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Journal of Virology, January 2001, p. 513-521, Vol. 75, No. 1
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.1.513-521.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Cooperative Transformation and Coexpression of
Bovine Papillomavirus Type 1 E5 and E7 Proteins
Joanna
Bohl,1
Bruce
Hull,2 and
Scott
B.
Vande Pol1,*
Institute of Pathology, Case Western Reserve
University, Cleveland, Ohio 44106,1 and
Department of Surgery, School of Veterinary Science, Ohio
State University, Columbus, Ohio 432102
Received 10 July 2000/Accepted 28 September 2000
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ABSTRACT |
Productively infected bovine fibropapillomas were examined for
bovine papillomavirus type 1 (BPV-1) E7 localization. BPV-1 E7 was
observed in the cytoplasm of basal and lower spinous epithelial cells,
coexpressed in the cytoplasm of basal cells with the E5 oncoprotein. E7
was also observed in nucleoli throughout the basal and spinous layers
but not in the granular cell layer. Ectopic expression of E7 in
cultured epithelial cells gave rise to localization similar to that
seen in productive fibropapillomas, with cytoplasmic and nucleolar
expression observed. Consistent with the coexpression of E7 and E5 in
basal keratinocytes, BPV-1 E7 cooperated with E5 as well as E6 in an
anchorage independence transformation assay. While E5 is expressed in
both basal and superficial differentiating keratinocytes, BPV-1 E7 is
only observed in basal and lower spinous epithelial cells. Therefore,
BPV-1 E7 may serve to modulate the cellular response of basal
epithelial cells to E5 expression.
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TEXT |
Papillomaviruses are causative agents
for a variety of epithelial neoplasms in vertebrates. Typically,
virus-induced epitheliomas are benign lesions containing episomal viral
DNA in proliferative basal epithelial cells, producing virus within
superficial differentiated epithelial cells. The role of virus-encoded
oncogenes (E5, E6, and E7) in the maintenance of viral DNA within
proliferative basal epithelial cells and/or the role of these oncogenes
in the transition to vegetative viral DNA amplification and virus
production has yet to be fully defined. The separation of
papillomavirus oncogenes into at least three separate polypeptides (E5,
E6, and E7) may reflect a requirement for separate regulation of
expression and activity of each product in the virus life cycle. One
area of uncertainty in papillomavirus biology has been the location of each oncoprotein within the stratified epithelium of a virus-induced epithelioma.
While bovine papillomavirus type 1 (BPV-1) encodes E5, E6, and E7
oncoproteins, only mutation of BPV-1 E5 decreases transformed cell
focus formation by BPV-1 viral DNA. Mutations in the BPV-1 E6 or E7
genes have little effect upon focus formation or viral DNA replication,
although there is a decrease in anchorage-independent growth and
tumorigenicity (12, 15, 20). The modest oncogenic potency
of BPV-1 E6 and BPV-1 E7 expressed from the wild-type BPV-1 genome is
due to repression by two separate repressor mechanisms that, when
mutated, reveal cooperative transformation by BPV-1 E6 and BPV-1 E7
(35). Since both BPV-1 E6 and BPV-1 E7 are thought to be
translated from the same mRNA transcript, it was unproven if BPV-1 E7
had a direct role in transformation together with BPV-1 E6. Correlating
with this observation, BPV-1 E6 strongly transforms murine C127 cells
when expressed from retroviral long terminal repeats, whereas E7 did
not (27).
The E6 proteins of human papillomavirus type 16 (HPV-16) (16E6) and
BPV-1 E6 bind to the cellular targets E6AP, ERC-55, and paxillin
through interaction with homologous peptide sequences found on the
target proteins (5, 8, 36), and transformation by E6 can
be repressed by competitive binding of E6 to peptides that interact
with E6 (3). The cancer-associated HPV E6 oncoproteins bind p53 together with the cellular ubiquitin ligase E6AP, resulting in
the degradation of p53 through ubiquitin-mediated proteolysis in vitro
(13). While BPV-1 E6 interacts with E6AP, targeted degradation of p53 by BPV-1 E6 has not been observed (21).
The BPV-1 E5 oncoprotein interacts with the platelet-derived growth
factor (PDGF) receptor to stimulate ligand-independent intracellular
activation of the PDGF receptor (23). BPV-1 E5 also
modulates epidermal growth factor (EGF) receptor downregulaton (6, 16), resulting in enhanced surface expression of the EGF receptor. It is as yet uncertain if E5 transformation is
exclusively through activation of receptor tyrosine kinases or if
additional activities contribute. BPV-1 E5 interacts with a 16-kDa
vacuolar ATPase (10), resulting in the alkalization of
intracellular compartments, which may contribute to its transforming
ability (22, 26, 29, 30, 32). Interactions between HPV E5
oncoproteins and receptor tyrosine kinases have varied between
different HPV types (7), but HPV E5 oncoproteins have been
found to synergize with HPV E6 and E7 in the immortalization of
keratinocytes (31).
There are striking differences between BPV-1 E7 and HPV E7
oncoproteins, most notably in the absence of a LXCXE interaction motif
in BPV-1 E7 for the retinoblastoma family of tumor suppressors that is
found in all HPV E7 oncoproteins. While HPV E7 proteins interact with
the retinoblastoma protein in vitro, BPV-1 E7 does not
(19).
Since BPV-1 E7 has not been described as having a strong independent
transforming activity, it may act to enhance or modulate transforming
activity of BPV-1 E5 or BPV-1 E6. In order to determine if BPV-1 E5 or
BPV-1 E6 might be coexpressed with BPV-1 E7, we determined the
predominant localization of BPV-1 E7 within productively infected
bovine fibropapillomas and found E7 coexpressed with E5 in the
cytoplasm of basal epithelial cells. Correlating with this observation,
BPV-1 E7 cooperated with both E5 and E6 in the transformation of rodent
cells to anchorage-independent growth.
Location of E7 in fibropapillomas.
Rabbit polyclonal
antibodies specific to nonoverlapping segments of the E7 amino terminus
or carboxy terminus were prepared and analyzed for specificity against
bacterially expressed fragments of E7 (Fig. 1A to
C). These antibodies immunoprecipitated
and identified on a Western blot a 15-kDa protein from a bovine
fibropapilloma that comigrated with E7 derived from transiently
transfected Cos-1 cells (Fig. 1D).
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FIG. 1.
Specificity of antibodies to E7. Antiserum to
bacterially expressed histidine-tagged full-length E7 and antiserum to
a synthetic peptide comprising the C-terminal 10 amino acids were
raised in rabbits. Antibodies were extensively adsorbed to fixed,
detergent-extracted bovine MDBK cells and then to fixed and
detergent-extracted normal bovine epidermis to remove antibodies
cross-reactive to bovine skin. The adsorbed antiserum raised against
full-length E7 was affinity purified by binding and elution from a
glutathione S-transferase (GST) fusion to the first 80 amino
acids of E7. The anti-peptide antibody was affinity purified by binding
and elution to immobilized E7 amino acids 80 to 127. (A, B, and C)
Lanes 1, 2, and 3 show bacterially expressed GST fusion proteins of
full-length E7 amino acids 1 to 127, 80 to 127, and 1 to 80, respectively. In panel A, the proteins are Coomassie blue stained,
while in panels B and C the proteins were analyzed by Western blot with
affinity-purified antibodies directed to the amino terminus of E7 (B)
and the carboxy terminus of E7 (C). (D) Analysis of E7 in
fibropapillomas. Approximately 50 mg of fibropapillomas or uninfected
bovine tissue were frozen sectioned and thawed into 1.0 ml of boiling
1% SDS. The sonicated, clarified lysate was diluted into 9 ml of
phosphate-buffered saline (PBS) containing 1% Triton X-100. E7 was
immunoprecipitated overnight with 10 µl of rabbit antibody to E7 and
50 µl of protein A-Sepharose. An SDS-15% polyacrylamide gel
electrophoresis gel was analyzed by immunoblot with affinity-purified
rabbit antibody to the E7 carboxy terminus and detected with
peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG) and enhanced
chemiluminescence. Lanes 1 and 2, 25 µg of protein from Cos-1 cells
transfected with E7 or empty control plasmid, respectively; lanes 3 and
4, 10 and 50 µg of bovine MDBK cell lysate, respectively; lanes 5 and
6, anti-E7 immunoprecipitates from bovine fibropapilloma (lane 5) or
uninfected bovine tissue (lane 6). Arrowheads indicate the positions of
E7 and rabbit IgG light chains.
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Immunolocalization of E7 showed two distinct patterns of expression in
naturally occurring fibropapillomas (Fig.
2). E7 was
principally localized to the
cytoplasm of basal and parabasal
epithelial cells (Fig.
2c). E7
cytoplasmic staining was strongest
in the basal cells (Fig.
2c and
3j) but
was not strictly localized
to those cells, since staining into the
lower spinous epithelial
cells three to five cell layers up from the
basement membrane
was observed. Sporadic cytoplasmic staining of
epithelial cells
within mid-spinous cells was seen (Fig.
3j). Normal
bovine epithelium
within the same tissue section but adjacent to the
fibropapilloma
showed no similar immunostaining (Fig.
2e and f). While
the most
intense staining for E7 was observed in basal cell cytoplasm,
scattered and less-intense punctate nuclear staining was observed
throughout the basal and spinous cell layers (best seen in Fig.
4d and
e). No E7 staining was observed in the
superficial granular
cell layer. E7 within the keratinized layer of
fibropapillomas
could not be assessed due to staining of the
keratinized layer
by the secondary goat anti-rabbit antibody (Fig.
2f).
E7 staining
of the basal and lower spinous cells was observed in four
fibropapillomas
from two separate animals; similar, although weaker,
epithelial
staining was also observed in an experimentally induced
BPV-1
fibropapilloma (generously provided by Alison McBride, National
Institute of Allergy and Infectious Diseases, Bethesda, Md.; data
not
shown). Two separate affinity-purified rabbit polyclonal antibodies
to
E7 gave the same pattern of E7 localization. In Fig.
2c,
4d,
and
4e
(see below), E7 immunostaining was performed with an antibody
specific
to the amino terminus of E7, while in Fig.
3j (see below),
E7 was
localized with an antiserum specific to the C terminus
of E7. E7
staining of the fibroma was not consistently higher
than background
staining of adjacent normal-appearing dermis.
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FIG. 2.
Immunolocalization of E7 in bovine fibropapillomas.
Bovine fibropapillomas were collected at the Ohio State University
veterinary surgical clinic from domestic cows. Four warts from two cows
were analyzed by immunostaining. Frozen sections were air dried, stored
at  85°C, and fixed for 6 min in PBS containing 3% freshly prepared
formaldehyde. Alexa-468-labeled goat anti-rabbit antibodies (Molecular
Probes) were extensively adsorbed with a fixed extract of a bovine
fibropapilloma and fixed human HaCat cells to remove antibodies
reactive to fibropapilloma or HaCat cells. Panels a and d are shown for
orientation and were taken from regions of the same fibropapilloma
similar to those of panels b, c, e, and f but are not adjacent
sections. In all of the sections, arrow DE indicates the
dermal-epidermal junction. E, epithelioma; F, fibroma. (a) Low-power
(×100) hematoxylin-and-eosin-stained section, shown for orientation.
(b) Low-power photomicrograph (×100) showing DAPI-stained nuclear DNA.
(c) Low-power photomicrograph (×100) of the same field as in panel b,
showing BPV-1 E7 cytoplasmic expression at the dermal-epidermal
junction. (d) High-power (×400) hematoxylin-and-eosin-stained normal
bovine skin, shown for orientation for panels e and f. (e) DAPI-stained
normal bovine skin adjacent to the fibropapilloma in the same tissue
section as panel c. (f) E7 immunostained normal bovine skin, showing
the same field as that shown in panel e.
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FIG. 3.
Comparison of E5 and E7 localization in fibropapillomas.
Panel pairs a and b, c and d, e and f, g and h, and i and j are
identical views stained with DAPI (a, c, e, g, and i), antibody to E5
(b, d, f, and h, gifts of Daniel DiMaio and Richard Schlegal), or
antibody to E7 (j, C-terminal peptide antibody affinity purified to the
E7 carboxy terminus), respectively. Annotations are as in Fig. 2.
Slides were treated with 0.2% SDS after fixation but prior to
immunofluorescence to enhance immunostaining signal. (a and b)
High-power (×400), same views of normal bovine skin adjacent to
fibropapilloma stained with DAPI (a) or immunostained with antibody to
E5 (b). (c and d) Same ×100 views of bovine fibropapilloma stained
with DAPI (c) or immunostained with antibody to E5 (d). (e and f) Same
×200 views of fibropapilloma, stained with DAPI (e) or immunostained
with antibody to E5 (f). (g and h) Same ×200 views of fibropapilloma,
stained with DAPI (g) or immunostained with antibody to E5 (h). The
arrow in panel h indicates sporadically positive E5-expressing cells in
the spinous layer. (i and j) Same ×200 views of fibropapilloma,
stained with DAPI (i) or immunostained with antibody to E7 carboxy
terminus (j).
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FIG. 4.
Localization of E7 within the cytoplasm and nucleolus of
fibropapilloma epithelial cells. (a and b) Same ×400 views of
DAPI-stained cell (a, blue) or cell stained with DAPI plus monoclonal
antibody 72B9 to fibrillarin (24) (green, b). (c and d)
Same views as in panels a and b but stained for E7 (red, c) or E7 plus
fibrillarin (fibrillarin is green, d). (e) A different bovine
fibropapilloma than that shown in panels a to d or Fig 1 and 2 was
immunostained with amino-terminal specific antibody to E7 (red), and
the nuclei were counterstained with DAPI (blue), showing basal and
parabasal E7 cytoplasmic staining, together with focal E7 nuclear
staining.
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Comparison of E5 and E7 expression in fibropapillomas.
BPV-1 E5 was localized utilizing a rabbit anti-BPV-1 E5 antiserum
previously used to localize BPV-1 E5 in bovine fibropapillomas (4) and a second rabbit antibody (generously provided by
Richard Schlegel). With both antibodies BPV-1 E5 was localized to basal cells and the superficial granular cell layer (Fig. 3d). Unlike staining of E7 where the basal cell layer was uniformly stained (Fig.
2c), prominent E5 staining was patchy along the basal cell layer, being
more intense at the base of dermal papilla (Fig. 3f). Sporadic E5
staining within mid-spinous cell layers was observed but was much less
intense than basal E5 staining (Fig. 3f and h). In tissue culture, E5
is expressed in the membranes of the Golgi and the endoplasmic
reticulum. The resolution of our photomicrographs on frozen sections
could not sublocalize E5 within the cytoplasm. In Fig. 3h an apparent
expansion of E5-expressing cells above the basal layer is due to
tangential sectioning of this portion of the fibropapilloma. In
comparison to E5, E7 was expressed only basally and within the lower
spinous cell layer (compare E7 in Fig. 3j to E5 in Fig. 3f and h).
While there was clear BPV-1 E5 staining in the granular cell layer
where viral capsid proteins are synthesized and virions are assembled,
BPV-1 E7 immunostaining was not observed in the granular layer (not
shown). We have not as yet been able to observe E6 in either BPV-1 or
BPV-2 fibropapillomas by immunofluorescent staining.
Figures
2 and
3 show immunostaining for BPV-2 fibropapillomas.
Fibropapillomas associated with BPV-1 and BPV-2 were similar
in both
histological appearance and immunostaining for E5 and
E7. Sequencing of
viral DNA isolated from the BPV-2 fibropapilloma
shown in Fig.
2
revealed three amino acid differences within the
E7 gene compared to
the prototype BPV-2 sequence (T6N, S62P, and
H84N).
In addition to the predominant cytoplasmic localization of E7 in
fibropapillomas, focal nuclear staining of E7 was also observed
(not
seen in Fig.
2 and
3). Figure
4 shows a section of fibropapilloma
immunostained for both the nucleolar protein fibrillarin and E7.
Fibrillarin was exclusively localized to foci within DAPI
(4',6'-diamidino-2-phenylindole)-stained
nuclei (Fig.
4a and b). Focal
E7 staining within the spinous cell
layer typically, but not
invariably, colocalized with fibrillarin
staining (Fig.
4c and d), with
46% of the 200 nuclei counted demonstrating
focal E7 nuclear
expression and with 12% of the spinous cells
showing only focal
nuclear E7. Figure
4e demonstrates focal E7
staining within
DAPI-stained nuclei in a separate fibropapilloma
than that shown in
Fig.
2 or
3. E7 nucleolar staining can vary
depending upon the
processing of the samples. Both fibrillarin
and E7 nucleolar staining
were reduced by sodium dodecyl sulfate
(SDS) denaturation of
fibropapilloma tissue sections prior to
immunostaining, while E7
cytoplasmic staining was unchanged. The
section in Fig.
3j was treated
with SDS prior to immunostaining
with antibodies to E7, while sections
in Fig.
4 were not treated
with
SDS.
Location of E7 in transiently transfected cells.
In order to
compare the experimental expression of BPV-1 E7 with that seen in the
fibropapilloma, BPV-1 E7 was transiently expressed in human HaCat
epithelial cells. The expression patterns of E7 in HaCat cells were
similar to that seen in the fibropapilloma. Figure
5a shows the DAPI-stained nuclei of five
cells, two of which express E7 (Fig. 5b). In the upper cell of Fig. 5b,
E7 is predominantly cytoplasmic with a fibrillar pattern, although some E7 is within the nucleus in a punctate distribution. In the
fibropapilloma, strong cytoplasmic expression is observed in basal and
parabasal epithelial cells (Fig. 3j and 4e). The lower E7-expressing
cell in Fig. 5b demonstrates E7 that is predominantly nuclear with focal concentrations, as is observed in some spinous cells of the
fibropapilloma (Fig. 4c and e). Comparison of Fig. 5c and d
demonstrates that focal concentrations of E7 in the HaCat cell nucleus
can colocalize with the nucleolar protein fibrillarin. However, the E7
concentration within nucleoli was not uniformly observed in HaCat
transfections, since some E7-expressing cells showed either no
nucleolar concentration of E7 or even occasionally perinucleolar
concentration of E7 (Fig. 5e and f). The percentage of E7-expressing
cells with nuclear localization-transfected cells varied from 18 to
54% in three separate transfections. The variable localization of E7
suggests that localization of E7 is subject to regulation but was not
related to cell density, time posttransfection, or the intensity of E7
expression, nor were cells expressing nuclear versus cytoplasmic E7
expression grossly altered in mophology. E7 localization was not
altered by treatment of transfected HaCat cells with leptomycin B, a
drug that blocks Crm1-mediated nuclear export of proteins that contain
leucine-rich export sequences (9), so it is unlikely that
E7 localization is regulated by a typical nuclear export signal (data
not shown). Despite numerous attempts, we have been unsuccessful in
obtaining cell lines that stably express E7 that can be observed by
immunofluorescence.
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FIG. 5.
Localization of BPV-1 E7 within the cytoplasm and
nucleolus of epithelial cells. HaCat cells lipofected with a plasmid
expressing BPV-1 E7 were fixed 24 h posttransfection and examined
by indirect immunofluorescence with rabbit polyclonal antibodies to E7
and mouse monoclonal antibody to fibrillarin. (a and b) Same views of
cells stained with DAPI (a) or immunostained for E7 (b). (c and d) Same
views immunostained for fibrillarin (c) and E7 (d). (e and f) Same
views immunostained for fibrillarin (e) and E7 (f).
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Synergistic transformation by BPV-1 E5 and E7.
Since BPV-1 E7
and BPV-1 E5 are coexpressed within the basal cell layer of
fibropapillomas, they might cooperate in controlling cell
proliferation. To investigate this possibility, we examined the
influence of E7 upon transformation by E5 and E6 of anchorage-dependent rodent cells. BPV-1 E6 and BPV-1 E7 open reading frames were cloned into retroviral expression plasmids with either neomycin or puromycin resistance genes to allow for positive selection of two separately transduced genes. BPV-1 E5 was cloned as an
EcoRI-to-BamHI fragment of BPV-1 containing a
translation termination linker within the E2 and E4 open reading
frames. This plasmid is predicted to express only the intact E5 polypeptides.
Murine C127 cells were transduced with replication-defective
retroviruses expressing BPV-1 E5, BPV-1 E6, BPV-1 E7, or dually
infected combinations so that every resulting cell line expressed
both
puromycin and neomycin resistance at the same cell culture
passage
history. C127 cells transduced with either E5 or E6 had
a typical
transformed appearance, while cells transduced with
E7 alone were
unchanged in appearance (data not shown). Pooled
drug-resistant
colonies (>10
4 colonies per infection) were tested for
anchorage-independent
colony formation. While both E5- and
E6-expressing retroviruses
induced anchorage independence in the
absence of E7, coexpression
of E7 together with E5 or E6 increased both
the frequency and
the size of colony formation (Fig.
6). Results from separate experiments
using independently transduced cells are shown in Table
1 (group
A). E7 enhanced focus formation
by E5 and E6 by ca. 4- to 10-fold.
Disruption of the E5 open reading
frame with a translation stop
codon eliminated anchorage-independent
colony formation (Table
1, group B). Similarly, disruption of the E7
open reading frame
eliminated synergistic transformation with both E6
and E5. E7
had a small direct oncogenic activity independent of E5 or
E6,
but the resulting colonies were infrequent and much smaller than
those produced by either E6 or E5 alone. No significant differences
in
E6 or E5 protein expression levels were observed by Western
blot or
immunoprecipitation when coexpressed with E7 (data not
shown).
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FIG. 6.
E7 synergizes with E6 and E5 in transformation. C127
cells retrovirally transduced with the indicated BPV-1 oncogenes were
assayed for anchorage-independent colony formation as described in the
legend to Table 1. Colonies were photographed 14 days after seeding
into agar.
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The separation of papillomavirus oncogenes into separate
polypeptides (E5, E6, and E7) allows for the separate regulation
of expression and activity of each product within the
differentiated
layers of a epithelioma. This prompted our
examination of fibropapillomas
for E5 and E7 proteins. Examination of
mRNA expression in both
human and animal epitheliomas has been
performed, but few studies
have examined protein expression. While we
were able to localize
E7 and E5 in this study, we were unable to
localize E6 in fibropapillomas,
indicating a more-restricted expression
of E6 than E7 in
fibropapillomas.
E7 protein has not been previously localized in HPV-induced
epitheliomas or in animal papillomaviruses that are similar to
HPV
papillomaviruses. E7 has been localized in BPV-4, a BPV that
is quite
different from BPV-1, BPV-2, and HPV viruses. While BPV-4
contains an
E7 protein with an LXCXE retinoblastoma protein binding
motif, it does
not encode an E6 protein (
14). In BPV-4 alimentary
epitheliomas, E7 was detected within the nucleus of basal and
parabasal
cells and within the cytoplasm of differentiated spinous
and granular
cells (
1). In contrast, in the present study we
observe
uniform BPV-1 E7 expression in the cytoplasm and nucleus
of basal and
lower spinous epithelial cells. Nuclear staining
that colocalized with
the nucleolus was sporadically observed
in basal and spinous epithelial
cells of fibropapillomas and was
also observed within transfected HaCat
epithelial cells in tissue
culture.
It is difficult to correlate the expression of E7 protein to viral
mRNAs. In BPV-1-induced fibropapillomas, in situ hybridizations
to
basal epithelial cells detected only mRNA from the P2443 and
P3080
promoters that are thought to make E5 and the E2 repressor,
respectively (
2). From these in situ hybridization studies
it is unclear which promoter(s) is responsible for the synthesis
of
either E1 (necessary for plasmid maintenance) or for E7 in
basal cells.
While it is presumed that E1, E6, and E7 proteins
are made in basal
cells from transcripts originating at promoter
P89, this is as yet
unproven. It is possible that an additional
viral promoter upstream of
E7 may be responsible for the expression
of E7 protein observed in this
study.
Subcellular HPV E7 localization within cultured cells has varied with
different studies, with both nuclear and cytoplasmic
distribution
observed, depending upon the expression system. A
recent study using
new monoclonal antibodies to HPV-16 E7 localized
E7 to nucleoli and to
the cytoplasm in HPV-transformed CaSki cells
(
38). In
overexpression experiments, HPV E7 cytoplasmic expression
can be
prominent, and E7 has been shown associate with actin stress
fibers in
the cytoplasm when expressed by vaccinia virus (
25).
We
have not observed actin colocalization of BPV-1 E7 either in
transfected HaCat cells or in vaccinia virus expression systems,
nor
does cytochalasin D alter the localization of BPV-1 E7 in
these
transfection systems (data not shown). The absence of a
retinoblastoma
protein binding motif on BPV-1 E7 indicates that
BPV-1 E7 and HPV-16 E7
are in some ways functionally dissimilar,
but it is possible the BPV-1
E7 and HPV E7 proteins may share
as-yet-unknown cytoplasmic functions
and nucleolar functions despite
their limited primary sequence
homology.
BPV-1 E5 was the first papillomavirus oncoprotein whose expression
pattern in a productive infection was described (
4).
As
documented in that study and here, E5 is strongly expressed
in the
basal and granular cell layers and is sporadically expressed
in the
spinous cell layer. Although all early region transcripts
in BPV-1
include the E5 open reading frame, the genetic evidence
indicates that
E5-dependent transformation results from a spliced
message originating
from the P2440 promoter and spliced from nucleotides
2505 to 3225 (
11). Correlating with this observation, only a
spliced
message from nucleotides 2505 to 3225 could be specifically
localized
to basal epithelial cells in BPV-1 fibropapillomas (
2).
It
is unclear which messages may give rise to E5 expression observed
in
the spinous and granular cell layers. A recent study of the
oncogenic
HPV-31 E5 showed a pattern of E5 expression similar
to that of BPV-1
(
17).
The cytoplasmic coexpression of BPV-1 E7 with BPV-1 E5 in bovine
fibropapillomas observed in this study prompted us to reexamine
cooperative transformation between E7 and E5 and between E7 and
E6.
BPV-1 E5 and BPV-1 E6 synergy in transformation has been previously
described (
37) and was not explored here. We observed
clear
synergy in transformation between E5 and E7 and between E6 and
E7. Synergy in transformation between BPV-1 E5 and BPV-1 E7 has
not
been previously reported. In the experiments described in
the present
study each oncoprotein was expressed from separately
transduced
retroviruses, and care was taken to prevent the dominance
of the
culture by infrequent but highly transformed clones of
cells. It is
possible that prior studies failed to identify the
activity of BPV-1 E7
due to the overgrowth of the culture by cells
highly transformed by
either E5 or E6. If that were the case,
cells fully transformed by E6
or E5 might mask the contribution
to anchorage-independent colony
formation by
E7.
The E5 immunofluorescence signal in fibropapillomas was much stronger
than that of E7 despite high-titer antibodies to E7,
suggesting low
levels of E7 in fibropapillomas. But localization
of neither E5 nor E7
within transformed murine C127 cells could
be unambiguously observed
utilizing the same antibodies, indicating
even lower expression levels
in transformed C127 cells (data not
shown). E5 and E7 are clearly
biologically active within transformed
C127 cells, as shown by the
transformation assay in Fig.
6. Thus,
our inability to observe E7 above
the lower spinous cell layer
in epithellomas or the inability to
observe E5 within most cells
of the spinous cell layer should not as
yet be interpreted as
strong evidence for an absence of biological
function for these
oncoproteins in these regions of a
fibropapilloma.
The colocalization of E7 with E5 within the cytoplasm of basal
epithelial cells and the failure to detect E7 in the differentiated
granular layer where E5 is expressed suggest the possibility that
E7
may act to modulate the response of basal keratinocytes to
E5
expression. As noted in the introduction, E5 activates receptor
tyrosine kinases and stimulates mitogen-activated protein kinase
activity. In neuronal and epithelial cell culture systems, transient
ERK activation results in cell proliferation, while sustained
ERK
activation can result in terminal differentiation (
33,
34).
Similar to these observations, sustained
ras
signaling in primary
fibroblasts results in terminal differentiation to
a senescent
phenotype (
28,
39). If keratinocytes might
differentiate as
a result of sustained E5 expression, modulation of
E5-induced
signaling might be necessary to sustain cellular
proliferation
within basal epithelial cells. We hypothesize that the
coexpression
of E7 and possibly E6 may act to modulate cellular
responses of
epithelial cells to sustained high-level expression of
E5.
| |
ACKNOWLEDGMENTS |
This study was supported by NIH grant CA69292 to S.B.V.P.
We thank Alison McBride for frozen sections of a BPV-1 bovine
fibropapilloma and Daniel DiMaio and Richard Schlegel for rabbit antibody to E5.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Pathology, Case Western Reserve University, 10900 Euclid Ave.,
Cleveland, OH 44106. Phone: (216) 368-1679. Fax: (216) 368-1300. E-mail: sbv{at}pop.cwru.edu.
| |
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Journal of Virology, January 2001, p. 513-521, Vol. 75, No. 1
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.1.513-521.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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