Cell, Vol.

63, 1129-1136, December 21, 1990, Copyright 0 1990 by Cell Press

The E6 Oncoprotein Encoded by Human Papillomavirus

Types 16 and 18 Promotes the Degradation of ~53

Martin Scheffner,” Bruce A. Werness, viruses encode oncoproteins. The E6 and E7 proteins of
Jon 54. Huibregtse,‘Arnold J. Levine,t the “high risk” viruses are regularly expressed in cervical
and Peter M. Howley’ cancers and in the derived cell lines. The E8 and E7 genes
* Laboratory of Tumor Virus Biology of these “high risk” viruses have been best studied for
National Cancer Institute HW-18 and HPV-18, and both E6 and E7 have been shown
Bethesda, Maryland 20892 to have transformation properties. E7 is sufficient for the
t Department of Biology transformation of established rodent cell lines such as
Princeton University NIH 3T3 (Kanda et al., 1988; Phelps et al., 1988; Vousden
Princeton, New Jersey 08540 et al., 1988; Watanabe and Yoshiike, 1988; Bedell et al.,
1989; Tanaka et al., 1989) and can cooperate with an acti-
vated ras oncogene to transform primary rat cells (Phelps
Summary et al., 1988; Storey et al., 1988). The transformation poten-
tial of the E8 gene was revealed by studies with primary
The E6 protein encoded by the oncogenic human human keratinocytes and fibroblasts which showed that
papillomavirus types 16 and 16 is one of two viral prod- E8 was required in combination with E7 for efficient im-
ucts expressed in HPV-associated cancers. E6 is an mortalization of the cells (Mijnger et al., 1989a; Hawley-
oncoprotein which cooperates with E7 to immortalize Nelson et al., 1989; Watanabe et al., 1989). An emerging
primary human keratinocytes. Insight into the mech- theme among DNA tumor viruses is that the viral-encoded
anism by which E6 functions in oncogenesis is pro- oncoproteins interact specifically with critical cellular reg-
vided by the observation that the E6 protein encoded ulatory proteins, and that the oncogenic effects of these
by HPV-16 and HPV-16 can complex the wild-type ~53 viruses are at least in part a consequence of these spe-
protein in vitro. Wild-type p53 gene has tumor sup- cific interactions. In this regard the oncogenic HPVs show
pressor properties, and is a target for several of the some parallels with the adenoviruses and SV40. All three
oncoproteins encoded by DNA tumor viruses. In this encode transforming proteins that can complex with pRB
study we demonstrate that the E6 proteins of the on- (the product of the retinoblastoma tumor susceptibility
cogenic HPVs that bind p53 stimulate the degradation gene) (Whyte et al., 1988; DeCaprio et al., 1988; Dyson et
of ~53. The EG-promoted degradation of ~53 is ATP al., 1989) and with ~53 (Lane and Crawford, 1979; Linzer
dependent and involves the ubiquitin-dependent pro- and Levine, 1979; Sarnow et al., 1982; Werness et al.,
tease system. Selective degradation of cellular pro- 1990) which is now also regarded as having properties of
teins such as ~53 with negative regulatory functions a tumor suppressor gene (Finlay et al., 1989; Eliyahu et
provides a novel mechanism of action for dominant- al., 1989).
acting oncoproteins. The E7 proteins encoded by the oncogenic genital
HPVs, adenovirus ElA, and the SV40 large T antigen all
Introduction localize to the nucleus and share certain characteristics.
Each can extend the life span of primary cells, cooperate
The papillomaviruses are small DNA viruses that cause with other cytoplasmic oncoproteins such as ras to fully
benign squamous epithelial tumors (warts and papil- transform primary rat cells, induce DNA synthesis in
lomas) in their natural hosts and have been etiologically growth-arrested cells, transform established rodent cells,
implicated in some cancers. There are over 60 different and modulate transcription from certain promoters (re-
human papillomaviruses (HPVs) associated with a variety viewed by Ruley, 1990). Regions of amino acid sequence
of clinical lesions (DeVilliers, 1989) and approximately 20 similarity exist among the E7, ElA, and large T antigens
of these are associated with anogenital tract lesions. of these different DNA tumor viruses (Phelps et al., 1988;
Some of these, such as HPV-6 and HPV-11, are considered Figge et al., 1988) and these conserved regions have
low risk viruses since they are associated with benign been shown to participate in the binding to a number of
proliferative lesions such as condyloma acuminata that in- these important cellular proteins, including pRB (DeCap-
frequently progress to malignancy. Others, including HPV- rio et al., 1988; Whyte et al., 1989; Ewen et al., 1989; Mijn-
16, HPV-18, and HW-33, are considered high risk because ger et al., 1989b). Thus the comparable biologic proper-
of their association with potentially preneoplastic lesions ties of these different oncoproteins may derive from their
and with certain anogenital carcinomas, most notably ability to target a similar set of cellular regulatory proteins.
cancer of the uterine cervix (zur Hausen and Schneider, The properties of the E8 proteins encoded by the genital
1987). Transcriptionally active DNAs of these “high risk” tract papillomaviruses have been less well studied. It has
viruses are found in a high percentage of cervical cancers been suggested that the HPV-16 and HW-18 proteins have
and in cell lines derived from cervical cancer tissues transcriptional activation properties (Gius et al., 1988;
(Schwarz et al., 1985; Yee et al., 1985; Smotkin and Wett- Lamberti et al., 1990). It was the oncogenic properties of
stein, 1988). the HPV-18 and HPV18 E8 proteins that prompted our
Further support for a causal role of HPV in associated studies to investigate whether E6 targeted ~53, similar to
cancers derives from the recognition that the “high risk” SV40 large T antigen and Ad5 ElB. An association of the
Cdl
1130
4% 25oc 37w
I-11,811-
-106
- 71
P53- -44
- 26
: :: *
: :-
- 18
E6 -
a bcdetghi
Figure 1. The E6 Protein of HPV-1s Induces Degradation of ~53 In
Vitro
Synthetic RNAs encoding human ~53 or the E6 protein of either HPV-
11 or HPV-1swere translated in rabbit reticulocyte lysate in the pres-
ence of [35S]cysteine.In vitro translated ~53 was incubated either in
the absence (lanes a, d, g) or in the presence of approximately equimo-
lar amounts of either HPV-11E6 (lanes b, e, h) or HPV-18E6 (lanes c,
f, i) in a total volume of 40 gl containing 10 pl of rabbit reticulocyte ly-
sate, 25 mM R&Cl (pH 7.5), 100 mM NaCI, and 3 mM DTT After a 3
hr incubation at the indicated temperatures, the total reaction mixtures
were separated on a 14% SDS-polyacrylamide gel and the proteins
visualized by fluorography. The positions of the human p53 and the
HPV E6 proteins are indicated on the left, and the positions of molecu-
lar weight markers (in kd) are shown on the right.
E6 proteins of the “high risk” HPV types but not of the “low
risk” HPV types with ~53 was demonstrated in vitro using
programmed rabbit reticulocyte lysates (Werness et al.,
1990). It was clear, however, that although the oncopro-
teins of these different DNA tumor viruses all targeted
~53, the consequence of the protein-protein interaction
was likely to,be quite different. In SV40- and AdB-trans-
formed cells, sufficient large T antigen and El6 55 kd pro-
tein, respectively, exists to form complexes with ~53; these
complexes result in an increased half-life and steady-state
levels of the protein in transformed cells (Oren et al., 1961;
Reich et al., 1983) and presumably inactivating its normal
function as a negative regdator of cell growth. In contrast,
levels of ~53 in many cervical carcinoma cell lines and in
HPV-16- and HPV-la-transformed cell lines are generally
quite low (our unpublished data). For example, in the HeLa
cell line, which is an HPV-la-positive human cervical car-
cinoma cell line, ~53 is reported to be undetectable de-
spite the presence of translatable mRNA (Matlashewski et
al., 1986). The studies reported in this article were de-
signed to investigate the functional consequence of the
association of E6 and ~53.
Results
The E6 Proteins Encoded by HPV-16 and HIV-18
Stimulate the Degradation of ~53
The specific association of the E6 proteins encoded by
HW-16 and HPV-18 with the cellular ~53 protein has been
demonstrated in vitro (Werness et al., 1990). This interac-
tion was shown by the coprecipitation of E6 with wild-type
~53 from mixed lysates of rabbit reticulocyte-translated
E6 and ~53 proteins using the p53-specific monoclonal
antibody PAb 421 at 4OC. In the course of extending these
studies, we noted that less ~53 was immunoprecipitated
from mixed lysates that contained either HPV-16 or -18 E6
than from mixed lysates containing HPV-8 or -11 E6, which
do not bind to ~53 (Werness et al., 1990; data not shown).
A possible explanation of this phenomenon was that bind-
ing of the E6 proteins to ~53 partially masked the epitope
recognized by the PAb 421 antibody. Another possibility
was that the presence of the E6 proteins capable of com-
plexing ~53 actually led to a degradation of ~53 in the rab-
bit reticulocyte system.
To distinguish between these possibilities, ~53 trans-
lated in reticulocyte lysate in the presence of [35S]cys-
teine was incubated in the absence or presence of labeled
HPV-18 or HFV-11 E6 proteins at 4OC. After 3 hr, the total
reaction mixtures were processed for SDS-PAGE and the
labeled proteins monitored by fluorography. As shown in
Figure 1, less ~53 was present after incubation with HPV-
18 E6 (compare lanes a and c), whereas no difference was
detected after incubation with HW-11 E6 (compare lanes
a and b). From these results, it was concluded that the
presence of HPV-18 E6 leads to the in vitro degradation
of p53.
Figure 1 also shows the temperature dependency of
p53 degradation. Under the conditions used, no signifi-
cant difference in the amount of ~53 was observed after
a 3 hr incubation at any temperature in the absence of E6
proteins (lanes a, d, and g) or in the presence of HPV-11
E8 (lanes b, e, and h). In the presence of HPV-18 E6, how-
ever, ~53 was degraded with the highest efficiency at
25OC (about 90% as determined by densitometry) and
with reduced efficiencies at rPC (80%-70%) and 4’X
(40%).
Translation of in vitro synthesized ~53 mRNA in the rab-
bit reticulocyte system resulted mainly in full-length ~53
protein that comigrated with in vivo labeled ~53 from
different cell lines (data not shown), although some minor
species of faster migrating ~53 proteins could also be de-
tected (Figure 1). It is not known if these species represent
proteins that are generated by premature translation ter-
mination or from internal initiation. Interestingly, some of
these minor truncated products were degraded while
others were not, which may prove useful in defining re-
gions of ~53 necessary for this specific degradation.
In the experiment shown in Figure 1, no degradation in-
termediates were detected after the 3 hr incubation.
Therefore, a time course experiment was performed to
see if degradation intermediates could be observed at
shorter incubation times. As expected, ~53 remained rela-
tively stable throughout the 3 hr incubation at 25°C in the
absence of HPV E6 proteins (Figure 2A) or in the presence
of HPV-11 E6 (not shown). No degradation was noted after
150 min, although there was a reduction of about 20% of
the full-length ~53 protein after 3 hr in the absence of E6
(Figure 2A). In contrast, in the presence of HPV-18 E6 the
amount of ~53 decreased rapidly and continuously with
time (Figure 26). After 30 min 40% of the input ~53 was
degraded, and by 3 hr no full-length ~53 protein was de-
tected. At no time, however, could degradation intermedi-
ates of ~53 be detected, suggesting that once ~53 mole-
cules were targeted, degradation occurred rapidly and
HPV E6 Protein Promotes Degradation of p53
1131

A 6 E6 11 E6
0' 30 60 90' 120' 156 160
-12
w--m- - -. w p53

e p53
..‘::’

B a b c d e f g
0' 30' 60 90' 120' 150 160

- p53
16E6 18E6

rh7

-16 E6

Figure 2. Time Course of the HPV16 B-Induced Degradation of p53

In vitro translated p53 was incubated in the absence (A) or in the pres-
ence of HPV-18 E6 (6) at 25% under the conditions described in the
legend to Figure 1. The reactions were stopped at different time points
and analyzed by gel electrophoresis followed by fluorography.
rows at the right indicate the positions of migration of p53 and of HPV-
16 E6.
completely. The level of HPV-18 E8 remained stable
throughout the time course (Figure 28) and at the different
incubation temperatures (Figure l), consistent with the
possibility that HPV-18 E6 may act catalytically for ~53
degradation.
To further examine whether the binding of an HPV E6
protein to ~53 correlated with its ability to stimulate the
degradation of ~53, the E6 proteins of different HPVs
which bind (HPV-16 and HPV-18) or do not bind (HPV-6 and
HPV11) to ~53 were incubated with ~53. As demonstrated
in Figure 3, ~53 remained stable in the presence of in-
creasing amounts of the E6 proteins of HPV-11 and HPV-6.
In the presence of HPV16 or HPV-18, however, ~53 was
degraded in a manner dependent on the concentration of
the E6 proteins. Interestingly, the HPV-16 E6 was 2- to
3-fold more efficient in promoting degradation than the
HPV-18 E6, which correlated with the previous finding that
HPV16 E6 has a higher binding affinity for ~53 than
HPV18 E6 (Werness et al., 1990). In the experiments de-
scribed below, the HW-18 E6 was used. Similar results
were obtained using HPV16 E6.
The Specificity of EG-Stimulated
Degradation of p53
Although the EG-dependent degradation of ~53 correlated
with the ~53 binding properties of the E6 proteins, the pos-
sibility could not be excluded that in the rabbit reticulocyte
system, the E6 proteins cause a general degradation of
proteins. To control for this, RNA from brome mo.saic virus
(BMV) was translated in reticulocyte lysate in the presence
of [35S]cysteine and the protein8 were examined for their
stability in the presence of HPV18 E6 (Figure 4). The BMV
The ar-
h i j k I m
Figure 3. Degradation of ~53 Is Correlated with Its Ability to Complex
the HFV E6 Proteins
Unlabeled E6 proteins of HPV-6b or HF’V-11, which do not bind to ~53,
or of HPV-16 or HPV18, which do bind to p53 (Werness et al., 1990)
were generated by in vitro translation in the absence of radioactively
labeled amino acids. The relative amount of each E6 protein was deter-
mined in a parallel in vitro translation reaction in the presence of 35S-
labeled cysteine (see Experimental Procedures). Approximately equal
amounts of the different E6 proteins were then incubated with radioac-
tively labeled p53 under standard reaction conditions (see Figure 1).
Lane a, incubation of p53 in the absence of E6 proteins. Lanes b-m,
incubation of p53 in the presence of increasing relative amounts (lx,
2x, or 4x) of HPV6b E6 (lanes b-d), HPV11 E6 (lanes e-g), HPV16
E6 (lanes h-i), and HPV18 E6 (lanes k-m).
proteins remained stable in the absence or presence of
HPV-18 E6 (lanes a-c), whereas under the same condi-
tions ~53 was degraded, even in the presence of labeled
BMV proteins, demonstrating the specificity of the E6-
induced degradation (lanes d-i). In experiments not
shown, in vitro translated SV40 large T antigen and in vitro
translated pRB were each shown to be stable in the pres-
ence of the HPV-18 E6 protein. Although these results es-
tablish specificity for ~53 in this EG-stimulated degrada-
tion, they do not exclude the possibility that other specific
cellular proteins may also be affected by E8.
SV40 Large T Antigen Does Not Stimulate
Degradation of p53
Oncoproteins encoded by other DNA tumor viruses,
namely the S/40 large T antigen and the Ad5 ElB 55 kd
protein, also bind to ~53. In vivo, the binding of ~53 by
SV40 large T antigen or by the Ad5 ElB 55 kd protein leads
to an increased half-life of ~53 (Oren et al., 1981). To exam-
Cell
1132

A A
mM AMP,+ E6 mM ATP.yS, + E6

- E6 E6 - - E6 E6
-196 - 1%
BMV = -106 - 105
- 71 - 71
-
P53 - -44 -44

a b c d e f g h i a b c d e 1 g h i I

B B
a b c d e

p53 -

- 28

a bcdefghi

Figure 4. The HPV-16 EG-Induced Degradation Is Specific for ~53 and Figure 5. Degradation of ~53 Is Dependent on ATP Hydrolysis
Cannot Be Conferred by Binding of SV40 Large T Antigen to p53
(A) Degradation reactions were performed in the presence of increas-
(A) RNA from BMV was translated in rabbit reticulocyte lysate in the ing concentrations of AMP or of the nonhydrolyzable ATP analog
presence of [%]cysteine. The resulting proteins were incubated with
ATPyS, as indicated. ~53 and the high molecular weight forms of ~53
unlabeled HFV-18 E6 under standard degradation conditions (see Ex- (~53’) are marked on the left; the running positions of molecular
perimental Procedures). Lanes a-c, incubation of BMV proteins in the
weight markers (in kd) are indicated on the right.
absence (lane a) or in the presence of increasing amounts of HPV-18
(8) Radioactively labeled HPV-18 E6 and ~53 were incubated in the ab-
E6 (lanes band c); lanes d-f, same as lanes a-c but with radioactively
sence or in the presence of AMP or of ATPyS under standard degrada-
labeled ~53 added; lanes g-i, incubation with ~53 in the absence of
tion conditions at 25%. The amount of HPV18 E6 bound to ~53 was
BMV proteins. The running position of the BMV proteins and ~53 are
determined bycoimmunoprecipitation using the p53-specific monoclo-
indicated at left. Molecular size markers are indicated on the right.
nal antibody PAb 421 followed by SDS-polyacrylamide gel electropho-
(B) Increasing amounts of immunopurified SW0 large T antigen (Tag)
resis and fluorography. In a parallel control experiment using the SW0
were incubated with ~53 under standard degradation conditions at the large T antigen-specific monoclonal antibody PAb 419, which does not
indicated temperatures. Lanes a and e, no T antigen; lanes b and f, recognize ~53, no HPV-18 E6 or ~53 could be detected (not shown).
125 ng T antigen; lanes c and g, 250 ng of T antigen; lanes d and h, Lane a, immunoprecipitation of ~53 in the absence of HPV18 E6; lane
500 ng of T antigen; lane i, no T antigen but with HPV-18 E6.
b, in the presence of HPV-18 E6; lane c, with 4 mM AMP; lane d, with
2 mM ATP$S; lane e, same as lane b, but incubation was performed
at 4%. The high molecular weight forms of ~53 seen in (A) were also
ine whether stimulation of the degradation of reticulocyte- detected in this immunoprecipitation analysis; however, in this experi-
translated ~53 by E6 was specific for E6 or would be a con- ment they were only visible at long exposure times.
sequence of any ~53 binding protein, the effect of large
T antigen on ~53 in vitro was tested. Increasing amounts
of immunopurified T antigen were incubated with ~53 un- Since it is a component of the reticulocyte lysate transla-
der the same conditions used in the experiments with E6 tion system, ATP was present in all reaction mixtures of
described above (Figure 48). In a parallel immunoprecipi- the experiments shown above. To determine if ATP was re-
tation assay, 60%-70%~ of the input ~53 was bound to quired for the EBinduced degradation of ~53, AMP or
T antigen at the highest T antigen concentration used ATPYS, a nonhydrolyzable ATP analog, was added to a
(data not shown). Degradation of ~53, however, could not standard degradation assay (Figure 5A).
be detected (Figure 48, lanes a-h), demonstrating that the Both AMP (Figure 5A, lanes c-e) and ATP+ (lanes f-h)
binding of E6 specifically targets ~53 for degradation in a inhibited the EG-dependent degradation of ~53, indicating
manner not conferred by binding of SV40 large T antigen. that the degradation is dependent on ATP hydrolysis. The
In another experiment (not shown) it was observed that presence of ATPyS not only inhibited the degradation of
the EG-stimulated degradation of ~53 was not inhibited by ~53, but caused much of the labeled ~53 to migrate as
SV40 large T antigen. This suggests that E6 and T antigen high molecular weight forms (marked as ~53’ in Figure
may bind to different regions on ~53 or that E6 has a 5A). ~53 was the only labeled protein in the reaction mix-
higher binding affinity for ~53 than T antigen does. tures, suggesting that the higher migrating proteins are
indeed modified ~53 molecules. This assumption was
EG-induced Degradation of p53 Is ATP Dependent supported by immunoprecipitation analysis using the
A property of most protein degradation systems in vivo p53-specific monoclonal antibody PAb 421, which showed
and in vitro is a requirement for energy in the form of ATP that the higher migrating forms contain ~53 (data not
7;3\ E6 Protein Promotes Degradation of p53
shown). Densitometric quantitation revealed that the pres-
ence of ATPyS completely inhibited the degradation,
since the normal migrating and high migrating forms to-
gether contained all of the radioactivity initially added to
the reaction. Furthermore, the occurrence of ~53 high mo-
lecular weight forms was dependent on the presence of
HPV-18 E8 (Figure 5A, lane j), since the higher molecular
forms were not observed when HPV-18 E6 was not in-
cluded in the incubations. Finally, E6 is unlikely to be part
of this slower migrating form, since this form was not ob-
served in experiments using unlabeled ~53 and labeled
HPV-18 E6 (data not shown).
The addition of AMP to the degradation reaction had a
similar effect to that of ATPyS, although more of the ~53
proteins migrated at the normal molecular size and less
migrated as higher molecular weight forms. In addition,
the higher migrating forms had a lower apparent molecu-
lar weight compared with the forms observed in the pres-
ence of ATP+.
To demonstrate that the inhibition of ~53 degradation by
AMP and ATPyS was not due to a reduced binding of HPV-
18 E6 to ~53, a coimmunoprecipitation analysis was per-
formed using PAb 421 (Figure 56). HPV-18 E6 was immu-
noprecipitable in the presence of AMP (lane c) or ATP$S
(lane d). Lane e shows coimmunoprecipitation of E6 under
standard degradation conditions at 4X, while lane b
again demonstrates that ~53 is nearly totally degraded at
25% in the absence of AMP or ATPyS. These results con-
firmed that the EG-induced degradation of p53 is depen-
dent on ATP hydrolysis.
The Ubiquitin-Dependent Protease System
Is Involved in ~53 Degradation
A well-characterized eukaryotic ATP-dependent proteo-
lytic pathway is mediated by the ubiquitin-dependent pro-
tease system (reviewed in Ciechanover and Schwartz,
1989). Most studies concerning this degradation pathway
have been done in reticulocyte lysate, since reticulocytes
contain large amounts of factors involved in ubiquitin-
dependent protein degradation. Prior to degradation, mul-
tiple moieties of ubiquitin are covalently linked to the tar-
get protein by specialized ubiquitin-conjugating enzymes.
The ubiquitination of a protein leads to its slower migration
in denaturing protein gels. The occurrence of ubiquiti-
nated protein forms as an intermediate step in degra-
dation has generally been established either by using
radioactively labeled ubiquitin or by immunoblots with an-
tibodies directed against ubiquitin.
Because of the high molecular weight forms of ~53 ob-
served in Figure 5A, it seemed possible that the ubiquitin
system may be involved in the EG-induced degradation of
~53. To determine if the higher migrating forms of p53
represented ubiquitinated ~53, unlabeled reticulocyte ly-
sate-translated ~53 and HPV18 E6 were incubated to-
gether in the presence of ATP$S. p53 was then immuno-
precipitated with PAb 421, and the immunoprecipitated
complex was probed with a ubiquitin-specific rabbit poly-
clonal antibody (Haas and Bright, 1985) in a Western blot
(Figure 6). Bound antibodies were visualized by 1251-la-
beled protein A; therefore, a strong signal, which was not
+ p53, + PAb 421 - p63, + PAb 421 + p53, + PAb 419
*
-W h-v
chain
a b c d e f g h i
Figure 6. The High Molecular Weight Forms of ~53 Are Ubiquitinated
Unlabeled ~53 and HPV-16 E6 were incubated in the absence or in the
presence of 2 mM ATPrS. After the reaction, p53 was immunoprecipi-
tated as described in Experimental Procedures using PAb 421 or PAb
419, as indicated. A Western blot was then performed using a ubiquitin-
specific polyclonal antibody (Haas and Bright, 1965). Bound anti-
ubiquitin antibodies were detected by 1251-labeled protein A followed
by autoradiography.
dependent on the addition of the monoclonal ubiquitin an-
tibody, was observed in all reactions at the size of the
heavy chain of the antibodies used in the immunoprecipi-
tation (PAb 421 or PAb 419). In the presence of ATPyS and
HPV18 E6, a signal could be detected that correlated well
in size with the high molecular weight forms of ~53 appar-
ent in Figure 5 (compare Figure 6, lane c, with Figure 5,
lanes e-g). This signal was dependent on the presence
of ~53 (Figure 6, lanes d-f) and could not be detected
when a control antibody (PAb 419) was used in the immu-
noprecipitation (lanes g-i). These results strongly suggest
that the ubiquitin-dependent protease system is involved
in the EG-induced degradation of ~53.
Discussion
The ~53 protein was initially identified as a cellular protein
that binds to SV40 large T antigen (Linzer et al., 1979;
Lane et al., 1979). Until approximately two years ago, ~53
was thought to be an oncogene, based on early studies
that used a clone of the cellular ~53 gene that contained
a mutation (Hinds et al., 1989). Mutant forms of ~53 can
immortalize primary rat embryo fibroblasts and cooperate
with an activated fas oncogene to transform primary cells
(Jenkins et al., 1984; Eliyahu et al., 1984; Parada et al.,
1984; Finlay et al. 1988, 1989; Eliyahu et al., 1988; Hinds
et al., 1989). The wild-type ~53 gene, however, appears to
have tumor suppressor properties. In transfection studies
of rodent cells, wild-type ~53 has been shown to reduce
the efficiency of transformation by certain oncogenes,
while the mutated ~53 augments transformation (Finlay et
al., 1989; Eliyahu et al.,1989). Furthermore, in keeping
with its proposed role as a tumor suppressor, the ~53 gene
has been found to be mutated in Friend virus-induced
leukemias (Mowat et al., 1985) in a high percentage of
human colon carcinomas (Vogelstein et al., 1989; Baker
et al., 1989) and in human lung cancers (Takahashi et al.,
1989). Finally, expression of the wild-type p53 gene can
specifically suppress the growth of human colon carci-
noma cells in vitro (Baker et al., 1990) and is antiprolifera-
Cdl
1134
tive in SV40-transformed hamster cells (Mercer et al.,
1990).
SV40 large T antigen and Ad5 ElB 55 kd protein bind
to ~53 in the cell. This association results in an increased
half-life and increased steady-state levels of ~53, and pre-
sumably inactivates its function as a negative regulator of
cell proliferation. This model may not accommodate the
HPV E6 proteins, which also bind ~53 in vitro but do not
cause an increase in the steady-state level of p53 in im-
mortalized cells or in cancers (our unpublished data). The
in vitro evidence presented in this article provides a model
whereby the interaction of E6 with ~53 leads to a rapid and
specific degradation of ~53. From a functional standpoint,
the consequence of this interaction would be equivalent
to the stoichiometrically binding and inactivating an intra-
cellular negative growth regulator, leading to unrestricted
cellular proliferation.
The targeted degradation of ~53 by the E6 oncoproteins
encoded by the “high risk” HPVs would account for the
lowered levels of ~53 protein found in some of the cervical
carcinoma cell lines and HPV-immortalized squamous ep-
ithelial cell lines (Matlashewski, 1986; our unpublished
data). The elimination of wild-type p53 would be predicted
to provide a growth advantage to those cells expressing
E6 and could account for at least part of the function of
this oncoprotein in its role of cooperating with E7 to immor-
talize primary human cells (Munger et al., 1989a; Hawley-
Nelson et al., 1989; Watanabe et al., 1989).
Sufficiently high levels of mutant forms of ~53 can act
in a &ens-dominant fashion to abrogate the growth-sup-
pressive properties of wild-type p53 (Finlay et al., 1989;
Vogelstein et al., 1989; Baker et al., 1989). These mutant
forms of ~53, which have an activated oncogene function,
have an altered conformation with a prolonged half-life.
The frans-dominant nature of the mutated ~53 can be ex-
plained by its ability to oligomerize the wild-type ~53 pro-
tein, drawing it into an inactive complex (Eliyahu et al.,
1988; Rovinski and Benchimol, 1988; Finlay et al., 1989).
There is some evidence, however, that mutated forms of
~53 may have transforming activity even in the absence
of wild-type p53 (Wolf et al., 1984). If this is the case, one
might anticipate that mutations in the p53 gene could pro-
vide a further growth advantage in HPV-associated carci-
nomas even in the presence of active viral E6 expression.
Such a growth advantage in the tumor could only occur,
however, if the mutated ~53 proteins were not substrates
for EG-catalyzed degradation. Experiments examining
these possibilities are in progress.
In this study we have shown that the EG-facilitated
degradation of p53 is specific and not a general degrada-
tion of all proteins in the rabbit reticulocyte lysate. It seems
likely that other cellular proteins in addition to ~53 may
also be targeted for this degradation. We have not yet ex-
amined whether other normally short-lived nuclear reg-
ulatory factors such as the myc, fos, or jun proteins may
also be specifically degraded in the presence of E6. E6
has been reported to have transcriptional activation prop-
erties (Gius et al., 1988; Lamberti et al., 1990). It is cer-
tainly possible that these characteristics may result from
the specific degradation of cellular regulatory proteins
that modulate transcription. Indeed, the loss of p53 itself
through E6 mediated degradation may result in an altered
transcriptional milieu within the cell, since recent studies
have suggested that the wild-type p53 has transcriptional
modulatory activity (Fields and Jang, 1990; Raycroft et al.,
1990).
The ubiquitin-dependent protease system is likely to be
involved in the EG-stimulated degradation of ~53, since
slower migrating forms of ~53 as ubiquitinated intermedi-
ates were observed in the presence of inhibitors of ATP hy-
drolysis. The Western blot results depicted in Figure 6
using a polyclonal antibody to ubiquitin establish that ubiq-
uitinated forms of p53 are generated in the presence of
HPV-18 E6 after incubation in the rabbit reticulocyte lysate.
The mechanism by which E6 stimulates the degradation of
~53 is not yet clear. SV40 large T antigen, which also com-
plexes with ~53, does not lead to its in vitro degradation,
indicating that the observed proteolysis of ~53 is not simply
due to the fact that it is in a complex in the rabbit reticulo-
cyte lysate.
The specific mechanism for the EG-targeted, ubiquitin-
dependent degradation of p53 in reticulocyte extracts has
not yet been determined. The accelerated degradation of
~53 does appear to require formation of a complex involv-
ing p53 and E6, however, and as such has some similari-
ties with a degradation pathway recently described by
Johnson et al. (1990). In this system a determinant for
ubiquitin-dependent degradation can be provided in Pans
by one protein component to a second in an oligomeric
complex, resulting in the targeted degradation of that pro-
tein. Whether or not such a mechanism underlies the E6-
facilitated proteolysis of p53 is being investigated.
The selective degradation of important cellular regula-
tory proteihs with tumor suppressor activity provides a
new mechanism whereby dominant-acting oncoproteins
may function. The general model based on the interac-
tions of pRB with viral oncoproteins is that the normal
regulatory function of the tumor suppressor gene product
is abrogated by its specific association with the viral on-
coprotein. The association of E6 with ~53, however, may
lead to a loss of function for p53 by eliminating the gene
product. The finding that E6 leads to the selective degra-
dation of a tumor suppressor gene product may have
more general implications for carcinogenesis in that it
suggests a possible role for cellular factors that may be in-
volved in affecting the stability of important regulatory pro-
teins involved in controlling cellular proliferation and dif-
ferentiation.
Experimental Procedures
Proteins
As a source of the human wild-type ~53 and HPV E6 proteins, we used
a combined in vitro transcription/translation system. pGEM (Promega)
clones containing the E6 open reading frame of HPV type 6b, 11, 16,
or 16, or a cDNA encoding the wild-type human ~63 cDNA (Zakut-Houri
et al., 1966) was transcribed under recommended conditions using T7
or SP6 RNA polymerase (Promega). The construction of these differ-
ent pGEM clones has been described previously (Werness et al.,
1990). The resulting mRNAs (from 3 wg of template DNA) were used
in a 100 pl translation reaction containing 70 ~1 of pretreated rabbit
reticulocyte lysate (Promega). To generate radioactively labeled pro-
teins, translations were performed in the presence of [L-%]cysteine
tir3\E6 Protein Promotes Degradation of p53

(X00 Cilmmol; Amersham). Nonradioactively labeled proteins were Ciechanover, A., and Schwartz, A. L. (1989). How are substrates rec-
generated in a parallel reaction in the presence of unlabeled cysteine. ognized by the ubiquitin-mediated proteolytic system? Trends Bio-
Approximate molar ratios of synthesized proteins were determined by them. Sci. 74, 463-488.
densitometry of fluorographs of SDS-polyacrylamide gels. DeCaprio, J. A., Ludlow, J. W., Figge, J., Shew, J.-Y., Huang, C-M., Lee,
lmmunopurified SV40 large T antigen was purchased from MBR, W.-H., Marsilio, E., Paucha, E., and Livingston, D. M. (1986). SV40 large
Inc. BMV proteins were generated by in vitro translation using RNA tumor antigen forms a specific complex with the product of the retino-
supplied by Promega. blastoma susceptibility gene. Cell 54, 275-283.
DeVilliers, E.-M. (1969). Heterogeneity of the human papillomavirus
Drgradatlon Aeeay group. J. Virol. 63, 4696-4903.
The degradation-stimulating function of the HPV E6 proteins was as- Dyson, N., Hawley, R M., Miinger, K., and Harlow, E. (1989). The hu-
sayed in 40 ul volumes containing 2 pl of radioactively labeled p53 man papillomavirus16 E7 oncoprotein is able to bind the retino-
(iO,OOO-20,000 cpm) and 0.5-6.0 pl of radioactively labeled or unla- blastoma gene product. Science 243, 934-937.
beled HPV E6 protein. The reactions contained 25 mM Tris-Cl (pH 7.5)
Eliyahu, D., Raz, A., Gruss, P., Givol, D., and Oren, M. (1964). Participa-
100 mM NaCI, and 3 mM DTT, unless stated otherwise. The total
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amount of rabbit reticulocyte lysate was adjusted in each reaction to
bryonic cells. Nature 312, 646-649.
10 PI using reticulocyte lysate that was not programmed with exoge-
nous RNA. Unless otherwise indicated, the reactions were performed Eliyahu, D., Goldfinger, N., Pinhasi-Kimhi, O., Shaulsky, G., Skurnik,
at 25OC and were stopped after 3 hr by the addition of 1 vol of 100 mM Y., Arai, N., Rotter, V., and Oren, M. (1986). Meth A fibrosarcoma cells
Tris-HCI (pH 6.6) 200 mM DTT, 4% SDS, 20% glycerol, and boiling express two transforming mutant p53 species. Cncogene 3, 313-321.
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on SDS-polyacrylamide gels and the radioactively labeled proteins M. (1969). Wild-type p53 can inhibit oncogene-mediated focus forma-
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Immunopreclpltetlon and Western Blot N-terminal transformation-governing sequence of SV40 large T anti-
For immunoprecipitations, 200 ul reactions were performed as de- gen contributes to the binding of both pllORb and a second cellular
scribed above using a molar ratio of p53 to HPV18 E6 of approximately protein, ~120. Cell 58, 257-267.
2:l. The mixtures were precleared by incubating with 50 pl of 3% pro-
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tein A-Sepharose (in 20 mM Tris-Cl [pH 8.01, 100 mM NaCI, 2 mM
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EDTA, 1% NP40) and then immunoprecipitated with the p53-specific
monoclonal antibody PAb 421 as described previously (Werness et al., Figge, J., Webster, T., Smith, T. F., and Paucha, E. (1986). Prediction
1990). In control reactions, the SV40 large T antigen-specific monoclo- of similar transforming regions in simian virus 40 large T, adenovirus
nal PAb 419 was used. The immunoprecipitated proteins were sepa- ElA, and myc oncoproteins. J. Virol. 62, 1614-1816.
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For Western blot analysis of the high molecular forms of ~53, pro- gene product that forms an hsc70-p53 complex with an altered half-life.
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manuscript. We are grateful to Dr. A. Haas for providing the rabbit poly-
Hawley-Nelson, P, Vousden, K. H., Hubbert, N. L., Lowy, D. R., and
clonal antibodies to ubiquitin. M. S. was supported by the Deutsche
Schiller, J. T (1989). HPV16 E6 and E7 proteins cooperate to immortal-
Forschungsgemsinschaft, and B. A. W. was supported by a National
ize human foreskin keratinocytes. EMBO J. 8, 3905-3910.
Research Council-NIH Research Associateship. We are grateful to
Carol Comlish for excellent editorial assistance in the preparation of Hinds, P., Finlay, C., and Levine, A. J. (1989). Mutation is required to
this manuscript. activate the p53 gene for cooperation with the ras oncogene and trans-
The costs of publication of this article were defrayed in part by the formation. J. Virol. 63, 739-746.
payment of page charges. This article must therefore be hereby Jenkins, J. R., Rudge, K., and Curries, G. A. (1984). Cellular immortal-
marked “advertisement” in accordance with 18 USC Section 1734 ization by a cDNA clone encoding the transformation-associated phos-
solely to indicate this fact. phoprotein ~53. Nature 312, 651-654.
Johnson, E. S., Gonda, D. K., and Varshavsky, A. (1990). Cistrans rec-
Received September 14, 1990; revised October 10, 1990 ognition and subunit-specific degradation of short-lived proteins. Na-
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