Journal of General Virology(1995), 76, 1815-1820. Printedin Great Britain
1815
Cell cycle-dependent disruption of E2F-pl07 complexes by
human papillomavirus type 16 E7
Karin Zerfass, 3 L a u r a M . Levy, 2 Caterina Cremonesi, 1 F r a n c e s c a Ciccolini, 1 Pidder Jansen-Diirr, 3
L i o n e l Crawford, 1 R o b e r t R a l s t o n z and M a s s i m o T o m m a s i n o ~*
aImperial Cancer Research Fund Tumour Virus Group, Department of Pathology, University of Cambridge,
Tennis Court Road, Cambridge CB2 IQP, UK, 2 Virology Department, Chiron Corporation,
4560 Horton Street, Emeryville, CA 94608, USA and aAngewandte Tumorvirologie, Abteilung 620,
Deutsches Krebsforschungszentrum, INF 242, D-69120, Heidelberg, Germany
The human papillomavirus type 16 (HPV-16) E7 and
adenovirus (Ad) EIA oncoproteins share a common
pathway of transformation. They disrupt the cell cycle
G~ phase-specific protein complex containing the E2F
transcription factor and the regulatory protein Rbl, the
retinoblastoma tumour suppressor gene product. In the
G1 and S phases of the cell cycle, E7 and E1A bind two
other cellular complexes containing the Rbl-related
protein p107 and E2F. Ad E1A disrupts both complexes
and releases active E2F. In contrast, HPV-16 E7,
although it efficiently binds both E2F-pl07 complexes,
causes dissociation of the G 1 phase complex only. Using
chimeric proteins of HPV-16 E7 and Ad E1A we were
able to demonstrate that the ability of E1A to disrupt
both G1 and S phase E2F-pl07 complexes is not due to
the higher concentration o f A d E1A in the cell, but is an
intrinsic property of the Ad E1A transforming region.
These data suggest that E1A and E7 may function in
cellular transformation in similar, but not identical ways.
There is a strong association between some types of
human papillomavirus (HPV) and cervical cancer. In
most cases, HPV-16 or HPV-18 DNA has been found in
malignant cells (for review see zur Hausen, 1991). The
two early genes, E7 and E6, are responsible for malignant
cellular transformation (Matlashewski et al., 1987). In
fact, both of these genes are consistently retained in the
DNA of cervical cancer cells and are actively transcribed.
Several interactions between the two viral proteins and
cellular proteins have been characterized. The HPV-16
E7 gene product, a small nuclear phosphoprotein, is the
major transforming protein of the virus. E7 cooperates
with the activated ras gene product in the transformation
of primary rodent cells or with E6 in the immortalization
of human keratinocytes, the natural host cell for the
virus (for review see Mansur & Androphy, 1993). E7 is
structurally and functionally related to adenovirus (Ad)
E1A protein (Phelps et al., 1988) and on this basis can be
divided into three domains: CR1 and CR2, which are in
the N-terminal region, and CR3, which is in the Cterminal region. CR2 contains the LXCXE domain
involved in the binding of the retinoblastoma protein
(Rbl). Both viral oncoproteins bind the Rb family
proteins Rbl, p107 and p130 (White et al., 1988, 1989;
Dyson et al., 1989; Davies et al., 1993). These cellular
proteins are involved in control of the cell cycle by
interacting with and inhibiting transcription factors such
as E2F (Bagchi et al., 1991; Bandara & La Thangue,
1991; Chellappan et al., 1991; Cobrinik et aI., 1993).
E2F, which so far comprises a family of six proteins, is
involved in controlling transcription of several genes
which are required in DNA replication. Some E2F
proteins bind Rbl whilst others bind only to Rbl-related
proteins, such as p107 (Beijersbergen et al., 1994;
Ginsberg et al., 1994; La Thangue, 1994). Rbl is
phosphorylated in a cell cycle-dependent manner and it
is the hypophosphorylated form which is associated with
the transcription factor E2F-1 in G 1 phase (for review see
Goodrich & Lee, 1993). Ad E1A and HPV-16 E7 bind
the hypophosphorylated form of Rbl with consequent
release of free E2F, which is then able to activate the
transcription of genes required in the GI-S transition
(Bagchi et al., 1991; Bandara & La Thangue, 1991;
Chellappan et al., 1991; Mudryj et al., 1991; Phelps et
al., 1991). The Rbl-related protein p107 is also associated
with E2F (Cao et at., 1992; Devoto et at., 1992; Pagano
* Author for correspondence.Fax +44 1223 333 346.
0001-3024 © 1995SGM
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1816
Short communication
(a)
(b)
o%
¢'~
t-e5
z
z
z
z
z
kDa
29--
•
, ,~
.., .
:
~"
kDa
69 -~*. . . . . . . :
46-18--?~
,<
.4F.E1A
4--E7
30--
Fig. 1. I m m u n o b l o t s of N I H 3T3 E1A- or E7-expressing cells. Cell
extracts (50 I~g) of N I H 3T3, N I H 3T3 E7 and N I H 3T3 E1A cells were
applied to SDS polyacrylamide gels, transferred to P V D F m e m b r a n e
(Du Pont) and reacted with (a) an anti-E7 monoclonal antibody
(Triton) or (b) an anti-E1A monoclonal antibody (M73).
et al., 1992a, Shirodkar et al., 1992), regulating its
transcription activity (Schwarz et al., 1993; Zhu et al.,
1993) and the E2F-pl07 complex has also been found
associated with cyclin E and cyclin A/cdk2 in the G 1 and
S phases, respectively (Lees et al., 1992). Ad E1A and
HPV-16 E7 bind p107, but the consequences of these
interactions are different. When Ad E1A binds p107 it
releases free active E2F from both G1 and S complexes,
whereas HPV-16 E7, although it associates with p107,
clearly does not cause dissociation of the E2F-pl07
complex in S phase (Arroyo et al., 1993; Pagano et al.,
1992b). Complexes containing E7, E2F, p107 and cyclin
A/cdk2 are therefore present in cells expressing the E7
gene. Nothing is known about the existence of the G1
E2F-pl07 complex in E7-expressing cells. Lam et al.
(1994) have shown that HPV-16 E7 activates the
transcription of B-myb, a gene which is required for G1 S
progression, apparently by binding to p107. We have
performed a detailed analysis of E2F-p 107 complexes by
gel retardation in extracts of cells containing Ad E1A or
HPV-16 E7. Retrovirus constructs expressing Ad E1A or
HPV-16 E7 genes were transfected using the calcium
phosphate coprecipitation method into a producer cell
line (GPE) and selected in G418 for 3-4weeks. The
progeny virus was then used to infect NIH 3T3 cells in
presence of polybrene (8 gg/ml). Cells were grown in
G418 and after 4 weeks colonies were plated in soft agar
to select high E7- or E1A-producer cells. The trans-
formed colonies were isolated and the presence of Ad
E1A or HPV-16 E7 in corresponding cell lines NIH 3T3
E1A and NIH 3T3 E7 was determined by direct
immunoblotting of total cell extracts (Fig. 1). We then
performed a gel retardation assay to analyse the
E2F-pl07 complexes in N I H 3T3, NIH 3T3 E1A and
NIH 3T3 E7 cells. Extracts from asynchronously
growing ceils were incubated with a 32P-labelled otigonucleotide specific for the E2F DNA-binding site from
the Ad E2 promoter. DNA-protein complexes were
resolved on native acrylamide gels and detected by
autoradiography. As can be seen in Fig. 2 (a), NIH 3T3
cell extract contained two complexes that were supershifted with an antibody specific to p107. Only the upper
pl07-containing band could be disrupted by an anticyclin A antibody. As expected, these two E2F-pl07
complexes are present at different times during the cell
cycle. NIH 3T3 cells were blocked in the G0-G 1 phase of
the cell cycle by serum starvation and subsequently
released by the addition of serum• Cells were collected
for E2F gel retardation analysis after 5 and 10 h, which
corresponds approximately to G a phase and the beginning of S phase, respectively, as shown by FACScan
analysis and pH]thymidine incorporation (data not
shown). Gel retardation assays with NIH 3T3 cells
extracted 5 and 10h after serum starvation release
demonstrated that the upper band, disrupted by an
anti-cyclin A antibody, was present in S phase (Fig. 2b).
The G 1 and S phase E2F p107 complexes are not
confined to NIH 3T3 cells; in fact the same complexes
were observed in cellular extracts of human primary
foreskin fibroblasts and HaCat cells by gel retardation
analysis (data not shown). In agreement with previous
data, E1A and E7 also have different activities in NIH
3T3 cells with respect to the disruption of the E2F-pl07
complexes (Arroyo et al., 1993; Pagano et al., 1992b). In
NIH 3T3 E1A cells both E2F-pl07 complexes were
absent (Fig. 2c), whereas in cell extract from E7expressing cells (NIH 3T3 E7) only one of the two
E2F p107 bands was detectable (Fig. 2c). This supershifted band comigrated with the S phase E2F-pl07cyclin A complex (Fig. 2 cO and was disrupted by an E7specific monoclonal antibody and by anti-cyclin A
antibody, suggesting that both proteins were present in
the E2F-pl07 complex (Fig. 2c; data not shown). The
G1 E2F p107 band was not visible in E7 extracts, even
after a long exposure of the film. Thus, although E7 is
synthesized at low levels in these cells (at least 5-10 times
lower than in CaSki cells; data not shown), it disrupted
the G a phase E2F-pI07 complex very efficiently but not
the S phase E2F pl07-cyclin A complex. There are two
possibilities why the two viral oncoproteins have such
different activities in the disruption of E2F-pl07
complexes: (i) the disruption of S phase E2F-pl07
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Short communication
(a)
<
.
O
z
.
<
<
~
+
.
.
<
<
(d)
(c)
(b)
<
~
~
.=
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.=
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.-
.-
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z
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<
a
~-N_
I-N__
I -N_
.............
ii - / - -
II f
1817
NIH 3T3
5
10
H_/-
NIH 3T3 E7
NIH 3T3 EiA
t'¢3
Time (h)
z
=
?-,
e~
~"
z
z
7
Fig. 2. E2F gel retardation assay of the NIH 3T3 cell extracts containing HPV-16 E7 or Ad E 1A289. Extracts were prepared from NIH
3T3 cells (a), synchronized NIH 3T3 cells 5 and 10 h after serum starvation release (b), NIH 3T3 E1A289 or NIH 3T3 E7 cells (c) and
NIH 3T3 or NIH 3T3 E7 cells (d), and incubated with z~P-labelled E2F binding site DNA. The mixture was separated on a native 4-5 %
polyacryamide gel (Jansen-Dfirr et al., 1993)• The specificity of the resulting complex was controlled by competition with a 20-fold
excess of unlabelled oligonucleotide of either tb_e wild-type or the mutated sequence. Tb_e amounts of different antibodies used were:
1 gl of the supernatant of polyclonal anti-cyclin A antibody, 1 lal of the supernatant of anti-pl07 monoclonal antibody (SD15), 1 gl
of the supernatant of E1A monoclonal antibody (M73) and 1 rtl of E7 antibody (Triton).
(a)
HPV-16E7
aD
¢'q
£-q
t"q
[I 1 eCK
1
98
M73
M37
1
289
kDa
E1A243
1
243
7- 7~ 7~ Z Z
kDa
68--
68--
43--
43--
29--
29--
18--
18--
7~7~ 7~ 7~ 7~
M73
E7/E1A289
1
37/138
189
M73
~i~;'~i~i~i~i~i~i~!,~i~i~i~ii'~l
E7/E1A243
1
37/186
M37
1
1 43
6[3
137138
198
M73
M37
Fig. 3. (a) Schematic representation of the chimeric constructs• Shaded areas indicate the positions of the Ad E1A and HPV- 16 E7 CRI
and CR2, the region containing the two CXXC motifs involved in the binding of zinc and the phosphorylation site of casein kinase
II. The numbers on the right indicate the amino acid residues in the proteins. The underlined numbers indicate the position of the
fusion in the amino acid sequence of Ad E 1A or HPV-16 E7. The black bars indicate the position of the monoclonal antibody epitopes
in Ad E1A, HPV-16 E7 and the chimeric proteins• (b) Immunoblots of NIH 3T3 cell extracts containing the chimeric proteins• NIH
3T3, NIH 3T3 E7/E1A289, NIH 3T3 E7/E1A243, NIH 3T3 EtA E7 and NIH 3T3 E1A cell extracts (50 ~tg) were applied to
SDS-polyacrylamide gels, transferred to PVDF membrane (Du Pont) and reacted with two different anti-EIA monoclonal antibodies
(M37 and M73). The same NIH 3T3 E1A cell extract has been included in both immunoblots as an internal standard.
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1818
Short communication
(a)
1
2
3
4
:!~2~
5
~°
-~.-p107
(b)
1
2
3
4
5
Cyclin A
(c)
1
2
3
~
4
5
~ - cdk2
Fig. 4. Association of the chimeric proteins with cellular proteins. Cell
extract (3 rag) containing the three chimeric proteins or Ad E1A was
immunoprecipitated as described by Tommasino et al. (1993).
Immunopellets were applied to SDS-polyacrylamide gels, transferred
to nitrocellulose and incubated with rabbit polyclonal antibodies
against p107 (a), cyclin A (b) or the PSTAIRE motif of cdk2 (c). Lane
1, NIH 3T3 cell extract immunoprecipitated with 50 gl of anti-E1A
monoclonal antibody M73; lane 2, NIH 3T3 E7/E1A289 cell extract
immunoprecipitated with 50 gl of M73; lane 3, NIH 3T3 E7/E1A243
cell extract immunoprecipitated with 50 lal of M73 ; lane 4, NIH 3T3
E1A-E7 cell extract immunoprecipitated with 1 gl of anti-E7 monoclonal antibody 6D; lane 5, NIH 3T3 E1A289 cell extract immunoprecipitated with 50 gl of M73.
complex occurs only because Ad E1A is present at a
much higher concentration than HPV-16 E7 in cells
(Fig. 1), or (ii) the ability to disrupt the S phase
E2F-p107 complex is an intrinsic property of Ad E1A.
To distinguish between these alternatives we constructed
three chimeric proteins, fusing the N- and C-terminal
regions of HPV-16 E7 and Ad E1A in all possible
combinations. As shown in Fig. 3(a), the junction
between the two viral proteins was positioned after the
casein kinase II site. Two E7-E1A proteins were
constructed: E7/E1A289, containing the E1A CR3
region, and E7/E1A243, which lacks the CR3 CXXC
motifs. The third chimeric protein was obtained by
fusing the N-terminal region of Ad E1A with the Cterminal region of HPV-16 E7. The chimeric genes were
generated by overlap PCR, cloned into a retrovirus
vector (pMXSV neo-18), sequenced and introduced in
NIH 3T3 cells as described above. All three chimeric
proteins retained the ability of native oncoproteins to
transform NIH 3T3 cells (data not show). Colonies were
isolated from soft agar plates and maintained under
selection of G418. The concentration of the different
proteins in the stably transfected NIH 3T3 cells was
determined by direct immunoblotting using two different
Ad E1A-specific monoclonal antibodies, M37 and M73,
which react with the N- and C-terminal regions of the
protein, respectively. Ad E1A was included in both
immunoblots as an internal standard. As shown in Fig.
3 (b) all proteins appeared to be synthesized at approximately the same level. Before analysing the E2F-pl07
complexes in cellular extracts containing the chimeric
proteins by gel retardation assay, it was important to
check that they retained the ability of the parental
proteins to interact with p107 and the other cellular
proteins. Cell extracts from NIH 3T3 cells and NIH 3T3
cells containing the three different chimeric proteins or
E1A289 were immunoprecipitated with E1A- or E7specific antibodies; the presence of p107, cyclin A and
cdk2 was determined by immunoblotting. As shown in
Fig. 4, all three cellular proteins were associated with
E7/E1A289, E7/E1A243 and E1A/E7, suggesting that
the main binding properties of Ad E1A and HPV-16 E7
are conserved in the chimeric products. Next we analysed
the E2F-p107 complexes in the cells containing the
chimeric proteins. Gel retardation analysis with cellular
extracts containing the E7 E1A chimeric proteins
(ET/E1A289 and E7/E1A243) showed the same slower
migrating band present in the E7 cellular extracts. This
band was super-shifted by the pl07-specific monoclonal
antibody (Fig. 5). Incubation of E7/E1A289 cellular
extracts with the 32P-labelled E2F DNA consensus site in
the presence of M73 antibody resulted in a reduction of
the E2F-p107 band, suggesting that the chimeric protein
was able to bind to the p107 complex without disrupting
it, as in the case of HPV-16 E7. In contrast the E1A-E7
cell extract did not contain any complex which was
super-shifted with p107 antibody. E1A-E7 protein,
although present in the cell at a much lower concentration than Ad E1A, was still able to disrupt both
E2F-pl07 complexes (Fig. 5). All NIH 3T3 cells
containing the chimeric proteins had approximately the
same level of p 107, as indicated by immunoblotting (data
not shown) and by analysis of the chimeric protein
immunopellets (Fig. 4). Thus, the absence of the
E2F-p107 complexes in NIH 3T3 E7-E1A cells was not
due to the lower concentration of p107.
These results show clearly that the different activity of
Ad E1A and HPV-16 E7 in disruption of the E2F-pl07
complex is not related to the concentration of the viral
oncoproteins in the cell, but is due to an intrinsic
property of the Ad E 1A N-terminal transforming region.
The G 1 phase E2F-pl07 complex may be involved in
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Short communication
1819
<
2
p.
o
t--
<
o
Z
<
<
o
<
,~
~,
<
Z
<
¢¢3
i
G
Z
<
E2F/pl07
mD
NIH 3T3
E7/EIA289
NIH 3T3
E 1A-E7
NIH 3T3
E7/E 1A243
Fig. 5. E2F gel retardation assay of the NIH 3T3 cell extracts containing the three chimeric proteins. Extracts were prepared from NIH
3T3 E7/E1A289, NIH 3T3 E1A-E7 or NIH 3T3 E7/E1A243 cells and processed as described in Fig. 2. The antibodies used in the gel
retardation assay are indicated.
deregulation of cellular growth control by HPV and Ad.
Both viruses have to disrupt the G 1 phase E2F-pl07
complex in order to stimulate progression of the ceil
cycle. Disruption of the E2F-pl07-cyclin A complex by
E1A may be required for a specific function of the Ad life
cycle, for instance transactivation of the E2 gene. The
difference between the two E2F-pl07 complexes in their
behaviour with E7 is not entirely clear. It is possible that
cyclin A/cdk2, when activated in S phase, phosphorylates the E2F-pl07 complex preventing the dissociation induced by E7. Alternatively the two E2F-p 107
complexes may contain different members of the E2F
protein family. The G 1 E 2 F ~ 1 0 7 complex does not
contain cyclin E, since we did not observe any band
super-shifted or disrupted by anti-cyclin E antibody in
our gel retardation assays (data not shown). The
physiological function of the interaction of E7 with the
E2F-pl07 complex in S phase is not yet clear but may
result in subtle differences between Ad and HPV in the
type of transformation produced. This could be linked to
the fact that only HPV is involved in human cancer.
We would like to thank Dr R.W. Tindle for the 6D anti-E7
antibody, Drs Michele Pagano and Giulio Draetta for anti-pl07, anticyclin A and cdk2 antibodies, Dr E. Harlow for the M37 and M73 antiE1A antibodies, Dr Jonathan Pines for the anti-cyclin A antibody, Dr
N. Dyson for the SD15 antibody, Dr Masakane Yamashita for the
monoclonal anti-PSTAIRE antibody and Almut Schulze for the
synchronized NIH 3T3 cell experiments. F.C. was supported by a
fellowship from Associazione Italiana per la Ricerca sul Cancro and
C.C. by an EC fellowship in Biomedicine. K.Z. and P.J.D. were
partially supported by a EC grant from the HCM programme.
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(Received 18 November 1994; Accepted 8 February 1995)
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