E6, not the relative genomic arrangement of E6 and E7 ORFs in the E6E7 fragment, is required for the enhanced focal transformation by the E6 and E7. Using pSRa-CAT as a reporter, we tested a possible transactivation function of the E6 protein on the SRa promoter, which could account for the synergetic transformation by E6 and E7. The CAT activity of the extract from 3Yl cells transfected with pSRol-CAT and pSRa-E6 (or pSRa-E7 or pSRa-E6E7) was marginally higher than that of those transfected with pSR&AT and pSRa-0 (data not shown), probably because the SRa promoter is by itself efficient and not transactivated much in 3Y 1 cells. The results strongly suggest that the transactivation by E6 is not responsible for the synergetic transformation. Foci produced by pSRa-E6, pSRt-v-E7, or pSRa-E6E7 were isolated and characterized. Two cell clones from transfection with pSRa-E6 alone were found to contain E6 DNA and mRNA, as assayed by the Southern (DNA) method (data not shown) and by the Northern (RNA) method (Fig. 1 B), respectively. Six clones obtained from transfection with pSRa-E-l or pSRor-E6E7 contained E7 mRNA and protein. The cell clones containing either E6 or E7 alone, or E6E7, when isolated and grown to confluence, showed slight but distinctive morphological difference among them (Fig. 2). At saturation, the E7-transformants appeared smaller and more densely packed than normal 3Yl cells and the E6transformants. The EG-transformants, under the same conditions, appeared thinner than normal 3Y1, and tended to appear packed somewhat in parallel. The double (E6 and E7) transformants, whether they were
ET AL.
from transfection with pSRa-E6E7 or the mixture of pSRa-E6 and pSRa-E7, or from two step transformation (the EG-transformants were transfected with pSRor-E7 and the resulting foci were isolated), had characteristics of the both transformants. The thin, small cells tended to parallel one another and formed collections of curved parallel lines like a part of whirlpool. In a soft agar medium, the E6E7 double transformants grew better than the E7-transformants, the E6transformants, and untransformed 3Yl cells. We tested for rat 3Y 1 cells the HPV 16 E7 genes with mutations in the N-terminal half (Watanabe eta/., 1990) and the E7 genes from HPVs 1, 6b, 18, and 33 under the control of SRa-promoter in conjunction with HPV 16 E6, because these conditions yield so far the highest efficiency for focal transformation (Table 4). Focal transformation by the HPV 16 mutants was enhanced by cotransfection with HPV 16 E6. Residual focusforming capacity of even nontransforming 7H02D (as assayed alone) was detectable as assayed in conjunction with the E6. The cotransformation increased focal transformation by the E7s from the high-risk HPVs, but failed to detect any focus-formation by the low-risk HPVs. Induction
of cell DNA synthesis
by E7
We previously showed that expression of HPV 16 E7 in rat 3Yl cells induces cell DNA synthesis and mitosis (Sato et a/., 1989a). In this study we tested the randomly selected G418-resistant clones, presumably containing the MMTV-LTR-controlled E7 genes from HPV 16 mutants or different HPVs, for their ability to induce cell DNA synthesis in response to the dexamethasone treatment (if integration occurs within the E7 expression unit, the E7 would be inactive). Induced cell DNA synthesis was expressed as a stimulation index, which is a ratio of radioactivity of dexamethasonetreated to nontreated cultures at 22 hr because the induced cell DNA synthesis reaches the highest level then (whereas induced E7 mRNA peaks at 7 to 10 hr) (Sato et al., 1989a). The number of cell clones was plotted against the intervals of stimulation indexes as shown in Fig. 3. Figure 3A shows the results obtained with cell clones from transfection with a backbone vector (pMSN), and pMSN plasmids containing the MMTVLTR controlled HPV 16 E7 or mutant E7s. The cell clones with the stimulation indexes higher than mean + 3 X SD for the cells containing the backbone vectors are considered to have the inducible E7 gene capable of inducing cell DNA synthesis in rat 3Yl cells, and the presence of such cell clones is regarded as evidence that the products of those E7 genes are mitogenic.
HPV E7 FUNCTIONS
IN RAT CELLS
111
FIG. 2. Rat 3Yl cell clones transformed by HPV 16 E6 or E7 and cotransformed by E6 and E7; morphology of cells (A through E) and anchorage-independent growth (F through J). (A, F) Untransformed cells; (6, G) EG-transformants from transfection with pSRa-E6; (C, H) E7-transformants from transfection with pSRa-E7; (D, I) E6E7 double transformants from transfection with pSRol-E6E7; (E, 1) E6E7 double transformants from transfection of EG-transformants with pSRa-E7. Phase-contrast photomicrographs (A through E) were taken at saturation of growing cells. Ratio of saturation densities for untransformed, EG-transformed, and E7-transformed cells was approximately 1:1.5:3. Pictures of cells in a 0.4% agarose medium (F through J) were taken 1 1 days after seeding. Bar, 100 pm.
Mutant 7SS, although it was less transforming than the wild type, particularly in the absence of E6, appeared to be capable of inducing cell DNA synthesis as efficiently as the wild type. Mutants 7H02D and 7C24G, which had only residual transforming activity in the presence of E6, were almost unable to induce cell DNA synthesis. A comparison of these mutant HPV 16 E7s suggests that the mitogenic ability tends to parallel the focus-forming capability. Figure 3B shows the results obtained with cell clones from transfection with the MMTV-LTR controlled E7 from HPVs 1, 6b, 16, and 18. The cell clones with HPV 16 El or E2 ORF controlled by the MMTVLTR did not respond to dexamethasone, indicating that these ORFs are probably not mitogenic for rat 3Yl cells. The cell clones with HPV 1 or HPV 6b E7 were found to contain some clones showing induced DNA synthesis in response to the hormone, less frequently than those with HPV 16 or 18 E7, but more frequently
than those with nontransforming HPV 16 E7s (7H02D and 7C24G). The cell clones containing the inducible HPV 33 E7, which were not included in Fig. 3B because they were fewer than those obtained with the other types, showed a distribution pattern similar to that of HPV 16 or 18. We further characterized the three cell clones presumably containing the HPV 6 E7 gene and showing the stimulation index higher than the mean + 3 X SD (Fig. 3B) to confirm that the induction of E7 mRNA synthesis (peaking at 7 hr) precedes the induction of DNA synthesis, as shown for HPV 16 E7. The HPV 6 E7 mRNA was shown to be induced in these cell clones before induction of DNA synthesis (Fig. 4). Despite the induction of abundant E7 mRNA in the HPV 6 clones, induction of cell DNA synthesis was less prominent in these cells than in the HPV 16 clones (Table 5). These data, togetherwith those in Fig. 3, indicate that the E7s of these HPVs were capable of inducing cell DNA syn-
WATANABE
112 TABLE 4
FOCALTRANSFORMATIONBY HPV 16 MUTANT E7 AND E7 FROM HPV TYPES IN CONJUNCTIONWITH HPV 16 E6” Rat 3Y 1 focus formation (No. of foci/l 0 pg of E7 DNA) in the presence of Plasmid
pSRa-0
pSRol-0 pSRol-HP16E7 pSRa-7H02Db pSRa-7L13S” pSRa-7C24Gd pSRcu-7SSe pSRa-HP1 E7 pSRa-HP6E7 pSRc+HP18E7 pSRa-HP33E7
0 480 0 350 5 150 0 0 340 170
a Mixture transfected b HPV 16 ’ HPV 16 d HPV 16 ’ HPV 16
pSRol-HP16E6 1 1400 50 1450 14 1010 0 0 1360 1160
ET AL.
the conditions used in this study, the efficiency of focal transformation by the E7s from HPVs 1 and 6b was estimated to be less than one-thousandth of that from HPV 16. Studies using synthetic E7 polypeptides have shown that transactivating function and mitogenic function are mapped to separate regions of the E7 polypeptide (Rawls et al., 1990). Whether or not the E7 transactivating function is required for focal transformation remains to be clarified. The HPV 16 E6 transforming function for rat 3Yl cells revealed by the SRcu-promoter was not only to raise efficiency of E7 focal transformation, but to give subtle but distinctive morphological changes to the transformed cells (Fig. 2). Presence of morphological difference between E6 and E7 transformants suggests that these two genes probably function differently in transformation. If anchorage-independent growth of
of E7 and backbone vector (pSRa-0) or HPV 16 E6 was to rat 3Yl cells for focal transformation. E7 with Asp substituting for His-2. E7 with Ser substituting for Leu-13. E7 with Gly substituting for Cys-24. E7 with Arg substituting for Ser-31 Ser-32.
6
1’
’
’
L
’
’
‘1
HPVldE2
_]
thesis, although HPVs 1 and 6b appeared to be less mitogenic than HPVs 16 and 18. HPVlE7
i
DISCUSSION Use of the SRa-promoter (Takebe et al., 1988) which enhanced expression of the HPV 16 oncogenes in monkey and rat cells, enabled us to compare the E7 functions of low-risk and high-risk HPVs in a single rat cell line in this study. The E7 transactivating activity in rat cells, which was low when controlled by the SV40promoter (Watanabe et al., 1990) could be readily measured and compared with those of other types. Increased levels of focal transformation by E7 made a quantitative comparison possible. Furthermore, the E6 transforming activity, which has been detectable only with primary rat cells (Kanda et al., 198813) became apparent in a rat cell line, probably owing to the increased level of expression. The putative oncogenicity of HPVs appears to be reflected in vitro by the capability of focal transformation, but not by the transactivating activity of E7. Whereas the high-risk HPVs (16, 18, and 33) were transactivating and transforming in rat 3Yl cells, the low-risk HPVs (1 and 6b) were transactivating but not transforming. The efficiency of E7-mediated focal transformation was increased when the E7 was expressed in conjunction with the E6, but no focal transformation was detectable with HPVs 1 and 6b. Under
HW16E7
_
I
I
rnp-,n
,rl,
,
HPVlSE7 -I t
123456 STIMULATION
INDEX
FIG. 3. Induction of cell DNA synthesis by the HPV 16 mutant E7s (A) and the E7s from HPV types (B). Stimulation index of DNA synthesis is a ratio of incorporated [3H]thymidine (at 22 hr) of dexamethasone-treated to nontreated cultures. pMSN is a nonmitogenic backbone vector. Cell clones were from transfection with the pMSN-plasmids containing the fragments indicated here and subsequent selection with G418. Description for the mutant E7s is given in the footnote for Table 4. For comparison, fragments El and E2 (HPV 16) have been included as nonmitogenic fragments. Arrow indicates the mean of the indexes in each group. Solid rectangles represent cell clones with the indexes within a range of the mean + 3 X SD of clones transfected with pMSN, and open rectangles represent cell clones with the indexes higher than this range.
HPV E7 FUNCTIONS
cells reflects malignancy, the E6E7 cotransformed cells were more malignant than those transformed by E7 or E6 alone. The sensitive cotransformation system (Table 4) revealed the residual transforming activities of HPV E7 mutants and showed that the E7 from HPVs 18 and 33 can work in conjunction with HPV 16 E6 in transformation, but failed to detect the transformation by the E7s from HPVs 1 and 6b. Probably, the capability to induce cell DNA synthesis is necessary but not sufficient for the E7-mediated focal transformation of rat 3Yl cells. Nontransforming E7 mutants of HPV 16,7H02D, and 7C24G did not induce cell DNA synthesis, but the E7 from low-risk HPVs incapable of focal transformation did. DNA synthesis induced by HPVs 1 and 6b was less marked than that by high-risk HPVs. Possibly, the mitogenic function of E7 is related to its capability to form a complex with the retinoblastoma tumor suppressor gene product (Rb), because the E7s from HPVs 6 and 11 can associate with Rb with a lower affinity than the E7 protein from HPV 16 (MOnger et a/., 1989b; Gage et al., 1990). It would be interesting to know whether the E7 from HPV 1 binds to Rb. In summary we found that the E7s from both low-risk and high-risk HPVs can transactivate a heterologous promoter and induce cell DNA synthesis in rat 3Yl cells, but that the E7s only from high-risk HPVs are capable of focal transformation. Thus, the focus assay in rat 3Yl cells, as well as the cotransformation assay with activated ras in primary rat cells (Storey et al., 1988), is probably useful to differentiate low-risk and high-risk types for new HPV isolates. We have found
IN RAT CELLS
113 TABLE 5
STIMULATIONOF DNA SYNTHESISBY DEXAMETHASONEIN RAT 3Yl CELLS CONTAINING INDUCIBLEHPV 6 E7 GENE
Cell clones
E7 gene from
Stimulation index of DNA synthesisb
1-7 l-14 2-21 2-23 2-27 3-42 3-45
None None HPV6 HPV 6 HPV6 HPV 16 HPV 16
0.98 0.78 2.5 4.8 2.8 6.2 6.9
a G418-resistant clones from transfection with pMSN-plasmids. b Stimulation index of DNA synthesis: a ratio of radioactivity dexamethasone-treated to nontreated cultures at 22 hr.
of
that the E7 ORF of HPV 58 isolated from cervical carcinoma (Matsukura and Sugase, 1990) is capable of focal transformation (unpublished data). Besides the E7 mitogenic function, a yet unidentified E7 function of high-risk HPVs is probably required the E7-mediated focal transformation. Perhaps this unidentified function plays an important role in carcinogenesis by high-risk HPVs. ACKNOWLEDGMENTS This work was supported in part by cancer research grants from the Ministry of Education, Science, and Culture and from the Ministry of Health and Welfare. H.S. was a fellow of the Japan Health Sciences Foundation.
REFERENCES 123456
78919
FIG. 4. Induction of HPV 6 E7 mRNA by dexamethasone in rat 3Y1 cells containing the inducible E7 gene. Total RNA was extracted from cells maintained for 7 hr without (lanes 1, 3, 5, 7, and 9) and with (lanes 2, 4, 6, 8, and IO) dexamethasone and was analyzed by the Northern (RNA) blot method. Filters with RNA were probed with ‘“P-labeled E7 fragments from HPV 6 (lanes 1 to 6) or 16 (lanes 7 to 10). Cell clones 2-21 (lanes 1 and 2), 2-23 (lanes 3 and 4), and 2-27 (lanes 5 and 6) contain the HPV 6 E7 gene. Cell clones 3-42 (lanes 7 and 8), and 3-45 (lanes 9 and 10) contain the HPV 16 E7 gene. Induction of cell DNA synthesis at 22 hr after addition of dexamethasone to these clones is shown in Table 5. E6 or E7 mRNA is denoted by arrow. Bars on the left indicate the positions for molecular size markers of 0.3, 1.4, and 2.4 kb (from the bottom).
COLE, S. T., and DANOS, 0. (1987). Nucleotide sequence and comparative analysis of the human papillomavirus type 18 genome. Phylogeny of papillomaviruses and repeated structure of the E6 and E7 gene products. J. Mol. Biol. 193, 599-608. COLE, S. T., and STREECK, R. E. (1986). Genome organization and nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer. J. Viral. 58, 991-995. DANOS, O., ENGEL. L. W., CHEN, E. Y., YANIV, M., and HOWLEY, P. M. (1983). Comparative analysis of the human type la and bovine type 1 papillomavirus genomes. J. Viral. 46, 557-566. DE VILLIERS,E.-M. (1989). Heterogeneity of the human papillomavirus group. J. Viral. 63, 4898-4903. DORST, M., GISSMANN, L., IKENBERG,H., andzv~ HAUSEN, H. (1983). A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions. Proc. Natl. Acad. Sci. USA 80, 3812-3815. DYSON, N., HOWLEY, P. M., MUNGER, K., and HARLOW, E. (1989). The human papilloma virus-l 6 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243, 934-937. EDMONDS, C., and VOUSDEN, K. H. (1989). A point mutational analysis of human papillomavirus type 16 E7 protein. J. Viral. 63, 26502656. GAGE, J. R., MEYERS,C., and WETBTEIN, F. 0. (1990). The E7 proteins
114
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of the nononcogenic human papillomavirus type 6b (HPV-6b) and of the oncogenic HPV-16 differ in retinobiastoma protein binding and other properties. J. Viral. 64, 723-730. GLUZMAN, Y. (1981). SV40-transformed simian ceils support the replication of early SV40 mutants. Cell 23, 175-l 82. GORMAN, C. M., MOFFAT, L. F., and HOWARD, B. H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. /3/o/. 2, 1044-l 051. GRAHAM, F. L., and VAN DER EB, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52,456467. HALBERT, C. L., DEMERS, G. W., and GALLOWAY, D. A. (1991). The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. 1. Vkol. 65, 473-478. HAWLEY-NELSON,P., VOUSDEN, K. H., HUBBERT,N. L., Lowv. D. R., and SCHILLER, J. T. (1989). HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J. 8, 39053910. KANDA, T., FURUNO, A., and YOSHIIKE, K. (1988a). Human papillomavirus type 16 open reading frame E7 encodes a transforming gene for rat 3Y 1 cells. J. Viral. 62, 61 O-61 3. KANDA, T., WATANABE, S., and YOSHIIKE,K. (1987). Human papillomavirus type 16 transformation of rat 3Yl cells. Jpn. 1. Cancer Res. (Gann) 78, 103-l 08. KANDA, T., WATANABE, S., and YOSHIIKE, K. (1988b). Immortalization of primary rat cells by human papillomavirus type 16 subgenomic DNA fragments controlled by the SV40 promoter. Krology 165, 321-325. KANDA, T., ZANMA, S., WATANABE, S., FURNO, A., and YOSHIIKE, K. (1991). Two immunodominant regions of the human papillomavirus type 16 E7 protein are masked in the nuclei of monkey COS-1 cells. Virology 182, 723-731. KIMURA, G., ITAGAKI,A., and SUMMERS, J. (1975). Rat cell line 3Yl and its virogenic polyoma- and SV40-transformed derivatives. Int J. Cancer 15,694-706. MANIATIS, T., FRITSCH, E. F., and SAMBROOK, J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. MATSUKURA,T., and SUGASE, M. (1990). Molecular cloning of a novel human papillomavirus (type 58) from an invasive cervical carcinoma. Virology 177, 833-836. MCCUTCHAN, J. H., and PAGANO, J. S. (1968). Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J. Nat/. Cancer Inst. 41, 351-357. M~~NGER,K., PHELPS,W. C., BUBB, V., HOWLEY, P. M., and SCHLEGEL, R. (1989a). The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J. Viral. 63, 4417-4421. MONGER, K., WERNESS, B. A., DYSON, N., PHELPS,W. C., HARLOW, E., and HOWLEY, P. M. (1989b). Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J. 8,4099-4105. MONGER, K., YEE, C. L., PHELPS,W. C., PIETENPOL,J. A., MOSES, H. L., and HOWLEY, P. M. (1991). Biochemical and biological differences between E7 and oncoproteins of the high- and low-risk human papillomavirus types are determined by amino-terminal sequences. J. Viral. 65, 3943-3948.
ET AL. PHELPS,W. C., YEE, C. L., MONGER, K., and HOWLEY,P. M. (1988). The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus ElA. Cell 53, 539-547. RAWLS, J. A., PUSTTAI, R., and GREEN, M. (1990). Chemical synthesis of human papillomavirus type 16 E7 oncoprotein: Autonomous protein domains for induction of cellular DNA synthesis and for trans activation. J. Viol. 64, 6121-6129. SATO, H., FURUNO,A., and YOSHIIKE,K. (1989a). Expression of human papillomavirus type 16 E7 gene induces DNA synthesis of rat 3Y 1 cells. Virology 168, 195-l 99. SATO, H., WATANABE, S., FURUNO, A., and YOSHIIKE, K. (1989b). Human papillomavirus type 16 E7 protein expressed in fscherichia co/i and monkey COS-1 ceils: lmmunofluorescence detection of the nuclear E7 protein. tirology 170, 31 l-31 5. SCHWARZ, E., D~~RsT,M., DEMANKOWSKI,C., LATTERMANN, O., ZECH, R., WOLFSPERGER,E., SUHAI, S., and ZUR HAUSEN, H. (1983). DNA sequence and genome organization of genital human papillomavirus type 6b. EMBO J. 2, 2341-2348. SEEDORF, K., KR&MMER, G.. DORST, M., SUHAI, S., and R~WEKAMP, W. G. (1985). Human papillomavirus type 16 DNAsequence. Viralogy 145, 181-185. SMOTKIN, D., PROKOPH, H., and WETTSTEIN, F. 0. (1989). Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms. J. Viral. 63, 1441-l 447. STOREY, A., ALMOND, N., OSBORN, K., and CRAWFORD, L. (1990a). Mutations of the human papillomavirus type 16 E7 gene that affect transformation, transactivation and phosphotylation by the E7 protein. J. Gen. Viral. 71, 965-970. STOREY,A., OSBORN, K.. and CRAWFORD, L. (1990b). Co-transformation by human papillomavirus types 6 and 1 1. J. Gen. Vkol. 71, 165-171. STOREY,A., PIM, D., MURRAY, A., OSBORN, K., BANKS, L., and CRAWFORD, L. (1988). Comparison of the in vitro transforming activities of human papillomavirus types. fMB0 J. 7, 1815-l 820. TAKEBE, Y., SEIKI, M., FUJISAWA,J., HOY, P., YOKOTA, K., ARAI, K., YoSHIDA, M., and ARAI, N. (1988). SRL~ promoter: An efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell. Biol. 8, 466-472. WATANABE, S., KANDA, T., SATO, H., FURUNO, A., and YOSHIIKE, K. (1990). Mutational analysis of human papillomavirus type 16 E7 functions. J. Vjrol. 64, 207-214. WATANABE, S., KANDA, T., and YOSHIIKE,K. (1989). Human papillomavirus type 16 transformation of primary human embryonic fibroblasts requires expression of open reading frames E6 and E7. 1. viral. 63, 965-969. WATANABE, S., and YOSHIIKE, K. (1989). Inefficient expression in rat cells of transforming gene of BK virus with 68.bp tandem repeats in the viral promoter-enhancer. Vjrology 169, 204-212. WERNESS,B. A., LEVINE,A. J., and HOWLEY, P. M. (1990). Association of human papillomavirus type 16 and 18 E6 proteins with ~53. Science 248,76-79. ZUR HAUSEN, H., and SCHNEIDER,A. (1987). The role of papillomaviruses in human anogenital cancer, p. 245-263. In “The Papovaviridae” (N. P. Salzman and P. M. Howley (Ed%), Vol. 2, pp. 245263. Plenum, New York.
187, 107-l 14 (1992)
The E7 Functions SUMIE WATANABE,’ Department
of Human Papillomaviruses
in Rat 3Yl Cells
HIRONORI SATO,’ NAOKI KOMIYAMA,‘TADAHITO of Enteroviruses,
National institute of Health, Kamiosaki,
Received July 8. 199 1; accepted
November
KANDA, AND KUNITO YOSHIIKE
Shinagawa-ku,
Tokyo 14 1, Japan
5, 199 1
Among more than 60 human papillomavirus (HPV) genotypes, several HPVs are believed to be high risk because they are found in close association with cervical carcinoma. We compared the E7 genes from HPVs 1,6b, 16, 18, and 33 for their transactivating, transforming, and mitogenic functions in a single cell line rat 3Yl. Whereas both the low-risk (1 and 6b) and the high-risk (16.18, and 33) HPVs were transactivating for the adenovirus E2 promoter, only the high-risk HPVs were capable of focal transformation as assayed by an efficient method using the SRLu-promoter and in conjunction with the HPV 16 E6 gene. The putative oncogenicity of HPVs appears to be reflected in vitro by the focal transformation, but not by the transactivation. Transient expression of the E7 genes controlled by the dexamethasone-responsive MMTV-LTR showed that the HPV 16 mutant E7s only with residual transforming activity were not mitogenic, but that, although the low-risk HPV E7s were less efficient, both the low-risk and high-risk HPV E7s were capable of inducing cellular DNA synthesis. Probably, the capability to induce cell DNA synthesis is necessary but not sufficient o 1992 Academic PDZSS, inc. for the E7-mediated focal transformation.
by mitosis (Sato et al., 1989a). Correlation of the E7 functions and transformation has been studied with HPV 16 E7 mutants (Edmonds and Vousden, 1989; Watanabe et al., 1990; Storey et a/., 1990a) and the synthetic polypeptides of E7 (Rawls et al., 1990), but has not been fully elucidated as yet. More than 60 different HPV genotypes have been isolated and identified to date (de Villiers, 1989) and a group of HPVs (including 16, 18, and 33), which have been frequently found in cervical carcinomas, are considered high-risk types for these cancers. The variety of HPV types has facilitated comparative studies of the E7 functions of the HPVs with and without putative oncogenic potential (Storey et al., 1989, 1990b; Smotkin eta/., 1989; Gage eta/., 1990; Mungeretal., 1991). In this study we examined transactivating and transforming capabilities and the capacity to induce cell DNAsynthesis of HPVs 1, 6b, 16, 18, and 33 in a single cell line, rat 3Y1, in an attempt to correlate the in vitro E7 functions to the oncogenicity of HPVs.
INTRODUCTION Human papillomavirus type 16 (HPV 16) (Dilrst eta/., 1983) a probable causative agent for carcinoma of the cervix in humans (zur Hausen and Schneider, 1987) encodes two viral oncoproteins E6 and E7 capable of binding to tumor suppressor gene products (Dyson et a/., 1989; Werness et a/., 1990). Their ability to immortalize or transform rodent and human cells is believed to reflect in vitro the putative oncogenicity of HPV 16. Whereas expression of the two genes is required for extension of the life span of human fibroblasts (Watanabe et al., 1989) and for immortalization (Mllnger et a/., 1989a; Hawley-Nelson et al., 1989) or efficient immortalization (Halbet-t eta/., 1991) of human keratinocytes, expression of the E7 alone is sufficient for focal transformation of rat cells (Kanda eta/., 1988a,b) or morphological transformation of mouse cells (Phelps et a/., 1988). Like adenovirus ElA and SV40 large-T, the nuclear (Sato eta/., 1989b) HPV 16 E7 oncoprotein of 98 amino acid residues long (Seedot-f et al., 1985) is multifunctional. Expression of HPV 16 E7 is capable of activating chloramphenicol acetyltransferase (CAT) expression controlled by the adenovirus E2 promoter (Phelps et a/., 1988) and of inducing cell DNA synthesis followed
MATERIALS Expression
AND METHODS
plasmids
Appropriate HPV DNA fragments with appropriate linkers were inserted into expression vectors, pSV2-0 (at HindIll site), pSRa-0 (at HindIll site), and pMSN (at Xbal site), in a sense orientation. The entire genomic DNA sequences have been determined for HPV 1 (Danos et al., 1983) HPV 6b (Schwarz et a/., 1983), HPV 16 (Seedot-f et al., 1985), HPV 18 (Cole and Danos, 1987) and HPV 33 (Cole and Streeck, 1986).
’ To whom reprint requests should be addressed. ’ Present address: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892. ’ Present address: Meiji Institute of Health Science, 540 Naruda, Odawara 250. Japan. 107
0042-6822192
$3.00
Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.
108
WATANABE
The HPV DNA fragments that contained only the E7 open reading frame (ORF) intact were HP1 E7 (HPV 1 fragment nucleotides (nt) 487 to 971) HP6bE7 (HPV 6b nt 474 to 1074), E7P or HP1 6E7 (HPV 16 nt 554 to 875) HP18E7 (HPV 18 nt 512 to 923) and HP33E7 (HPV 33 nt 531 to 1077). Fragment E6 (HPV 16 nt 25 to 657) contained E6 ORF alone. Fragment E6E7 (HPV 16 nt 25 to 875) contained the E6 and E7 ORFs intact. E6(f)E7 and E6(/V)E7 have a frameshift mutation at ,Fnu4HI and lvcfel sites, respectively, within E6 ORF in the E6E7 (Watanabe et al., 1989). Fragments El (HPV 16 nt 686 to 321 1) and E2 (nt 2713 to 4470) contained El and E2 ORFs intact, respectively. Construction of HPV 16 E7 mutants 7H02D (His-2 replaced with Asp), 7L13S (Leu-13 replaced with Ser), 7C24G (Cys-24 replaced with Gly), and 7SS (Ser-31 -Ser-32 replaced with Arg) had been described previously (Watanabe et al., 1990). Expression vector pSV2-0 (Kanda et a/., 1988a) has the SV40 promoter and transcriptional regulatory elements, pSRar-0 (Kanda et al., 1991) has the SRorpromoter (Takebe et a/., 1988) consisting of SV40 ori and the HTLVl -LTR R-U5 sequence and the SV40 regulatory elements, and pMSN (Sato et al., 1989a), derived from pMDSG, has a dexamethasone-inducible promoter (MMTV-LTR) and the SV40-derived transcriptional elements and an independent transcriptional unit for the neomycin-resistance gene for dominant selection. Construction and preparation of plasmids and DNA were according to standard methods (Maniatis et a/., 1982). Detection
of HPV 16 E7 protein
For detection of steady-state E7 expression, monkey COS-1 (Gluzman, 1981) cells on coverslips were transfected by the DEAE-dextran method (McCutchan and Pagano, 1968) with pSV2-E7P or pSRor-E7 (1 pg of DNA per coverslip). At 48 hr after transfection, the transfected cells were processed as described previously with the anti-lac-E7 serum (Sato et a/., 198913) and fluorescent cells were scored. For detection of pulse-labeled E7, the COS-1 cells transfected with pSV2-E7P or pSRcu-E7or the E-/-transformed rat cells were labeled with [35S]methionine-cysteine mixture (trans35S-label; ICN Radiochemical, Inc., Irvine, CA). The labeled cells were processed as described previously (Watanabe et a/., 1990). Detection
of focal transformation
Subconfluent rat 3Yl (Kimura et al., 1975) cells (30mm dish) were transfected by the calcium coprecipitation method (Graham and van der Eb, 1973) with plasmid DNA (0.3 to 10 pg per culture). Transfected cells were replated at a split ratio of 1:30 and maintained for
ET AL
4 to 5 weeks as described previously (Kanda et al., 1987). Foci were scored with the cultures stained with 5% Giemsa. Before staining some foci were isolated for characterization. Anchorage-independent growth was tested as described (Kanda et a/., 1987). Detection
of transactivation
Rat 3Yl cells (60-mm dish) were transfected with pE2CAT (5 pg) and pSRa-plasmids with HPV E7 (5 pg) under the conditions used previously (Watanabe et a/., 1990). CAT activities were assayed for [14C]chloramphenicol (Amersham International plc., Buckinghamshire, England) with the cell extracts prepared 48 hr after transfection, as described by Gorman et al. (1982). Reporter plasmid pSRa-CAT (pSRa-0 containing CAT gene) was used for the test of transactivating function of pSRa-E6. Analyses
of RNA
Total RNA extracted from 3Y 1 cells was analyzed by the Northern (RNA) blot method as described previously (Watanabe et a/., 1990). RNA on membrane filters was probed with HPV 16 E6 or HPV 6 E7 fragments isolated from pSV2-expression plasmids and labeled with 32Pby nick translation. Induction
of ceil DNA synthesis
Rat 3Y 1 cells were transfected with pMSN plasmids containing HPV DNA fragments and were grown in the presence of G418 (400 pg/ml), as described previously (Sato et al,, 1989a). Randomly selected G418 resistant clones were maintained in the medium without serum for 2 days and, then, dexamethasone (lop6 M) was added to the medium. Twenty-two hours after the addition of the hormone, at which time the induced cell DNA synthesis reaches the highest level, cells were labeled with [3H]thymidine (Amersham) (2 &i/ml) for 2 hr and acid-insoluble fractions were measured for radioactivity. The ratio of radioactivity of dexamethasone-treated to nontreated cultures was used as an index for stimulation of DNA synthesis. RESULTS Expression of HPV 16 E7 ORF controlled SRa-promoter
by the
Use of the SRa-promoter in place of the SV40 promoter markedly enhanced expression of the HPV 16 E7 ORF placed under its control in monkey COS-1 cells and rat 3Yl cells (Table 1). The number of immunofluorescent cells (cells transiently producing E7 protein at a level detectable by immunofluorescence staining) at 48 hr after transfection increased by 20-fold. Increase
HPV E7 FUNCTIONS
109
IN RAT CELLS
TABLE 1
TABLE 2
COMPARISONOF pSV2 AND PSRL~EXPRESSIONPLASMIDSFOR HPV 16 E7
E7 TRANSACTIVATINGAND TRANSFORMINGFUNCTIONSOF HPVs
Plasmid
Steady-state E7 protein in monkey COS-1 cell9 No. of nuclei/fig DNA
Transforming activity for rat 3Yl cell9 No. of foci/l 0 pg DNA
pSV2-E7P pSRa-HP16E7
3.5 x lo3 7.9 x lo4
39 630
a Transfected COS-1 cells were stained with anti&c-E7 48 hr after transfection. ’ Number of foci of transformed cells scored 4 weeks after transfection.
in 35S-labeled E7 protein was also confirmed by the immunoprecipitation of the pulse-labeled proteins in the transfected cultures (data not shown). The efficiency of focus formation by the HPV 16 E7 gene in rat 3Yl cells was increased by 15-fold. Within a range of 0.3 to 10 gg DNA per culture, the number of foci increased proportionally with the increasing amount of transfecting DNA (data not shown). Several clones of pSRa-E7-transformed cells were isolated and examined for the presence of E7 mRNA (data not shown) and protein (Fig. 1A). The levels of expression in these clones were, on the average, higher than those of the cell clones obtained by transfection with pSV2-E7P. The results show that pSRor-0 is an efficient vehicle for expressing HPV 16 E7 transiently or permanently.
A
B 12
34
5
12
Plasmid
1
2
1
2
pSRor-0 pSRol-HP1 E7 pSRa-HPGbE7 pSRa-HP16E7 pSRa-HP18E7 pSRa-HP33E7
1.0 7.6 4.0 4.6 NT” 6.3
1 .o 8.4 4.4 5.2 3.4 6.0
0 0 0 590 330 140
0 0 0 340 180 50
a CAT activities (percentage conversion to acetylated form per protein content) of pE2CAT with each plasmid were normalized to that with pSRa-0. which is a nontransactivating backbone vector. ’ Number of foci scored 4 weeks after transfection. ’ Not tested.
E7 transactivating
and transforming
functions
Using the pSRa-expression vector, we compared HPVs 1, 6b, 16, 18, and 33 for their E7 transactivating and transforming functions in rat 3Yl cells (Table 2). The E7s from all the HPV types tested enhanced expression of CAT placed under the control of adenovirus E2 promoter in rat cells, but only HPVs 16, 18, and 33, which are from cervical carcinoma and thus considered to be high risk, were capable of focal transformation of rat 3Yl cells. Apparently, the two E7 functions in rat cells can be differentiated among HPV types. Synergetic
FIG. 1. Expression of HPV 16 E7 or E6 gene in transformed rat cells. (A) Expression of E7 protein under the control of the SV40 promoter and SRol promoter. Protein was immunoprecipitated with anti-lac-E7 from cell clones containing pSV2-E7P (lanes 1 and 2) and pSRti-E-I (lanes 3, 4, and 5). E7 protein is denoted by arrow. Bars on the left indicate the positions for molecular markers of 15, 20,28, 47, and 74 kDa (from the bottom). (B) Expression of E6 mRNA in rat cells isolated from foci of morphologically transformed cells in transfection with pSRa-E6. Total RNAs from cell clone E6-236 (lane 1) and clone E6-224 (lane 2) were examined by the Northern (RNA) blot method. E6 mRNA is denoted by arrow. Bars on the left indicate the positions for molecular size markers of 0.3, 1.4. and 2.4 kb (from the bottom).
Transforming activity for rat 3Yl cell@ No. of foci/l 0 pg DNA in Expt
Transactivation for Ad E2 promoter in rat 3Yl cell9 in Expt
transforming
function
of E7 and E6
Use of the SRa-promoter revealed the E6 transforming function, which is undetectable when placed under the control of the SV40 promoter (Kanda et al., 1988a), for rat 3Yl cells (Table 3). First, foci of the morphologically transformed cells were found at a very low frequency in the cultures transfected with the E6 alone (pSRa-E6). Second, the HPV 16 DNA fragment containing both E6 and E7 ORFs intact (pSRa-E6E7) generated significantly more foci than the DNA fragment containing E7 ORF alone (pSRa-E-l). Two HPV 16 DNA fragments, E6(F)E7 and E6(/V)E7, containing a frameshift mutation within the E6 ORF (at fnu4HI site and at lvo’el site, respectively) were tested for their transforming activities. The two fragments with mutated E6 failed to show the enhanced transforming activity (Table 3). Furthermore, the enhanced focal transformation was also noted when the mixture of ~SRCX-E6and pSRor-E7 was used, in place of pSRa-
110
WATANABE TABLE 3 COTRANSFORMATIONOF RAT 3Yl CELLS BY HPV 16 E6 AND E7 Transforming activity for rat 3Yl cells No. of foci/ 10 fig DNA in Expt Plasmid
1
2
pSRol-E6 pSRol-E7 pSRcu-E6E7’ pSRa-EG(F)E7* PSR~-EG(N)E~~
1 840 1980 500 210
1 660 2100 450 450
aTwo ORFs E6 and E7 are placed under a single set of SRapromoter. b E6E7 with a frameshift mutation at Fnu4HI site in E6 ORF. ’ E6E7 with a frameshift mutation at Mel site in E6 ORF.
E6E7, for transfection (data not shown). The results indicate that expression of the E6, not the relative genomic arrangement of E6 and E7 ORFs in the E6E7 fragment, is required for the enhanced focal transformation by the E6 and E7. Using pSRa-CAT as a reporter, we tested a possible transactivation function of the E6 protein on the SRa promoter, which could account for the synergetic transformation by E6 and E7. The CAT activity of the extract from 3Yl cells transfected with pSRol-CAT and pSRa-E6 (or pSRa-E7 or pSRa-E6E7) was marginally higher than that of those transfected with pSR&AT and pSRa-0 (data not shown), probably because the SRa promoter is by itself efficient and not transactivated much in 3Y 1 cells. The results strongly suggest that the transactivation by E6 is not responsible for the synergetic transformation. Foci produced by pSRa-E6, pSRt-v-E7, or pSRa-E6E7 were isolated and characterized. Two cell clones from transfection with pSRa-E6 alone were found to contain E6 DNA and mRNA, as assayed by the Southern (DNA) method (data not shown) and by the Northern (RNA) method (Fig. 1 B), respectively. Six clones obtained from transfection with pSRa-E-l or pSRor-E6E7 contained E7 mRNA and protein. The cell clones containing either E6 or E7 alone, or E6E7, when isolated and grown to confluence, showed slight but distinctive morphological difference among them (Fig. 2). At saturation, the E7-transformants appeared smaller and more densely packed than normal 3Yl cells and the E6transformants. The EG-transformants, under the same conditions, appeared thinner than normal 3Y1, and tended to appear packed somewhat in parallel. The double (E6 and E7) transformants, whether they were
ET AL.
from transfection with pSRa-E6E7 or the mixture of pSRa-E6 and pSRa-E7, or from two step transformation (the EG-transformants were transfected with pSRor-E7 and the resulting foci were isolated), had characteristics of the both transformants. The thin, small cells tended to parallel one another and formed collections of curved parallel lines like a part of whirlpool. In a soft agar medium, the E6E7 double transformants grew better than the E7-transformants, the E6transformants, and untransformed 3Yl cells. We tested for rat 3Y 1 cells the HPV 16 E7 genes with mutations in the N-terminal half (Watanabe eta/., 1990) and the E7 genes from HPVs 1, 6b, 18, and 33 under the control of SRa-promoter in conjunction with HPV 16 E6, because these conditions yield so far the highest efficiency for focal transformation (Table 4). Focal transformation by the HPV 16 mutants was enhanced by cotransfection with HPV 16 E6. Residual focusforming capacity of even nontransforming 7H02D (as assayed alone) was detectable as assayed in conjunction with the E6. The cotransformation increased focal transformation by the E7s from the high-risk HPVs, but failed to detect any focus-formation by the low-risk HPVs. Induction
of cell DNA synthesis
by E7
We previously showed that expression of HPV 16 E7 in rat 3Yl cells induces cell DNA synthesis and mitosis (Sato et a/., 1989a). In this study we tested the randomly selected G418-resistant clones, presumably containing the MMTV-LTR-controlled E7 genes from HPV 16 mutants or different HPVs, for their ability to induce cell DNA synthesis in response to the dexamethasone treatment (if integration occurs within the E7 expression unit, the E7 would be inactive). Induced cell DNA synthesis was expressed as a stimulation index, which is a ratio of radioactivity of dexamethasonetreated to nontreated cultures at 22 hr because the induced cell DNA synthesis reaches the highest level then (whereas induced E7 mRNA peaks at 7 to 10 hr) (Sato et al., 1989a). The number of cell clones was plotted against the intervals of stimulation indexes as shown in Fig. 3. Figure 3A shows the results obtained with cell clones from transfection with a backbone vector (pMSN), and pMSN plasmids containing the MMTVLTR controlled HPV 16 E7 or mutant E7s. The cell clones with the stimulation indexes higher than mean + 3 X SD for the cells containing the backbone vectors are considered to have the inducible E7 gene capable of inducing cell DNA synthesis in rat 3Yl cells, and the presence of such cell clones is regarded as evidence that the products of those E7 genes are mitogenic.
HPV E7 FUNCTIONS
IN RAT CELLS
111
FIG. 2. Rat 3Yl cell clones transformed by HPV 16 E6 or E7 and cotransformed by E6 and E7; morphology of cells (A through E) and anchorage-independent growth (F through J). (A, F) Untransformed cells; (6, G) EG-transformants from transfection with pSRa-E6; (C, H) E7-transformants from transfection with pSRa-E7; (D, I) E6E7 double transformants from transfection with pSRol-E6E7; (E, 1) E6E7 double transformants from transfection of EG-transformants with pSRa-E7. Phase-contrast photomicrographs (A through E) were taken at saturation of growing cells. Ratio of saturation densities for untransformed, EG-transformed, and E7-transformed cells was approximately 1:1.5:3. Pictures of cells in a 0.4% agarose medium (F through J) were taken 1 1 days after seeding. Bar, 100 pm.
Mutant 7SS, although it was less transforming than the wild type, particularly in the absence of E6, appeared to be capable of inducing cell DNA synthesis as efficiently as the wild type. Mutants 7H02D and 7C24G, which had only residual transforming activity in the presence of E6, were almost unable to induce cell DNA synthesis. A comparison of these mutant HPV 16 E7s suggests that the mitogenic ability tends to parallel the focus-forming capability. Figure 3B shows the results obtained with cell clones from transfection with the MMTV-LTR controlled E7 from HPVs 1, 6b, 16, and 18. The cell clones with HPV 16 El or E2 ORF controlled by the MMTVLTR did not respond to dexamethasone, indicating that these ORFs are probably not mitogenic for rat 3Yl cells. The cell clones with HPV 1 or HPV 6b E7 were found to contain some clones showing induced DNA synthesis in response to the hormone, less frequently than those with HPV 16 or 18 E7, but more frequently
than those with nontransforming HPV 16 E7s (7H02D and 7C24G). The cell clones containing the inducible HPV 33 E7, which were not included in Fig. 3B because they were fewer than those obtained with the other types, showed a distribution pattern similar to that of HPV 16 or 18. We further characterized the three cell clones presumably containing the HPV 6 E7 gene and showing the stimulation index higher than the mean + 3 X SD (Fig. 3B) to confirm that the induction of E7 mRNA synthesis (peaking at 7 hr) precedes the induction of DNA synthesis, as shown for HPV 16 E7. The HPV 6 E7 mRNA was shown to be induced in these cell clones before induction of DNA synthesis (Fig. 4). Despite the induction of abundant E7 mRNA in the HPV 6 clones, induction of cell DNA synthesis was less prominent in these cells than in the HPV 16 clones (Table 5). These data, togetherwith those in Fig. 3, indicate that the E7s of these HPVs were capable of inducing cell DNA syn-
WATANABE
112 TABLE 4
FOCALTRANSFORMATIONBY HPV 16 MUTANT E7 AND E7 FROM HPV TYPES IN CONJUNCTIONWITH HPV 16 E6” Rat 3Y 1 focus formation (No. of foci/l 0 pg of E7 DNA) in the presence of Plasmid
pSRa-0
pSRol-0 pSRol-HP16E7 pSRa-7H02Db pSRa-7L13S” pSRa-7C24Gd pSRcu-7SSe pSRa-HP1 E7 pSRa-HP6E7 pSRc+HP18E7 pSRa-HP33E7
0 480 0 350 5 150 0 0 340 170
a Mixture transfected b HPV 16 ’ HPV 16 d HPV 16 ’ HPV 16
pSRol-HP16E6 1 1400 50 1450 14 1010 0 0 1360 1160
ET AL.
the conditions used in this study, the efficiency of focal transformation by the E7s from HPVs 1 and 6b was estimated to be less than one-thousandth of that from HPV 16. Studies using synthetic E7 polypeptides have shown that transactivating function and mitogenic function are mapped to separate regions of the E7 polypeptide (Rawls et al., 1990). Whether or not the E7 transactivating function is required for focal transformation remains to be clarified. The HPV 16 E6 transforming function for rat 3Yl cells revealed by the SRcu-promoter was not only to raise efficiency of E7 focal transformation, but to give subtle but distinctive morphological changes to the transformed cells (Fig. 2). Presence of morphological difference between E6 and E7 transformants suggests that these two genes probably function differently in transformation. If anchorage-independent growth of
of E7 and backbone vector (pSRa-0) or HPV 16 E6 was to rat 3Yl cells for focal transformation. E7 with Asp substituting for His-2. E7 with Ser substituting for Leu-13. E7 with Gly substituting for Cys-24. E7 with Arg substituting for Ser-31 Ser-32.
6
1’
’
’
L
’
’
‘1
HPVldE2
_]
thesis, although HPVs 1 and 6b appeared to be less mitogenic than HPVs 16 and 18. HPVlE7
i
DISCUSSION Use of the SRa-promoter (Takebe et al., 1988) which enhanced expression of the HPV 16 oncogenes in monkey and rat cells, enabled us to compare the E7 functions of low-risk and high-risk HPVs in a single rat cell line in this study. The E7 transactivating activity in rat cells, which was low when controlled by the SV40promoter (Watanabe et al., 1990) could be readily measured and compared with those of other types. Increased levels of focal transformation by E7 made a quantitative comparison possible. Furthermore, the E6 transforming activity, which has been detectable only with primary rat cells (Kanda et al., 198813) became apparent in a rat cell line, probably owing to the increased level of expression. The putative oncogenicity of HPVs appears to be reflected in vitro by the capability of focal transformation, but not by the transactivating activity of E7. Whereas the high-risk HPVs (16, 18, and 33) were transactivating and transforming in rat 3Yl cells, the low-risk HPVs (1 and 6b) were transactivating but not transforming. The efficiency of E7-mediated focal transformation was increased when the E7 was expressed in conjunction with the E6, but no focal transformation was detectable with HPVs 1 and 6b. Under
HW16E7
_
I
I
rnp-,n
,rl,
,
HPVlSE7 -I t
123456 STIMULATION
INDEX
FIG. 3. Induction of cell DNA synthesis by the HPV 16 mutant E7s (A) and the E7s from HPV types (B). Stimulation index of DNA synthesis is a ratio of incorporated [3H]thymidine (at 22 hr) of dexamethasone-treated to nontreated cultures. pMSN is a nonmitogenic backbone vector. Cell clones were from transfection with the pMSN-plasmids containing the fragments indicated here and subsequent selection with G418. Description for the mutant E7s is given in the footnote for Table 4. For comparison, fragments El and E2 (HPV 16) have been included as nonmitogenic fragments. Arrow indicates the mean of the indexes in each group. Solid rectangles represent cell clones with the indexes within a range of the mean + 3 X SD of clones transfected with pMSN, and open rectangles represent cell clones with the indexes higher than this range.
HPV E7 FUNCTIONS
cells reflects malignancy, the E6E7 cotransformed cells were more malignant than those transformed by E7 or E6 alone. The sensitive cotransformation system (Table 4) revealed the residual transforming activities of HPV E7 mutants and showed that the E7 from HPVs 18 and 33 can work in conjunction with HPV 16 E6 in transformation, but failed to detect the transformation by the E7s from HPVs 1 and 6b. Probably, the capability to induce cell DNA synthesis is necessary but not sufficient for the E7-mediated focal transformation of rat 3Yl cells. Nontransforming E7 mutants of HPV 16,7H02D, and 7C24G did not induce cell DNA synthesis, but the E7 from low-risk HPVs incapable of focal transformation did. DNA synthesis induced by HPVs 1 and 6b was less marked than that by high-risk HPVs. Possibly, the mitogenic function of E7 is related to its capability to form a complex with the retinoblastoma tumor suppressor gene product (Rb), because the E7s from HPVs 6 and 11 can associate with Rb with a lower affinity than the E7 protein from HPV 16 (MOnger et a/., 1989b; Gage et al., 1990). It would be interesting to know whether the E7 from HPV 1 binds to Rb. In summary we found that the E7s from both low-risk and high-risk HPVs can transactivate a heterologous promoter and induce cell DNA synthesis in rat 3Yl cells, but that the E7s only from high-risk HPVs are capable of focal transformation. Thus, the focus assay in rat 3Yl cells, as well as the cotransformation assay with activated ras in primary rat cells (Storey et al., 1988), is probably useful to differentiate low-risk and high-risk types for new HPV isolates. We have found
IN RAT CELLS
113 TABLE 5
STIMULATIONOF DNA SYNTHESISBY DEXAMETHASONEIN RAT 3Yl CELLS CONTAINING INDUCIBLEHPV 6 E7 GENE
Cell clones
E7 gene from
Stimulation index of DNA synthesisb
1-7 l-14 2-21 2-23 2-27 3-42 3-45
None None HPV6 HPV 6 HPV6 HPV 16 HPV 16
0.98 0.78 2.5 4.8 2.8 6.2 6.9
a G418-resistant clones from transfection with pMSN-plasmids. b Stimulation index of DNA synthesis: a ratio of radioactivity dexamethasone-treated to nontreated cultures at 22 hr.
of
that the E7 ORF of HPV 58 isolated from cervical carcinoma (Matsukura and Sugase, 1990) is capable of focal transformation (unpublished data). Besides the E7 mitogenic function, a yet unidentified E7 function of high-risk HPVs is probably required the E7-mediated focal transformation. Perhaps this unidentified function plays an important role in carcinogenesis by high-risk HPVs. ACKNOWLEDGMENTS This work was supported in part by cancer research grants from the Ministry of Education, Science, and Culture and from the Ministry of Health and Welfare. H.S. was a fellow of the Japan Health Sciences Foundation.
REFERENCES 123456
78919
FIG. 4. Induction of HPV 6 E7 mRNA by dexamethasone in rat 3Y1 cells containing the inducible E7 gene. Total RNA was extracted from cells maintained for 7 hr without (lanes 1, 3, 5, 7, and 9) and with (lanes 2, 4, 6, 8, and IO) dexamethasone and was analyzed by the Northern (RNA) blot method. Filters with RNA were probed with ‘“P-labeled E7 fragments from HPV 6 (lanes 1 to 6) or 16 (lanes 7 to 10). Cell clones 2-21 (lanes 1 and 2), 2-23 (lanes 3 and 4), and 2-27 (lanes 5 and 6) contain the HPV 6 E7 gene. Cell clones 3-42 (lanes 7 and 8), and 3-45 (lanes 9 and 10) contain the HPV 16 E7 gene. Induction of cell DNA synthesis at 22 hr after addition of dexamethasone to these clones is shown in Table 5. E6 or E7 mRNA is denoted by arrow. Bars on the left indicate the positions for molecular size markers of 0.3, 1.4, and 2.4 kb (from the bottom).
COLE, S. T., and DANOS, 0. (1987). Nucleotide sequence and comparative analysis of the human papillomavirus type 18 genome. Phylogeny of papillomaviruses and repeated structure of the E6 and E7 gene products. J. Mol. Biol. 193, 599-608. COLE, S. T., and STREECK, R. E. (1986). Genome organization and nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer. J. Viral. 58, 991-995. DANOS, O., ENGEL. L. W., CHEN, E. Y., YANIV, M., and HOWLEY, P. M. (1983). Comparative analysis of the human type la and bovine type 1 papillomavirus genomes. J. Viral. 46, 557-566. DE VILLIERS,E.-M. (1989). Heterogeneity of the human papillomavirus group. J. Viral. 63, 4898-4903. DORST, M., GISSMANN, L., IKENBERG,H., andzv~ HAUSEN, H. (1983). A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions. Proc. Natl. Acad. Sci. USA 80, 3812-3815. DYSON, N., HOWLEY, P. M., MUNGER, K., and HARLOW, E. (1989). The human papilloma virus-l 6 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243, 934-937. EDMONDS, C., and VOUSDEN, K. H. (1989). A point mutational analysis of human papillomavirus type 16 E7 protein. J. Viral. 63, 26502656. GAGE, J. R., MEYERS,C., and WETBTEIN, F. 0. (1990). The E7 proteins
114
WATANABE
of the nononcogenic human papillomavirus type 6b (HPV-6b) and of the oncogenic HPV-16 differ in retinobiastoma protein binding and other properties. J. Viral. 64, 723-730. GLUZMAN, Y. (1981). SV40-transformed simian ceils support the replication of early SV40 mutants. Cell 23, 175-l 82. GORMAN, C. M., MOFFAT, L. F., and HOWARD, B. H. (1982). Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol. Cell. /3/o/. 2, 1044-l 051. GRAHAM, F. L., and VAN DER EB, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52,456467. HALBERT, C. L., DEMERS, G. W., and GALLOWAY, D. A. (1991). The E7 gene of human papillomavirus type 16 is sufficient for immortalization of human epithelial cells. 1. Vkol. 65, 473-478. HAWLEY-NELSON,P., VOUSDEN, K. H., HUBBERT,N. L., Lowv. D. R., and SCHILLER, J. T. (1989). HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J. 8, 39053910. KANDA, T., FURUNO, A., and YOSHIIKE, K. (1988a). Human papillomavirus type 16 open reading frame E7 encodes a transforming gene for rat 3Y 1 cells. J. Viral. 62, 61 O-61 3. KANDA, T., WATANABE, S., and YOSHIIKE,K. (1987). Human papillomavirus type 16 transformation of rat 3Yl cells. Jpn. 1. Cancer Res. (Gann) 78, 103-l 08. KANDA, T., WATANABE, S., and YOSHIIKE, K. (1988b). Immortalization of primary rat cells by human papillomavirus type 16 subgenomic DNA fragments controlled by the SV40 promoter. Krology 165, 321-325. KANDA, T., ZANMA, S., WATANABE, S., FURNO, A., and YOSHIIKE, K. (1991). Two immunodominant regions of the human papillomavirus type 16 E7 protein are masked in the nuclei of monkey COS-1 cells. Virology 182, 723-731. KIMURA, G., ITAGAKI,A., and SUMMERS, J. (1975). Rat cell line 3Yl and its virogenic polyoma- and SV40-transformed derivatives. Int J. Cancer 15,694-706. MANIATIS, T., FRITSCH, E. F., and SAMBROOK, J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. MATSUKURA,T., and SUGASE, M. (1990). Molecular cloning of a novel human papillomavirus (type 58) from an invasive cervical carcinoma. Virology 177, 833-836. MCCUTCHAN, J. H., and PAGANO, J. S. (1968). Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J. Nat/. Cancer Inst. 41, 351-357. M~~NGER,K., PHELPS,W. C., BUBB, V., HOWLEY, P. M., and SCHLEGEL, R. (1989a). The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J. Viral. 63, 4417-4421. MONGER, K., WERNESS, B. A., DYSON, N., PHELPS,W. C., HARLOW, E., and HOWLEY, P. M. (1989b). Complex formation of human papillomavirus E7 proteins with the retinoblastoma tumor suppressor gene product. EMBO J. 8,4099-4105. MONGER, K., YEE, C. L., PHELPS,W. C., PIETENPOL,J. A., MOSES, H. L., and HOWLEY, P. M. (1991). Biochemical and biological differences between E7 and oncoproteins of the high- and low-risk human papillomavirus types are determined by amino-terminal sequences. J. Viral. 65, 3943-3948.
ET AL. PHELPS,W. C., YEE, C. L., MONGER, K., and HOWLEY,P. M. (1988). The human papillomavirus type 16 E7 gene encodes transactivation and transformation functions similar to those of adenovirus ElA. Cell 53, 539-547. RAWLS, J. A., PUSTTAI, R., and GREEN, M. (1990). Chemical synthesis of human papillomavirus type 16 E7 oncoprotein: Autonomous protein domains for induction of cellular DNA synthesis and for trans activation. J. Viol. 64, 6121-6129. SATO, H., FURUNO,A., and YOSHIIKE,K. (1989a). Expression of human papillomavirus type 16 E7 gene induces DNA synthesis of rat 3Y 1 cells. Virology 168, 195-l 99. SATO, H., WATANABE, S., FURUNO, A., and YOSHIIKE, K. (1989b). Human papillomavirus type 16 E7 protein expressed in fscherichia co/i and monkey COS-1 ceils: lmmunofluorescence detection of the nuclear E7 protein. tirology 170, 31 l-31 5. SCHWARZ, E., D~~RsT,M., DEMANKOWSKI,C., LATTERMANN, O., ZECH, R., WOLFSPERGER,E., SUHAI, S., and ZUR HAUSEN, H. (1983). DNA sequence and genome organization of genital human papillomavirus type 6b. EMBO J. 2, 2341-2348. SEEDORF, K., KR&MMER, G.. DORST, M., SUHAI, S., and R~WEKAMP, W. G. (1985). Human papillomavirus type 16 DNAsequence. Viralogy 145, 181-185. SMOTKIN, D., PROKOPH, H., and WETTSTEIN, F. 0. (1989). Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms. J. Viral. 63, 1441-l 447. STOREY, A., ALMOND, N., OSBORN, K., and CRAWFORD, L. (1990a). Mutations of the human papillomavirus type 16 E7 gene that affect transformation, transactivation and phosphotylation by the E7 protein. J. Gen. Viral. 71, 965-970. STOREY,A., OSBORN, K.. and CRAWFORD, L. (1990b). Co-transformation by human papillomavirus types 6 and 1 1. J. Gen. Vkol. 71, 165-171. STOREY,A., PIM, D., MURRAY, A., OSBORN, K., BANKS, L., and CRAWFORD, L. (1988). Comparison of the in vitro transforming activities of human papillomavirus types. fMB0 J. 7, 1815-l 820. TAKEBE, Y., SEIKI, M., FUJISAWA,J., HOY, P., YOKOTA, K., ARAI, K., YoSHIDA, M., and ARAI, N. (1988). SRL~ promoter: An efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell. Biol. 8, 466-472. WATANABE, S., KANDA, T., SATO, H., FURUNO, A., and YOSHIIKE, K. (1990). Mutational analysis of human papillomavirus type 16 E7 functions. J. Vjrol. 64, 207-214. WATANABE, S., KANDA, T., and YOSHIIKE,K. (1989). Human papillomavirus type 16 transformation of primary human embryonic fibroblasts requires expression of open reading frames E6 and E7. 1. viral. 63, 965-969. WATANABE, S., and YOSHIIKE, K. (1989). Inefficient expression in rat cells of transforming gene of BK virus with 68.bp tandem repeats in the viral promoter-enhancer. Vjrology 169, 204-212. WERNESS,B. A., LEVINE,A. J., and HOWLEY, P. M. (1990). Association of human papillomavirus type 16 and 18 E6 proteins with ~53. Science 248,76-79. ZUR HAUSEN, H., and SCHNEIDER,A. (1987). The role of papillomaviruses in human anogenital cancer, p. 245-263. In “The Papovaviridae” (N. P. Salzman and P. M. Howley (Ed%), Vol. 2, pp. 245263. Plenum, New York.