Mar 5, 1991 - HAROLD L. MOSES,3 AND PETER M. HOWLEY'. Laboratory of ..... Huang, W.-H. Lee, E. Marsilio, E. Paucha, and D. M. Living- ston. 1988.
JOURNAL OF VIROLOGY, July 1991, p. 3943-3948
Vol. 65, No. 7
0022-538X/91/073943-06$02.00/0 Copyright X3 1991, American Society for Microbiology
Biochemical and Biological Differences between E7 Oncoproteins of the High- and Low-Risk Human Papillomavirus Types Are Determined by Amino-Terminal Sequences MUNGER,'*
CAROLE L. YEE,' WILLIAM C. PHELPS,2 JENNIFER A. PIETENPOL,3 HAROLD L. MOSES,3 AND PETER M. HOWLEY' Laboratory of Tumor Virus Biology, National Cancer Institute, Bethesda, Maryland 20892'; Division of Virology, Burroughs Wellcome Co., Research Triangle Park, North Carolina 277092; and Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 372323 KARL
Received
5
March 1991/Accepted 12 April 1991
Differences in the biological characteristics of the high-risk human papillomavirus type 16 (HPV-16) and the low-risk HPV-6 E7 proteins were analyzed and shown to correlate with certain biochemical properties. To ascertain which region of E7 conferred these properties, chimeric E7 genes were constructed by the exchange of the amino and carboxyl coding halves of the HPV-6 and HPY-16 E7 genes. The amino-terminal half of E7 determined the affinity for binding to the retinoblastoma protein pRB, the transformation properties, and the ability to abrogate transforming growth factor j-mediated repression of the c-myc promoter. This region of E7 is therefore responsible for the biological and biochemical differences between the E7 proteins of the low-risk and the high-risk HPVs and consequently is one of the critical determinants distinguishing these two groups of viruses. Transcriptional transactivation of the adenovirus E2 promoter, in contrast, was a property shared by E7 proteins of both low-risk and high-risk HPVs.
Of the more than 60 different human papillomavirus types (HPVs) which have been identified, approximately 20 have been associated with anogenital lesions (8). These genital tract HPVs can be further subdivided into two groups: those such as HPV type 6 (HPV-6) and HPV-11 which are associated with benign lesions, including condylomata acuminata, which are at low risk for malignant progression, and those such as HPV-16 and HPV-18 which are considered high risk in that they are associated with intraepithelial neoplastic lesions which are precursors for cervical cancers (40). DNAs of high-risk HPVs have been found in more than 85% of human cervical carcinomas (27). In the cervical cancers the viral DNA is usually integrated into the host chromosome in a pattern indicating that the stable association of the DNA with the tumor preceded its clonal expansion. The HPV genomes are transcriptionally active in the cancers, and a subset of the viral genes, E6 and E7, are consistently expressed (1, 29, 30). E6 and E7 each encode oncoproteins, and these gene products together are sufficient for the efficient transformation of primary human squamous epithelial cells (18, 19, 21). Both E6 and E7 are relatively small proteins with no known enzymatic activities. The E6 proteins of the high-risk HPV types can form a complex with p53 (37), a cellular protein with tumor suppressor activity (13, 15), and can promote the degradation of p53 in vitro (28). The HPV-16 E7 open reading frame (ORF) encodes a nuclear 21-kDa phosphoprotein (17, 30). The E7 proteins encoded by the highrisk HPVs are sufficient for transformation of established rodent fibroblasts such as NIH 3T3 cells (5, 20, 24, 33, 34, 36). The E7 protein shares amino acid sequence similarity with portions of the adenovirus (Ad) ElA proteins and with the large tumor antigen (TAg) of simian virus 40 (SV40) (24). E7 is functionally related to the Ad ElA in that it can
*
Corresponding author.
transactivate the Ad E2 promoter (24) and cooperate with an activated ras oncogene to transform primary baby rat kidney (BRK) cells (24, 32). Additional evidence for a functional similarity among Ad ElA, SV40 TAg, and HPV-16 E7 was provided by a recent study demonstrating that each of the three viral oncoproteins can abrogate the transforming growth factor a (TGF-,B)-induced transcriptional repression of the c-myc promoter in keratinocytes (25). E7, SV40 TAg, and Ad ElA each associate with pRB, the protein encoded by the retinoblastoma susceptibility gene, through amino acid sequences conserved among these oncoproteins (2, 7, 11, 22, 38, 39). The E7 proteins encoded by the high-risk HPVs bind to pRB with higher affinity than the E7 proteins encoded by the low-risk HPVs (2, 17, 22). For Ad ElA and SV40 TAg it has been shown that these conserved sequences are also necessary for the binding of other host cellular proteins, such as p107 (10, 14, 39). In order to directly compare the biological and biochemical properties of HPV-16 E7 with those of HPV-6 E7, the HPV-6 E7 gene was inserted in place of the HPV-16 E7 gene in a recombinant plasmid, p858 (24), containing the SV40 early promoter and the HPV-16 early region downstream of the E7 ORF (Fig. 1B). This permits expression of both of the different E7 proteins in the same transcriptional and genetic background. To determine whether differences in activities mapped to the amino-terminal half of the E7 proteins which contains regions of similarity with the Ad ElA proteins and SV40 TAg, chimeric E7 genes were constructed. Portions (amino acid residues I to 48 or 49 to 98) of the HPV-16 E7 ORF were exchanged with corresponding portions of HPV-6 E7 sequences, as shown in Fig. 1B. The initial cloning steps were performed in a previously described plasmid which contains the HPV-16 E7 coding sequence (nucleotide [nt] 562 to 879) in a pGEM1 vector (22). The unique BanII site in the HPV-16 E7 sequence (nt 711) was used to generate the chimeric E7 ORFs. These chimeric E7 genes were also inserted in place of the HPV-16 E7 gene in the plasmid p858 3943
3944
J. VIROL.
NOTES
A
5 E44855
-1LCR
1
65 559
7152
2
I
> LI
3
I
6150
7000
4097/4135
3807
3850
2726
E6|
E2
r
j LI AE
P97
II
I 1000
7905/0
B
3000
2000
4000
5000
6000
4468
505
p858
5527
|
3333 3617
AAL l
21l1
El59
E2/E4
El
SV40 Pe E7 855
562 1
E5
-
/
Plasmid
p858
I
16 E7
98
1
pl498
6b E7 ... ...... -.... .. -............~~~~~~~~~~~~~~~~~~~~~~~..... .. Bnl98 . .... Ban 11 1'5051I98 . ,.... ..C-X.X-C...(-9. 15/1637/348 49 98
1,. ..... ... .. , .:::
p1499
6b/16 E7
1
:: .::: :::::.:::: :...::.: ,: :: ..::::,
:.
Ban 11
p1 500
.f
16/6b E7
s.s.s. .. ....
51 ~~~~ ~ 98 ~~50
1 l
l
1
::sss
........
15/16
CR 1
37/38
C-X-X-C ...(29 AA)..C-X-X-.d..| ~~~~~... 94 98 58
CR2
Ad ElA sequence similarity
FIG. 1. (A) Linear map of the HPV-16 genome. The long control region (LCR) and the major ORFs (E, early; L, late) are indicated. The approximate position of the first methionine codon in each ORF is indicated by the broken vertical line, and the sequence positions of the first and the last nucleotides are indicated. The positions of the promoter P97 and the early (AE) and late (AL) polyadenylation sites are given. The parental plasmid used in these studies was the original isolate described by Durst et al. (9), which was subsequently shown to contain a frameshift mutation at sequence position 1138 (*) leading to disruption of the El ORF (1). (B) Schematic representation of the E7 expression plasmids used in this study. The HPV-6b and the chimeric HPV-6b/HPV-16 and HPV-16/HPV-6b E7 genes were generated by polymerase chain reaction and built into plasmid p858 (24) by replacing the corresponding sequences of HPV-16 E7. A BanII restriction endonuclease cleavage site (located at nt 711 in the HPV-16 sequence) was used as the junction for constructing the chimeric E7 genes. Structural features of the HPV E7 proteins are shown at the bottom of the figure. The amino-terminal 37 amino acids of E7 are similar in sequence to portions of Ad ElA conserved region 1 (CR1) and CR2 (24). The sequence similar to Ad ElA CR2 contains the pRB binding site (22) as well as the casein kinase II consensus sequence (2, 16). The carboxy-terminal portion of E7 contains two Cys-X-X-Cys sequence motifs (6) which are likely involved in its zinc-binding property (3).
(Fig. 1B). The resulting plasmids, p1498, p1499, and p1500, contain the HPV-6 E7 gene (p1498) in place of the HPV-16 E7 gene or the chimeric E7 genes, as shown in Fig. 1. In the p1498, p1499, and p1500 plasmids, the E7 genes are cloned directly downstream of the SV40 early promoter (nt 562) rather than at nt 505, as in plasmid p858. The chimeric E7 proteins were assayed and compared with the HPV-16 and HPV-6 E7 proteins for binding to pRB,
previously described (11). For these experiments, the pGEM-based plasmids containing the various E7 genes were used to synthesize cRNAs to serve as templates for in vitro translation of the E7 proteins. Equimolar amounts of these proteins were mixed with unlabeled lysates from human neuroblastoma NGP cells, and the amount of E7 in complex with pRB was determined by immunoprecipitation with the C36 monoclonal antibody to pRB (38) (Fig. 2). The HPV-6/ as
VOL. 65,
1991
NOTES
TABLE 1. Transformation of BRK cells with E7 and pEJrasa
+NGP lysate and apRB
No. of transformed foci
Transfected DNAs rP*-
IL
HPV16E7
HPV
6 E7
p858 (16 E7) + pEJras p1498 (6b E7) + pEJras p1499 (6b/16 E7) + pEJras p1500 (16/6b E7) + pEJras pEJras alone
LU
Co
to
cotD
'D-
to
m-
c
3945
r_
co
C0 r. a.
-
29
-
18
-
14
Expt 1
Expt 2
15, 1, 0, 11, 0,
16, 0, 2, 6, 0,
16 0 0 12 0
19 0 0 3
Expt 3
Expt 4
>100 2, 0 1, 0 45, 66 0 O,
16, 19 3, 0 2, 1 27, 12 0O, 0
a Primary BRK cells were transfected with a total of 10 JLg of plasmid DNA (5 ,ug of E7 expression plasmid plus 5 pLg of pEJras) as previously described (24). Foci were counted at 12 to 16 days after transfection. The results from duplicate plates from each experiment are indicated. Transfections with the individual E7 expression plasmids alone did not yield any transformed foci.
-
IN
3o
6
FIG. 2. Coprecipitation of HPV-16, HPV-16/HPV-6b, HPV-6b/ HPV-16, and HPV-6b E7 proteins with pRB. The proteins were synthesized by in vitro translation in the presence of [35S]cysteine from the corresponding cRNAs transcribed with either SP6 or T7 DNA polymerase and were quantitated by trichloroacetic acid precipitation. The programmed rabbit reticulocyte lysates containing equimolar amounts of the E7 proteins were mixed with unlabeled extract from the human neuroblastoma NGP cell line, which contains normal pRB. Immunoprecipitations with the C36 monoclonal antibody to pRB (38) were performed, and the E7 proteins in complex with pRB were analyzed by sodium dodecyl sulfate (SDS)16% polyacrylamide gel electrophoresis followed by autofluorography. The positions of molecular size markers are indicated on the right and the positions of the E7 proteins are indicated by arrows. The dashed arrow marks the position of a truncated HPV-16 E7 protein initiated from methionine codon 12 in the E7 sequence. The four separate lanes shown in the figure are from the same experiment and were exposed for the same time.
HPV-16 chimeric E7 protein had the same low affinity for binding pRB as the HPV-6 E7 protein and also displayed an electrophoretic mobility similar to that of wild-type HPV-6 E7. In contrast, the HPV-16/HPV-6 chimeric E7 protein bound to pRB with an affinity similar to that of HPV-16 E7 and migrated with an aberrant, slow mobility similar to that of the wild-type HPV-16 E7 protein. Thus, both the relative in vitro binding affinity to pRB and the electrophoretic mobility are determined by amino acid sequences in the amino-terminal half of the E7 proteins. These E7 proteins were next tested for their ability to cooperate with the activated ras oncogene in the transformation of BRK cells. In agreement with previous results (24), p858, which contains the HPV-16 E7 gene as well as the other early-region ORFs 3' to E7 (Fig. 1A), was active in this transformation assay. Plasmid p1498, which contains the entire HPV-6 E7 ORF in the context of the HPV-16 early region of p858, was weakly transforming and induced only rare foci (Table 1). Similar differences in the transformation potential of E7 proteins of high-risk and low-risk HPVs have been reported in other studies (4, 31). Plasmid p1499, which encodes the chimeric HPV-6/HPV-16 E7 protein, had a low transforming activity similar to that of the HPV-6 E7 plasmid. In contrast, the p1500 plasmid encoding the HPV-16/
HPV-6 E7 protein induced foci at levels comparable to that of p858 encoding the full-length HPV-16 E7 protein. The presence of the corresponding E7 proteins in representative established BRK cell lines was demonstrated by immunoprecipitation (Fig. 3) with HPV-16 and HPV-6 E7-specific antibodies. The electophoretic mobilities of the E7 proteins were similar to those observed with the in vitro-translated proteins (Fig. 2). These results indicate that efficient transformation by E7 is a function determined by the aminoterminal portion of the molecule and furthermore that this transformation capacity correlates with the relative binding affinity of E7 for pRB. The HPV-16 E7 protein can abrogate the TGF-,-induced transcriptional repression of c-myc in primary squamous epithelial cells (25). To determine whether this characteristic would distinguish the high-risk HPVs from the low-risk HPVs, individual E7 expression plasmids were transfected into cultures of human foreskin keratinocytes together with the plasmid pPLFCAT-100, a TGF-,-sensitive reporter plasmid in which the chloramphenicol acetyltransferase (CAT) gene is expressed from the human c-myc P2 promoter. In agreement with a previous study (25), HPV-16 E7 abrogated the TGF-p-mediated repression of the c-myc promoter. Plasmid p1498, which encodes HPV-6 E7, had no significant effect on the TGF-,-mediated repression of this c-myc promoter (Table 2). In this assay, the chimeric HPV-6/ HPV-16 E7 protein encoded by plasmid p1499 had no measurable effect, whereas the HPV-16/HPV-6 E7 protein encoded by plasmid p1500 abrogated the TGF-p-mediated repression of the c-myc promoter in a manner similar to that of the HPV-16 E7 protein (p858). Therefore, the ability of E7 to annul the TGF-p-mediated repression of the c-myc promoter correlated with the transformation and pRB binding properties of the E7 proteins. These data are in good agreement with previous analyses of mutant Ad ElA and SV40 TAg proteins which established that the capacity of these oncoproteins to counter the TGF-,-mediated repression of c-myc mapped to regions of Ad ElA and SV40 TAg involved in complex formation with pRB (25). HPV-16 E7 can transactivate the Ad E2 promoter (24). Mutational analyses of HPV-16 E7 have mapped this transactivation function to the amino-terminal portion of HPV-16 E7 and have suggested that the transactivation function was overlapping and not fully separable from the transformation property of E7 (12, 23, 35). By contrast, a recent study of Ad E2 transactivation by microinjection of synthetic E7 peptides is not in agreement with these genetic studies and has suggested that the carboxy-terminal portion of E7 was sufficient for Ad E2 transactivation (26). The HPV-16 and HPV-6 E7 constructs were compared for their ability to
3946
J. VIROL.
NOTES r'l-
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(D
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EL I ?S
EL
TABLE 2. Abrogation of TGF-3-mediated transcriptional repression of c-myc transcriptiona
wU
LL
to _~
I Mf
Plasmid and treatment
co
aC
o
In
I.
Kr. cn
LO o
p858 (16 E7)
a:
cr
cr
c
+TGF-P
co
m
m
-TFG-P
az
m
Expt 2
Expt 1
m I M
CAT expression
(pg of protein/ml)
% Inhibi tion
CAT expression
% Inhibition
(pg of protein/ml)
257, 201 226, 201
7
76, 73 73, 77
0
210, 190 105, 116
45
93, 62 29, 12
74
194, 193 118, 107
42
65, 143 25, 29
74
291, 271 269, 277
3
103, 88 82, 118
0
p1498 (6b E7)
-TGF-P
+TGF-P 43
p1499 (6b/16 E7) -TGF-,3
+TGF-P 29
p1500 (16/6b E7)
-TGF-P
+TGF-P
HPV-16 E7
HPV-6 E7
-
18
-
14
_-
pUC18
-TGF-P +TGF-P a
o-fife~
FIG. 3. Immunoprecipitation of HPV E7 proteins from transformed BRK cell lines. The cell lines are designated by the plasmid number used in cooperation with pEJras to transform the BRK cells. Cells were plated in 100-mm dishes and labeled for 3 h with 1 mCi of [35S]cysteine per plate. The cells were lysed by treatment for 30 min at 0°C with 250 mM NaCl-50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.0)-0.1% Nonidet P-40 as previously described (22). The HPV-16 E7-specific monoclonal antibody 100201 was from Triton Biosciences (Alameda, Calif.), and the HPV-6 E7-specific rabbit antiserum was kindly provided by Denise Galloway. The positions of molecular size markers are shown, and the positions of the E7 proteins are indicated by arrows. The amount of cellular lysate used in the immunoprecipitation reactions was not normalized; therefore, the individual E7 signals detected are not directly related to the amounts of E7 produced in these cell lines.
transactivate the Ad E2 promoter and were found to have similar activities, indicating that transactivation of the Ad E2 promoter is a common property of E7 proteins of both the low-risk and high-risk HPV types (Fig. 4). This result is in agreement with a recent study in which both the HPV-6 and HPV-11 E7 proteins were found to be able to transactivate the Ad E2 promoter (31). Therefore, as expected, each of the two chimeric E7 proteins could efficiently transactivate the Ad E2 promoter. The high-risk HPV E7 proteins and Ad ElA share certain biological characteristics, including the ability to transactivate the Ad E2 promoter and to cooperate with ras in the transformation of primary rat cells (24). These functions have been mapped to the amino-terminal portion of the E7 molecule (12, 23, 36), where there is homology with conserved regions 1 and 2 of Ad ElA. Although the studies
72, 138 73 27, 29 Secondary cultures of human foreskin keratinocytes were transfected by 187, 215 98, 111
48
lipofection with 20 ,ug of E7 expression plasmid plus 20 pg of pPLFCAT-100 and 5 ,ug of a Rous sarcoma virus-driven ,3-galactosidase expression plasmid. One day posttransfection, half the cultures were treated with TGF-1 at a concentration of 10 ng/ml. At 24 h after TGF-P treatment, cell extracts were prepared from treated and untreated cultures and CAT assays were performed on duplicate plates by using a nonradioactive enzyme-linked immunosorbent assay (ELISA) (25). The amount of protein extract used in the assay was normalized to the P-galactosidase activity.
presented here do not yet permit a precise mapping of the Ad E2 transactivation property to a specific portion of E7, they do suggest that these two Ad ElA-like functions of E7 can be separated. The level of transactivation of the Ad E2 promoter by HPV-6 E7 is only moderately decreased compared with that of HPV-16 E7. This contrasts with the marked differences in transformation potential between the E7 proteins of these HPVs. The differences in Ad E2 transactivation potential of the HPV-6 and HPV-16 E7 proteins again map to the amino-terminal portion of E7 (Fig. 4). The carboxy-terminal portion of the E7 protein is neutral for all these properties, in that it is interchangeable between the E7 proteins of the low-risk and the high-risk genitalassociated HPVs. This region of E7 contains a structural unit that consists of two copies of a Cys-X-X-Cys sequence motif which are separated by 29 amino acids. The E6 protein contains two copies of this same structural unit (6). The only biochemical property of E7 which has been tentatively assigned to this region is the ability to chelate Zn2+ ions (3). The structural integrity of this carboxy-terminal region, however, clearly is important for E7 function, since premature termination of E7 at the BanlI site (Fig. 1) abrogates any biological activity of E7 (24, 25). Some of the major biological and biochemical differences between E7 proteins of low-risk and high-risk HPVs can therefore be assigned to the amino-terminal region of E7. This portion of E7 contains amino acid sequences which are similar to portions of conserved region 1 and 2 of Ad ElA and SV40 TAg sequences (24), including the pRB binding site (22) and the casein kinase II recognition sequence (2, 16).
VOL. 65, 1991
NOTES
i ~
3947
i
..
0
Plasmid
pUC-8 L
E7 protein % chloramphenio|
0
1.0
|
p1498
pl1499
p1500
p858
HPV-6
HPV-6/1 6
HPV-1 6/6
HPV-1 6
14.8
|
14.3
|
28.5
l
23.0
l
FIG. 4. Transactivation of the Ad E2 promoter. Transactivation assays were performed as previously described (24). CV-1 cells were transfected with 5 ,ug of the Ad E2 CAT plasmid pEC113 plus 5 p.g of the E7 expression plasmid p858, p1498, p1499, or p1500. At 48 h after transfection, the cells were lysed and CAT assays were performed with equal amounts (15 ,ug) of protein. The numbers given represent the average of duplicate plates from a single experiment. Similar numbers were obtained in several independent experiments. We thank Denise Galloway for the HPV-6 E7 antibody, Ed Harlow for the C36 antibody, Janet Byrne for oligonucleotide synthesis, and Francoise Thierry for valuable discussions throughout the course of this work. We are grateful to Jon Huibregtse and Martin Scheffner for critical reading of the manuscript. K.M. was supported by an advanced training grant from the Swiss National Science Foundation. This work was supported in part by grant CA-42572 (to H.L.M.) from the U.S. Public Health Service.
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29.
NOTES
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