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J Virol, January 1998, p. 476-482, Vol. 72, No. 1
Department of
Pathology1 and
Department of Cell
Biology and Center for Blood Research,2
Harvard Medical School, Boston, Massachusetts 02115
Received 18 August 1997/Accepted 7 October 1997
The E6 gene of the bovine papillomavirus type 1 (BPV-1) is
expressed in fibropapillomas caused by BPV-1 and in tissue culture cells transformed by BPV-1. It encodes one of the two major
oncoproteins of BPV-1. In this study, we demonstrate an interaction
between the BPV-1 E6 protein and AP-1, the TGN (trans-Golgi
network)-specific clathrin adaptor complex. AP-1 is a four-subunit
protein complex required for clathrin-mediated cellular transport from
the TGN. The AP-1/E6 interaction was observed in vitro and in cells.
The E6 binding site on AP-1 was mapped to the N-terminal trunk domain of the The bovine papillomavirus type 1 (BPV-1) is a small DNA virus that can induce proliferation of dermal
fibroblasts and squamous epithelial cells of the skin, causing
fibropapillomas in cattle. It can also induce fibroblastic tumors in
hamsters, rabbits, and mice and neoplastically transform a variety of
rodent cells in tissue culture (for a review, see reference
16). BPV-1 has served as the prototype for the
studies of various aspects of papillomavirus biology, such as cellular
transformation, viral transcriptional regulation, and viral DNA
replication. Genetic studies have mapped the BPV-1 transforming genes
to two regions of the viral genome: the E5 gene (14, 35) and
the E6 and E7 genes (34, 44). The transforming activity of
the BPV-1 E5 protein is principally mediated through the constitutive
activation of growth factor receptors. It can directly bind and
activate the platelet-derived growth factor The BPV-1 E6 gene product is a relatively basic, 137-amino-acid
protein. It contains four Cys-X-X-Cys motifs which are conserved among
all papillomavirus E6 proteins (Fig. 1A).
Expression of BPV-1 E6 by itself can lead to transformation of mouse
C127 cells (25, 34). Unlike the E6 proteins of human
papillomavirus types 16 and 18, BPV-1 E6 does not bind to the tumor
suppressor protein p53 (43) or stimulate E6AP-mediated
ubquitination and degradation of p53 (39). BPV-1 E6 has been
shown to bind in vitro to a 55-kDa putative calcium-binding protein
(ERC-55) (4), although the biological significance of this
interaction remains to be established. Recently, we have provided
evidence that BPV-1 E6 transformation is mediated through its
interaction with the focal adhesion protein paxillin (38).
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Interaction of the Bovine Papillomavirus E6 Protein
with the Clathrin Adaptor Complex AP-1
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
subunit. BPV-1 E6 preferentially associated with
membrane-bound AP-1 in cells but not with free cytosolic AP-1. BPV-1 E6
was further shown to be recruited to isolated Golgi membranes and to
copurify with clathrin-coated vesicles. The recruitment of BPV-1 E6 to Golgi membranes was AP-1 independent, but the E6 interaction with AP-1
was required for its association with clathrin-coated vesicles. Furthermore, AP-1 proteins could compete with BPV-1 E6 for binding to
Golgi membranes, suggesting that the recruitment of BPV-1 E6 and AP-1
to Golgi membranes involves a common factor. Taken together, our
results suggest that cytosolic BPV-1 E6 is first recruited to the TGN,
where it is then recognized by membrane-bound AP-1 and subsequently
recruited into TGN-derived clathrin-coated vesicles. We propose that
BPV-1 E6, through its interaction with AP-1, can affect cellular
processes involving clathrin-mediated trafficking pathway.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
receptor (11,
27) and the epidermal growth factor receptor (6, 23).
BPV-1 E5 also interacts with the 16-kDa subunit of the vacuolar
H+-ATPase (12, 13), suggesting that it can also
regulate the acidification of intracellular compartments such as
endosomes, lysosomes, and the Golgi apparatus, thereby affecting the
trafficking of cellular proteins.
View larger version (42K):
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FIG. 1.
(A) Schematic of the BPV-1 E6 protein structure. The E6
protein has four Cys-X-X-Cys motifs and is predicted to form two
zinc-binding sites (41). Xn indicates
the number of residues between the cysteines. The E6 mutants used in
this study are also indicated. (B) Identification of BPV-1
E6-associated proteins. 35S-labeled cell lysates from mouse
C127 cells were incubated with wild-type (wt) and various mutant GST-E6
fusion proteins. The bound cellular proteins were separated by SDS-PAGE
and visualized by autoradiography. The transforming activity (TF) of
each E6 mutant and the positions of size standards (in kilodaltons) and
of p110, p100, and p50 are indicated. na, not applicable.
So far, the studies of BPV-1 E6 have been largely focused on its
transforming mechanism, and little is known about other potential functions of E6 in the papillomavirus life cycle. In this report, we
identify the TGN (trans-Golgi network)-specific clathrin adaptor (AP-1)
as a cellular target of BPV-1 E6. Clathrin-coated pits and
clathrin-coated vesicles (CCV) are important both in endocytosis and in
regulated secretion via the TGN. The assembly of clathrin into coated
pits on the TGN and the plasma membrane requires the interaction with a
heterotetrameric protein complex called clathrin adaptor (AP). Two
types of APs have been identified based on their localization: those
localized on the TGN (AP-1) and those localized on the plasma membrane
(AP-2). AP-1 and AP-2 are related heterotetramers; AP-1 consists of
1, , µ1, and 
1 subunits, whereas AP-2 consists of 2, 
,
µ2, and 2 subunits. The 1 and 2 chains are closely related
(89% similarity) (20). AP-2 is directly involved in endocytosis. AP-1, which is localized at the TGN, facilitates the
transport of newly synthesized proteins to intracellular compartments such as endosomes and lysosomes (for reviews see references
18 and 32). In addition to
promoting clathrin assembly, AP-1 and AP-2 are involved in the sorting
step where cargo proteins are recruited into coated pits which lead to
their directed vesicular traffic.
The BPV-1 E6-AP-1 interaction described here is the first example of a viral protein directly interacting with a component of the clathrin-dependent sorting machinery. Our data suggest a model of how BPV-1 E6 proceeds in the TGN-derived trafficking pathway. BPV-1 E6 is first recruited to the TGN membrane; it is then recognized by membrane-bound AP-1 and subsequently recruited into TGN-derived CCV. We propose that the interaction of E6 with AP-1 may affect a vesicular trafficking pathway that could be important to E6 transformation as well as to other aspects of viral pathogenesis such as evasion of the host immune system.
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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GST fusion protein affinity binding. Wild-type and mutant glutathione S-transferase (GST)-E6 fusion proteins were constructed by PCR using pXH800-E6 plasmids (42) as templates and cloned into pGEX-2TK, which contains a cyclic AMP-dependent protein kinase site for in vitro phosphorylation (Pharmacia). To identify BPV-1 E6-associated proteins, C127 cells were labeled with [35S]cysteine-methionine overnight and lysed in lysis buffer (20 mM HEPES [pH 8.0], 1% Nonidet p-40 [NP-40], 150 mM NaCl, 2 mM CaCl2, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 µg of aprotinin per ml). Lysates were centrifuged at 14,000 rpm for 15 min, and the supernantants were precipitated with about 2 µg of GST-E6 fusion proteins for 1 h at 4°C. Bound proteins were washed in lysis buffer plus 0.1% sodium dodecyl sulfate (SDS) and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and autoradiography. To obtain p110, lysates from 50- by 150-mm dishes of mouse L cells were purified by GST-E6 affinity column. Bound proteins were eluted with 20 mM glutathione and further purified by a source 15Q column (Pharmacia). Proteins were eluted with 250 mM NaCl and separated by SDS-PAGE. After transfer to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad), about 5 µg of p110 was excised and subjected to microsequencing at the Harvard microchemistry facility.
Binding of E6 to AP-1 in vitro and in cells.
To confirm E6
binding to the AP-1 complex, 0.5-µg aliquots of AP-1 and AP-2
proteins purified from bovine brain (24) were incubated with
2 µg of various GST-E6 fusion proteins. Bound AP-1 and AP-2 were
detected by immunoblotting for the subunit, using antibody 9A
(5). The primary antibody was visualized by enhanced chemiluminescence as instructed by the manufacturer (DuPont NEN). The
efficiency of binding was quantitated by using NIH image 1.5 software.
To immunoprecipitate E6, Cos-7 cells were transfected with pSGFLAG-E6
plasmids encoding wild-type or mutant E6 proteins (38) by
electroporation. Cells were labeled by
[35S]cysteine-methionine and harvested 40 h after
transfection and lysed in lysis buffer. E6 was precipitated with
monoclonal antibody M2 against the FLAG epitope (IBI), and the AP-1 and
AP-2 complexes were precipitated with antibody 9A against the subunit (5).
In vitro translation of AP subunits.
All four subunits were
in vitro transcribed and translated by using the TNT T7 polymerase as
instructed by the manufacturer (Promega). The 1 subunit was
generated from pRSET (10), the and µ1 subunits were
from BSK (
) (1, 31), and the 1 subunit was from pET5a,
which was derived from its cDNA clone (19).
Protein binding of immobilized AP complexes. Aliquots of 5 µg of AP-1 and AP-2 complexes purified from bovine brain were digested with serial dilutions of 0.25% trypsin (GIBCO/BRL) for 15 min at room temperature, separated by SDS-PAGE, and transferred to a PVDF membrane. The blots were hybridized with 32P-labeled GST-E6 fusion protein in 20 mM HEPES (pH 7.7)-75 mM KCl-0.1 mM EDTA-2.5 mM MgCl2-1% bovine serum albumin-0.05% NP-40 as described previously (17).
Cell fractionation.
Cell fractionation was carried out as
previously described (2), with the following modifications.
Cells were washed once with cold phosphate-buffered saline and lysed on
plate in STM buffer (20 mM HEPES [pH 8.0], 0.25 M sucrose, 10 mM
MgCl2, 1 mM phenylmethylsulfonyl fluoride, 2 µg of
aprotinin per ml, 1 mM dithiothreitol). The cells were disrupted with
30 strokes in a glass Wheaton Dounce homogenizer (VWR). Fraction a
(crude cytosol) was prepared by centrifuging the homogenate at 14,000 rpm for 20 min in a microcentrifuge. The membrane fraction (fraction b) was prepared by extracting the pellet from fraction a with STM buffer
plus 0.05% NP-40 and centrifuging at 14,000 rpm. The nuclear fraction
(fraction c) was prepared by extracting the insoluble material from the
membrane fraction with lysis buffer plus 0.1% SDS. The high-speed
cytosol was made by further centrifuging the crude cytosol at
100,000 × g for 1 h in a TLA 100.4 rotor
(Beckman). All fractions were adjusted to lysis buffer plus 0.1% SDS
before immunoprecipitation. The E6 protein and the AP-1 complex were detected by immunoblot analysis using antibody M2 against the FLAG tag
and antibody 100/3 against the subunit of AP-1 (Sigma) (1).
Isolation of CCV. CCV were isolated as described previously (24). Briefly, cells were lysed on plate in buffer A (100 mM morpholine ethanesulfonic acid [pH 6.5], 1 mM EGTA, 0.5 mM MgCl2, 0.05% Triton X-100) and sonicated. The lysates were centrifuged at 15,000 rpm in TLA 100.4 rotor (Beckman) for 10 min. The supernatants were further centrifuged at 85,000 rpm for 15 min. The high-speed supernatants (HSS) were saved for immunoblot analysis, and the high-speed pellet was resuspended in buffer A, mixed with Ficoll-sucrose (12.5% each in buffer A), and centrifuged at 30,000 rpm for 12 min. The supernatants were diluted with 6× volume of buffer A and centrifuged at 85,000 rpm for 20 min. The final pellet contained partially purified CCV and was resuspended in buffer A followed by SDS-PAGE and immunoblot analysis.
Golgi membrane binding assay.
Crude Cos cell cytosol was
made by freezing-thawing cells in binding buffer (20 mM HEPES [pH
8.0], 5 mM MgCl2, 1 mM dithiothreitol, 125 mM potassium
acetate) and centrifuging at 14,000 rpm for 15 min. The high-speed
cytosol was made by further centrifuging the crude cytosol at
100,000 × g for 1 h in a TLA 100.4 rotor
(Beckman). For each binding reaction, 200 µl of cytosol (3 mg/ml), 2 µl of Golgi membrane from rat liver (7.5 mg/ml) (40), and
100 µM GTPS or 100 µg of brefeldin A per ml, when indicated,
were added and incubated at 37°C for 15 min. The membrane-bound
proteins were retrieved by centrifuging at 10,000 rpm for 10 min and
subjected to SDS-PAGE and immunoblot analysis.
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RESULTS |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The AP-1 complex is a cellular target for BPV-1 E6.
To
identify potential BPV-1 E6 cellular targets, BPV-1 E6 was fused to GST
and used to bind cellular proteins from 35S-labeled cell
extracts of C127 cells. E6 mutants which had been tested for the
ability to transform C127 cells (42) were included in the
experiment as controls of specificity (Fig. 1A). In the GST-E6 affinity
binding assay (Fig. 1B), three cellular proteins with apparent
molecular masses of 50 kDa (p50), 100 kDa (p100), and 110 kDa (p110)
bound to wild-type GST-E6. A transformation-competent E6 mutant (I41T)
also bound to the three cellular proteins. Five of the seven
nontransforming E6 mutants (C20S, C53R, C90S, H105D, and C124V) did not
bind to p50, p100, or p110, whereas the remaining two mutants (R116S
and 
134-137) did bind. Although the ability of BPV-1 E6 to bind to
these three cellular proteins did not correlate with the ability of E6
to transform, these interactions could be necessary though not
sufficient for the E6 transformation function, since each of the
transformation-competent E6 proteins did bind to p50, p100, and p110.
Alternatively, these interactions could be important for other
functions of E6 in the papillomavirus life cycle that are not related
to its transforming activity. Therefore, we pursued the identification
of these three cellular proteins.
1
subunit of AP-1. The p50 and p100 polypeptides were further shown by
immunoblotting to be the µ1 and subunits, respectively (data not
shown; the 1 subunit is a small protein of 19 kDa which ran out of
the gel in Fig. 1B). An antibody against the subunit of AP-2
(29) failed to detect any band in the same experiment (data
not shown). We therefore conclude that GST-E6 can interact with the
intact AP-1 complex but not with AP-2 from cell lysates.
BPV-1 E6 interacts with AP-1 in vitro and in cells.
To confirm
the interaction between BPV-1 E6 and the AP-1 complex, equal amounts of
AP-1 and AP-2 purified from bovine brain coated vesicles
(24) were incubated with GST-E6, and the bound proteins were
analyzed by immunoblotting with a monoclonal antibody which recognizes
the 1 and 2 subunits of AP-1 and AP-2 (5). Wild-type
BPV-1 E6 bound to about 30% of input AP-1 and about 10% of input AP-2
(Fig. 2). Consistent with the results
shown in Fig. 1B, mutant GST-E6 (H105D), which did not precipitate
p110, p100, or p50, failed to bind to either AP-1 or AP-2 (Fig. 2). Similarly, GST-E6 (134-137), which did interact with p110, p100, and
p50 in Fig. 1B, bound to about 10% of input AP-1 but not to AP-2.
Although some interaction between GST-E6 and AP-2 was detected in this
assay, the significance of this observation is not clear, since the
AP-2 complex was not present in the cellular proteins retrieved by
GST-E6 or by E6 coimmunoprecipitation (Fig. 1 and 3).
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1 and 2 subunits
(5). AP-1 and AP-2 proteins purified from bovine brain were
run on the same gel and stained to serve as standards in order to
identify the position of each subunit (data not shown). Wild-type E6
and mutant E6 (134-137), which had been shown to bind to AP-1 in the
GST affinity assay, specifically coprecipitated the AP-1 but not the
AP-2 complex from Cos cells (Fig. 3). The 1 subunit comigrated with
wild-type E6 but was readily visible when the smaller mutant E6
(134-137) was used. Paxillin, a previously identified E6-binding
protein (38), was also coprecipitated by wild-type E6 (Fig.
3). Mutant E6 (H105D), which did not bind AP-1 in the in vitro assays
(Fig. 1 and 2), failed to coprecipitate the AP subunits (Fig. 3).
The AP-1-E6 interaction is mediated through the N-terminal trunk
of the subunit of AP-1.
We next used several approaches to
determine which one of the AP-1 subunits was responsible for the
interaction with BPV-1 E6. In the first experiment, each subunit of the
AP-1 complex was 35S labeled by in vitro translation and
tested in the GST-E6 binding assay. Wild-type E6 bound to about 20% of
input and 1 subunit but did not bind to the µ1 or the 1
subunit. As a control for specificity, we used mutant E6 (H105D), which
does not interact with AP-1. We found that E6 (H105D) did not bind to
any of the AP-1 subunits in this assay (Fig.
4).
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subunit of AP-1 and weakly to the 1
subunit (control lane). In contrast, GST-E6 bound only weakly to the
2 subunit of AP-2 and no other subunits (Fig. 5B, control lane). No
binding to any AP subunits was detected when the nonbinding mutant
GST-E6 (H105D) was used instead (data not shown). These results
indicate that the subunit is able to directly mediate the
interaction between the AP-1 complex and BPV-1 E6. Although the subunit could also bind E6 in vitro (Fig. 4 and 5), it is unlikely to
mediate the AP-1-E6 interaction in vivo, since it could not account
for the specificity of E6 for AP-1 in cells (Fig. 1 and 3). The
observed binding of BPV-1 E6 to the subunit could be due to
improper folding or denaturation of the polypeptide in vitro, which may
be responsible for the binding of AP-2 to E6 in vitro as shown in Fig.
2. However, the significance of the in vitro binding observed between
AP-2 and BPV-1 E6 is unclear since we did not detect it in the cells
(Fig. 1 and 3).
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subunits and the 70-kDa N-terminal trunk of 1 and (for AP-1)
or 2 and (for AP-2); the ear is to the 30-kDa C-terminal portion
of each of the large subunits. To determine which domain of the subunit mediated E6 binding, purified AP complexes were partially
digested with a serial dilution of trypsin, immobilized on a PVDF
membrane, and probed with 32P-labeled GST-E6. As shown in
Fig. 5, GST-E6 bound to a 70-kDa proteolytic polypeptide corresponding
to the N-terminal trunk of the subunit. The identity of the 70-kDa
band was confirmed by immunoblot analysis using an antibody against the
subunit (data not shown) (1). In contrast, GST-E6 failed
to bind to any proteolytic products of either the 1 or 2 subunit
(Fig. 5), suggesting that the binding site on these subunits may be destroyed by the protease treatment.
BPV-1 E6 coimmunoprecipitates with the membrane-associated AP-1
complex.
In cells, the AP-1 complex cycles between two pools: the
crude cytosolic AP-1 (which consists of free AP-1 and AP-1 assembled into CCV) and the TGN membrane-bound AP-1 (30). To determine with which cellular AP-1 fraction E6 is associated, Cos cells were
transfected with FLAG-tagged E6 and fractionated into a crude cytosolic
fraction (fraction a, obtained by centrifuging cell lysates at 14,000 rpm for 30 min, containing free AP-1 and AP-1 in CCV), a membrane
fraction (fraction b, obtained by extracting the pellet from fraction a
in 0.05% Triton X-100, containing TGN membrane-bound AP-1), and a
nuclear fraction (fraction c, obtained by extracting the pellet from
fraction b in 1% Triton X-100 plus 0.1% SDS) (2). Extracts
from each fraction were subjected to immunoprecipitation and immunoblot
analysis. The majority of AP-1 complex was present in the crude
cytosolic and membrane-bound fractions, as judged by
immunoprecipitation of AP-1 (Fig. 6A, upper panel). The results were confirmed by direct immunoblot of the
lysates from each fraction, using an antibody against the subunit
(data not shown). Furthermore, transfection of either wild-type E6 or
mutant E6 does not change AP-1 levels or distribution in the cell (Fig.
7A and data not shown). The distribution
of E6 was similar to what has been previously reported in
E6-transformed C127 cells (2) (Fig. 6A, middle panel),
although we did observe an increased amount of cytosolic E6, perhaps
due to the high level of E6 expression in transfected Cos cells.
Importantly, AP-1 in the membrane fraction coprecipitated with
wild-type E6 but not with the nonbinding mutant E6 (C90S) (Fig. 6A,
lower panel, lane b), further demonstrating an specific in vivo
interaction between AP-1 and E6. In contrast, immunoprecipitation of
cytosolic wild-type E6 brought down very little of the cytosolic AP-1
complex (Fig. 6A, lower panel, lane a). This was unexpected since
similar amounts of AP-1 and E6 were present in the cytosolic fraction
and the membrane fraction. Since the crude cytosol used in this
experiment contained free cytosolic AP-1 as well as AP-1 assembled into
CCV, we next tested E6-AP-1 interaction in high-speed cytosols which had been centrifuged at 100,000 × g for 1 h to
obtain free cytosolic AP-1. As shown in Fig. 6B, AP-1 was readily
immunoprecipitated from the high-speed cytosol, using an antibody
against its subunit. However, E6 failed to coprecipitate with AP-1
under such conditions. These results suggest that in cells E6 cannot
bind efficiently to free cytosolic AP-1 and that their interaction
probably occurs more favorably when both proteins are on the membranes.
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BPV-1 E6 copurifies with clathrin-coated vesicles.
Even though
the E6 antibody failed to retrieve any cytosolic AP-1, it was possible
that E6 was associated with AP-1 in CCV but that the presence of the
clathrin coats masked the accessibility of the antibody to epitopes
localized within the coats. To test this possibility, CCV were purified
from Cos cells transfected with either wild-type E6 or mutant E6 (C90S)
following standard procedures (24). Fractions containing
purified CCV were analyzed by immunoblotting for the AP-1 subunit
and the E6 protein. As shown in Fig. 7A, AP-1 was found in the HSS,
which contains free cytosolic AP proteins, and in the pellet, which is
enriched for CCV (24). The enrichment was estimated to be
15-fold by comparing the amount of AP-1 found in the HSS to that found
in CCV fractions after normalizing the total protein concentration
(data not shown). The distributions of AP-1 were similar in cells
transfected with wild-type E6 and mutant E6 (C90S). A large proportion
of wild-type E6 was also found in the CCV fraction. In contrast, very
little of the nonbinding mutant E6 (C90S) was detected in the CCV
fraction (Fig. 7A). From these data, we conclude that BPV-1 E6
copurifies with CCV and that interaction with AP-1 is probably required
for its assembly into CCV.
subunit of AP-1 and
autoradiography for E6. The clathrin antibody coprecipitated the AP-1
complex but not the E6 protein (Fig. 7B). Therefore, under these
conditions, BPV-1 E6 is unable to bind to AP-1 when it is enclosed in
the CCV, suggesting that E6 interacts with AP-1 before it is assembled
into CCV.
BPV-1 E6 can be recruited to isolated Golgi membranes. One possible location for the AP-1-E6 interaction to occur is the TGN membrane. AP-1 is abundant on the TGN membrane and is required for the formation of CCV derived from the TGN. Therefore, we investigated whether BPV-1 E6 and AP-1 were colocalized at the TGN by immunofluorescence. Unfortunately, due to the low level of E6 protein expressed in mouse C127 cells stably transformed by FLAG-tagged E6, the antibody did not detect any E6 signal by immunofluorescence. Transient overexpression of FLAG-tagged BPV-1 E6 in Cos cells and in C127 cells resulted in diffused cytoplasmic staining even after mild detergent treatment (reference 38 and data not shown), which made it difficult to assess its colocalization with AP-1. We also attempted to study E6 localization by immunoelectron microscopy, but the antibody failed to recognize the E6 protein in the electron microscopic experiments.
As an alternative approach, we tested biochemically whether BPV-1 E6 can be recruited onto the TGN membrane in vitro. It has been shown that AP-1 complex can bind to TGN membranes in the presence of cytosol and GTPS. One characteristic of this reaction is that it can be
inhibited by the fungal metabolite brefeldin A, indicative of an
ADP-ribosylation factor (ARF)-dependent process (33, 37,
40). Thus, purified Golgi membrane from rat liver was incubated
with crude cytosol made from Cos cells expressing E6. The crude cytosol
(Fig. 6A) was used as a source for both AP-1 and E6 proteins. After
incubation at 37°C, the Golgi membranes were pelleted by
centrifugation, and the AP-1 and E6 recruited to the membrane were
detected by immunoblotting. Incubation of cytosol at 37°C caused a
small amount of AP-1 to precipitate out of the solution as previously
reported (40) (Fig. 8A, lane
1). Addition of GTPS and Golgi membrane to cytosol resulted in an increase of AP-1 in the pellet, presumably due to AP-1 binding to the
Golgi membrane (Fig. 8A, lane 2). As expected, the binding was blocked
by brefeldin A (Fig. 8A, lane 3). In the same experiment, wild-type E6
was also recovered from the Golgi membrane upon addition of GTPS,
and treatment with brefeldin A prevented its association with the
membrane. These results suggest that the wild-type BPV-1 E6 protein can
be recruited to the Golgi membrane.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Here we demonstrated an interaction between the BPV-1 E6 protein and the AP-1 complex both in vitro and in cells. The interaction is highly specific in that certain single-point mutations in BPV-1 E6 greatly impair the binding. BPV-1 E6 preferentially interacts with the AP-1 complex but not with the AP-2 complex despite the extensive homology between the subunits of the two protein complexes. Several lines of evidence suggest a model of how BPV-1 E6 may proceed in the TGN-derived trafficking pathway. We have found that (i) BPV-1 E6 preferentially interacts with membrane-bound AP-1 but does not bind to free cytosolic AP-1 in cells, (ii) it can bind to isolated Golgi membrane independent of AP-1, and (iii) it copurifies with CCV but is unable to bind to AP-1 once AP-1 is incorporated into CCV. Based on these observations, we propose the following sequence of events. BPV-1 E6 is first recruited to the TGN membrane in an AP-1-independent manner; it is then recognized by the membrane-bound AP-1. Upon assembly of the clathrin coat, it is recruited into TGN-derived CCV together with AP-1. It should be noted that although E6 preferentially interacts with membrane-bound AP-1 in cells, GST-E6 can bind to purified AP-1 in vitro. Thus, the absence of the interaction of E6 with cytosolic AP-1 is likely due to the function of an inhibitory factor or the relative low concentration of the two proteins in the cytosol, and the enrichment of AP-1 and E6 on the Golgi membrane is a prerequisite for their interaction. It is unclear how BPV-1 E6 is recruited to the Golgi membrane. The fact that its recruitment is sensitive to brefeldin A suggests that like AP-1 (37), an ARF may be involved in the process. Even though AP-1 and E6 bind to Golgi membranes independently, they may require a common factor for their membrane recruitment, since excess of AP-1 can compete E6 in the Golgi membrane binding assay. The ARFs, as mentioned above, could be limiting factors; another possibility is that AP-1 and E6 compete for the same binding sites on the Golgi membranes.
The E6-AP-1 interaction is the first example of a viral protein
interacting with a component of the clathrin-dependent sorting machinery, and this interaction could have important functional consequences. Genetic evidence has indicated that the AP-1 complex may
play an important role in the regulation of cell proliferation and
differentiation. In Caenorhabditis elegans, mutation in the TGN µ1-chain gene (UNC-101) results in poor viability and
uncoordinated movements. UNC-101/µ1 is proposed to be
involved in the regulation of LET-23 (21), a tyrosine kinase
related to the epidermal growth factor receptor. In humans, a member of
the -subunit gene family localized on chromosome 22q12 is deleted in
a number of meningiomas (28). Inactivation of these genes
may lead to a loss of growth control due to disruption of growth
hormone receptor internalization and/or degradation. In this context,
BPV-1 E6 could affect signal transduction by disrupting AP-1 function
and thus interfering with the AP-1-dependent delivery of lysosomal
enzymes that are required for receptor downregulation.
Alternatively, BPV-1 E6 could affect the biosynthesis and delivery of cell proteins which utilize the vesicular trafficking pathway. One candidate could be the major histocompatibility complex (MHC) class II complex. Natural infection of papillomavirus is poorly immunogenic, possibly reflecting the ability of the virus to somehow evade the host immune system (9). Furthermore, human warts are more prevalent in conditions that depress T-cell functions, and regression of warts is associated with infiltration by CD4+ T cells (7, 8). One possible mechanism for BPV-1 to downregulate the CD4+ T-cell response could involve interference with the MHC class II-restricted antigen presentation. In fact, the biosynthesis of class II molecules includes many components of the endocytic pathway. The cytoplasmic tail of the class II Ii chain and HLA-DM, an accessory molecule required for the formation of antigen-presenting class II molecules, each contain sorting signals which could mediate their efficient delivery to the endocytic pathway (22, 26, 36). Similar signals have been shown to mediate AP-1-dependent endocytic transport (15). Therefore, it is possible that BPV-1 E6 affects the antigen presentation of virus-infected cells by impairing the maturation of MHC class II molecules through its interaction with the AP-1 complex.
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ACKNOWLEDGMENTS |
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We are grateful to H. Ploegh and T. Rapoport for helpful suggestions and critically reading of the manuscript.
X.T. is supported by the cancer research fund of the Damon Runyon-Walter Winchell Foundation. This research was supported by NIH grants P01CA50661-08 (P.M.H.) and GM36548 (T.K.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pathology, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115. Phone: (617) 432-2884. Fax: (617) 432-2882. E-mail: phowley{at}warren.med.harvard.edu.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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|---|
| 1. | Ahle, S., A. Mann, U. Eichelsbacher, and E. Ungewickell. 1988. Structural relationships between clathrin assembly proteins from the Golgi and the plasma membrane. EMBO J. 7:919-929[Medline]. |
| 2. |
Androphy, E. J.,
J. T. Schiller, and D. R. Lowy.
1985.
Identification of the protein encoded by the E6 transforming gene of bovine papillomavirus.
Science
230:442-445 |
| 3. |
Brodsky, F. M.
1985.
Clathrin structure characterized with monoclonal antibodies. I. Analysis of multiple antigenic sites.
J. Cell Biol.
101:2047-2054 |
| 4. |
Chen, J. J.,
C. E. Reid,
V. Band, and E. J. Androphy.
1995.
Interaction of papillomavirus E6 oncoproteins with a putative calcium-binding protein.
Science
269:529-531 |
| 5. | Clairmont, K. B., W. Boll, M. Ericsson, and T. Kirchhausen. The hinge-ear domain of the -chains is required
for incorporation into clathrin-coated pits and coated vesicles in
cells. Submitted for publication.
|
| 6. |
Cohen, B. D.,
D. J. Goldstein,
L. Rutledge,
W. C. Vass,
D. R. Lowy,
R. Schlegel, and J. T. Schiller.
1993.
Transformation-specific interaction of the bovine papillomavirus E5 oncoprotein with the platelet-derived growth factor receptor transmembrane domain and epidermal growth factor receptor cytoplasmic domain.
J. Virol.
67:5303-5311 |
| 7. | Coleman, N., H. D. L. Birley, A. M. Renton, N. F. Hanna, B. K. Ryatt, M. Byrne, D. Taylor-Robinson, and M. A. Stanley. 1994. Immunological events in regressing genital warts. Am. J. Clin. Pathol. 102:768-774[Medline]. |
| 8. | Coleman, N., and M. A. Stanley. 1994. Analysis of HLA-DR expression on keratinocytes in cervical neoplasia. Int. J. Cancer 56:314-319[Medline]. |
| 9. | Frazer, I. H. 1996. Immunology of papillomavirus infection. Curr. Opin. Immunol. 8:484-491[Medline]. |
| 10. |
Gallusser, A., and T. Kirchhausen.
1993.
The 1 and 2 subunits of the AP complexes are the clathrin coat assembly components.
EMBO J.
12:5237-5244[Medline].
|
| 11. | Goldstein, D. J., T. Andresson, J. J. Sparkowski, and R. Schlegel. 1992. The BPV-1 E5 protein, the 16 kDa membrane pore-forming protein and the PDGF receptor exist in a complex that is dependent on hydrophobic transmembrane interactions. EMBO J. 11:4851-4859[Medline]. |
| 12. | Goldstein, D. J., M. E. Finbow, and T. Andersson. 1991. Bovine papillomavirus E5 oncoprotein binds to the 16K component of the vacuolar H-ATPase. Nature (London) 352:347-349[Medline]. |
| 13. | Goldstein, D. J., and R. Schlegel. 1990. The E5 oncoprotein of bovine papillomavirus binds to a 16 kd cellular protein. EMBO J. 9:137-145[Medline]. |
| 14. | Groff, D. E., and W. D. Lancaster. 1986. Genetic analysis of the 3' early region transformation and replication functions of bovine papillomavirus type 1. Virology 150:221-230[Medline]. |
| 15. | Honing, S., J. Griffith, H. J. Geuze, and W. Hunziker. 1996. The tyrosine-based lysosomal targeting signal in lamp-1 mediates sorting into Golgi-derived clathrin-coated vesicles. EMBO J. 15:5230-5239[Medline]. |
| 16. | Howley, P. M. 1996. Papillomavirinae: the viruses and their replication, p. 2045-2076. In B. N. Fields, P. M. Howley, and D. M. Knipe (ed.), Virology, 3rd ed. Lippincott-Raven Publishers, Philadelphia, Pa. |
| 17. | Kaelin, W. J., D. C. Pallas, J. A. DeCaprio, F. J. Kaye, and D. M. Livingston. 1991. Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product. Cell 64:521-532[Medline]. |
| 18. |
Kirchhausen, T.
1993.
Coated pits and coated vesicles sorting it all out.
Curr. Opin. Struct. Biol.
3:182-188.
|
| 19. |
Kirchhausen, T.,
A. Davis,
S. Frucht,
B. Greco,
G. Payne, and B. Tubb.
1991.
AP17 and AP19, the mammalian small chains of the clathrin-associated protein complexes show homology to Yap17p, their putative homolog in yeast.
J. Biol. Chem.
266:11153-11157 |
| 20. |
Kirchhausen, T.,
K. L. Nathanson,
W. Matsui,
A. Vaisberg,
E. P. Chow,
C. Burne,
J. H. Keen, and A. E. Davis.
1989.
Structural and functional division into two domains of the (100- to 115-kDa) chains of the clathrin-associated protein complex AP-2.
Proc. Natl. Acad. Sci. USA
86:2612-2616 |
| 21. |
Lee, J.,
G. D. Jongeward, and P. W. Sternberg.
1994.
unc-101, a gene required for many aspects of Caenorhabditis elegans development and behavior, encodes a clathrin-associated protein.
Genes Dev.
8:60-73 |
| 22. |
Marks, M. S.,
P. A. Roche,
E. V. Donselaar,
L. Woodruff,
P. J. Peters, and J. S. Bonifacino.
1995.
A lysosomal targeting signal in the cytoplasmic tail of the chain directs HLA-DM to MHC class II compartments.
J. Cell Biol.
131:351-369 |
| 23. | Martin, P., W. C. Vass, J. T. Schiller, D. R. Lowy, and T. J. Velu. 1989. The bovine papillomavirus E5 transforming protein can stimulate the transforming activity of EGF and CSF-1 receptors. Cell 59:21-32[Medline]. |
| 24. | Matsui, W., and T. Kirchhausen. 1990. Stabilization of clathrin coats by the core of the clathrin-associated protein complex AP-2. Biochemistry 29:10791-10798[Medline]. |
| 25. |
Neary, K., and D. DiMaio.
1989.
Open reading frames E6 and E7 of bovine papillomavirus type 1 are both required for full transformation of mouse C127 cells.
J. Virol.
63:259-266 |
| 26. |
Odorizzi, C. G.,
I. S. Trowbridge,
L. Xue,
C. R. Hopkins,
C. D. Davis, and J. F. Collawn.
1994.
Sorting signals in the MHC class II invariant chain cytoplasmic tail and transmembrane region determine trafficking to an endocytic processing compartment.
J. Cell Biol.
126:317-330 |
| 27. |
Petti, L., and D. DiMaio.
1992.
Stable association between the BPV E5 transforming protein and activated platelet-derived growth factor in transformed mouse cells.
Proc. Natl. Acad. Sci. USA
89:6736-6740 |
| 28. |
Peyrard, M.,
I. Fransson,
Y. G. Xie,
F. Y. Han,
M. H. Ruttledge,
S. Swahn,
J. E. Colins,
I. Dunham,
V. P. Collins, and J. P. Dumanski.
1994.
Characterization of a new member of the human -adaptin gene family from chromosome 22q12, a candidate meningioma gene.
Hum. Mol. Genet.
3:1393-1399 |
| 29. |
Robinson, M. S.
1987.
100-kD coated vesicle proteins: molecular heterogeneity and intracellular distribution studied with monoclonal antibodies.
J. Cell Biol.
104:887-895 |
| 30. | Robinson, M. S. 1992. Adaptins. Trends Cell Biol. 2:293-297. |
| 31. |
Robinson, M. S.
1993.
Assembly and targeting of adaptin chimeras in transfected cells.
J. Cell Biol.
123:67-77 |
| 32. | Robinson, M. S. 1994. The role of clathrin, adaptors and dynamin in endocytosis. Curr. Opin. Cell Biol. 6:538-544[Medline]. |
| 33. | Robinson, M. S., and T. E. Kreis. 1992. Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effects of brefeldin A and G protein activators. Cell 69:129-138[Medline]. |
| 34. |
Schiller, J. T.,
W. C. Vass, and D. R. Lowy.
1984.
Identification of a second transforming region in bovine papillomavirus DNA.
Proc. Natl. Acad. Sci. USA
81:7880-7884 |
| 35. |
Schiller, J. T.,
W. C. Vass,
K. H. Vousden, and D. R. Lowy.
1986.
E5 open reading frame of bovine papillomavirus type 1 encodes a transforming gene.
J. Virol.
57:1-6 |
| 36. | Schmid, S. L., and M. R. Jackson. 1994. Making class II presentable. Nature (London) 369:103-104[Medline]. |
| 37. | Stamnes, M. A., and J. E. Rothman. 1993. The binding of AP-1 clathrin adaptor particles to Golgi membranes requires ADP-ribosylation factor, a small GTP-binding protein. Cell 73:999-1005[Medline]. |
| 38. |
Tong, X., and P. M. Howley.
1997.
The bovine papillomavirus E6 oncoprotein interacts with paxillin and disrupts the actin cytoskeleton.
Proc. Natl. Acad. Sci. USA
94:4412-4417 |
| 39. | Tong, X. 1997. Unpublished data. |
| 40. |
Traub, L. M.,
J. A. Ostrom, and S. Kornfeld.
1993.
Biochemical dissection of AP-1 recruitment onto Golgi membranes.
J. Cell Biol.
123:561-573 |
| 41. |
Ullman, C. G.,
P. I. Haris,
D. A. Galloway,
V. C. Emery, and S. J. Perkins.
1996.
Predicted -helix/-sheet secondary structures for the zinc-binding motifs of human papillomavirus E7 and E6 proteins by consensus prediction averaging and spectroscopic studies of E7.
Biochem. J.
319:229-239.
|
| 42. |
Vousden, K. H.,
E. J. Androphy,
J. T. Schiller, and D. R. Lowy.
1989.
Mutational analysis of bovine papillomavirus E6 gene.
J. Virol.
63:2340-2342 |
| 43. |
Werness, B. A.,
A. J. Levine, and P. M. Howley.
1990.
Association of human papillomavirus types 16 and 18 E6 proteins with p53.
Science
248:76-79 |
| 44. |
Yang, Y.-C.,
H. Okayama, and P. M. Howley.
1985.
Bovine papillomavirus contains multiple transforming genes.
Proc. Natl. Acad. Sci. USA
82:1030-1034 |
J Virol, January 1998, p. 476-482, Vol. 72, No. 1
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