 |
INTRODUCTION |
Adenoviruses, as well as other DNA viruses, have developed
distinct strategies to counteract host immune defenses and cellular responses to viral infection. These strategies include blocking cellular apoptosis at critical junctions in the death-signaling cascade, suppressing the interferon response, and inhibiting
presentation of viral antigens (reviewed in Refs. 1 and 2). The E3
region of the adenovirus genome contains seven expressed open reading frames, most of which encode proteins with immunomodulatory functions, and viral deletion mutants lacking E3 genes induce stronger
pro-inflammatory responses in animal models (3). Although the E3 region
is dispensable for viral replication in tissue culture, emerging data
reveal that several E3 genes are involved in the evasion of host immune defenses. Uncovering the mechanism by which E3 genes temper host immune
responses could lead to the development of novel anti-inflammatory therapeutic strategies.
The E3-10.4K and 14.5K open reading frames encode type 1 transmembrane
glycoproteins that form a heteromeric complex (4-6). Both viral 10.4K
and 14.5K proteins are required to down-regulate epidermal growth
factor receptor (EGF-R)1 and
some related tyrosine kinase receptors from the surface of infected
cells (5, 7). More recently, the E3-10.4K/14.5K complex has been shown
to mediate the down-regulation of cell surface Fas (8-10), a
proapoptotic member of the tumor necrosis factor receptor (TNFR)
superfamily. Other members of the TNFR family, such as the LT
R and
TNFR1 were not modulated by the 10.4K/14.5K complex. Loss of cell
surface Fas results in the desensitization of virus-infected cells to
apoptosis induced by Fas signaling, thus counteracting a key defense
pathway of cytotoxic T cells and NK cells. It is not clear whether the
targeting of both EGF-R and Fas, which show no primary sequence
homology, reflects independent or linked functions of the
E3-10.4K/14.5K complex. This issue of receptor specificity exhibited by
the E3 protein complex prompted us to address whether additional death
domain-containing receptors in the TNFR family are targeted by adenovirus.
Cell surface receptors for the TNF-related apoptosis-inducing ligand
(TRAIL) are members of the TNFR superfamily, and currently four
membrane-anchored TRAIL receptors have been described (reviewed in
Refs. 11 and 12). TRAIL-R1 (DR4) and TRAIL-R2 (DR5) both contain a
cytoplasmic death domain that when ligated or overexpressed recruits
the adaptor FADD, allowing direct activation of the caspase cascade and
apoptosis (13-15). In contrast, neither TRAIL-R3 (DcR1) nor TRAIL-R4
(DcR2) induce cell death because they lack a death domain. TRAIL-R3 is
linked to the cell surface via a glycosylphosphatidylinositol (GPI)
anchor (15-17), whereas ligation of TRAIL-R4, which is unable to
recruit FADD, can initiate anti-apoptotic signals through the activation of the transcription factor NF-
B (18). However, TRAIL-R3
and TRAIL-R4 expression correlates poorly with the sensitivity of tumor
cell lines to TRAIL-mediated death (19). These findings, and the fact
that TRAIL-R1 and TRAIL-R2 can activate NF-
B in addition to inducing
apoptosis (16, 20), suggest a complex hierarchy in the regulation of
TRAIL signaling. Additionally, TRAIL has been implicated in both CTL
and NK cell killing of target cells (21-23), suggesting a possible
role for this cytokine in the innate response to viral pathogens.
Here we report that adenovirus infection results in the down-regulation
of TRAIL-R1 and TRAIL-R2 from the cell surface causing desensitization
of infected cells to TRAIL-mediated apoptosis. The E3-6.7K protein (24,
25) is required in addition to the E3-10.4K/14.5K proteins for TRAIL
receptor down-regulation. These results identify a strategy for viral
modulation of TRAIL receptor-mediated apoptosis and suggest the E3
trimolecular complex has evolved to regulate the signaling of selected
cytokine receptors.
 |
EXPERIMENTAL PROCEDURES |
Cell Lines, Viruses, and Reagents
HT29.14S cells are a human colon adenocarcinoma line that are
sensitive to the death inducing activities of TNF-related ligands (26).
293T-Fas cells were generated by transfecting a Fas expression vector
into 293T cells (from ATCC), and drug selecting cells that stably
expressed a "noncytotoxic" level of Fas on their surface (provided by P. Schneider and J. Tschopp). HeLa cells were
obtained from the ATCC. Primary small airway epithelial and normal
human bronchial epithelial cell lines were acquired from Clonetics (San Diego, CA) and were propagated in the company's recommended medium. All other cell lines were propagated in Dulbecco's modified Eagles medium (Life Technologies, Inc.) supplemented with 10 mM
glutamine and 10% fetal calf serum (HyClone, Logan, UT).
All viruses used in these experiments have been described in detail
previously (27, 28) and were kind gifts of W. Wold (St. Louis
University, St. Louis, MO). Briefly, rec700 is a recombinant Ad5
subtype virus containing the Ad2 10.4K protein (EcoRI-D
fragment, map position 73-86) and the Ad5 14.5K and 14.7K proteins.
All viral deletion mutants were generated from this parent recombinant "wild-type" virus. dl752 lacks the first 5 amino acids of the 10.4K
protein; dl759 deletes the N-terminal 103 amino acids of 14.5K
resulting in a nonfunctional 10.4K/14.5K fusion protein; dl762 is
deleted for 14.7K but expresses wild-type levels of 10.4K/14.5K, and
dl799 is deleted for both 10.4K and 14.5K.
For production of anti-TRAIL-R1, -R2, and -R3 antibodies, a custom
antibody production service was utilized (Eurogentec, Seraing, Belgium). Rabbits were immunized with TRAIL-R1:Fc, TRAIL-R2:Fc, and
TRAIL-R3:Fc (Alexis Biochemicals, San Diego, CA). For antibody purification, the various TRAIL receptors were coupled to HighTrap NHS-Sepharose (Amersham Pharmacia Biotech) according to the
manufacturer's protocol. Fc-specific antibodies were first depleted by
repeated passage over human IgG1-agarose (Sigma). TRAIL-R1, -R2, and
-R3 specific antibodies were then purified on TRAIL-R1(R2 and
R3)-Fc-Sepharose, eluted in 50 mM Tris-HCl, pH 2.7, neutralized with citrate NaOH, pH 9, and dialyzed against PBS. The
anti-10.4K polyclonal antiserum was generated by immunizing rabbits
with keyhole limpet hemocyanin-coupled peptide corresponding to amino
acids 60-91 of Ad2-10.4K. Total IgG was purified from crude rabbit
serum using protein G-Sepharose, eluted in 50 mM glycine,
pH 3.0, neutralized with Tris, pH 8.0, and dialyzed against PBS. The
anti-TRAIL-R4 goat polyclonal antibody was obtained from R & D Systems
(Minneapolis, MN). FLAG-tagged FasL and TRAIL were from Alexis
Biochemicals (San Diego, CA); the anti-EGF-R antibody (clone Ab-1) was
from Calbiochem (Cambridge, MA); the anti-Fas antibody (clone DX2) was
obtained from PharMingen (San Diego, CA), and the anti-FLAG and
anti-VSV antibody were obtained from Sigma.
Plasmids
Plasmids Used in 293T Transfections--
The pBluescriptII-Ad2E3
region plasmid (Ad2-E3) has been described previously (9) and was a
generous gift from Hans-Gerhard Burgert (Max Von Pettenkofer-Institut,
Munich, Germany). The E3-10.4K expression plasmid was generated by
excising the Ad2 10.4K coding sequence from pMAM-10.4K (gift of W. Wold) with SalI and ligating the fragment into
pcDNA3.1(
) (Invitrogen, Carlsbad, CA) cut with XhoI.
The E3-14.5K expression plasmid was generated by amplifying the 14.5K
coding sequence from isolated Ad5 genomic DNA using Pfu
polymerase (Stratagene, San Diego, CA) (primers,
5'-ggactatagctgatcttctc-3' and 5'-cgggatccatccaattctagatctag-3'),
digesting with BamHI and EcoRI, and ligating the
fragment into pcDNA3.1(
). FLAG-14.5K was generated by
amplification of the mature 14.5K coding sequence from E3-14.5K
(primers, 5'-acgtgaattcccgacctccaagcctcaa-3' and 5'acgtgaattcctatcagtcatctcctcctg-3') with Pfu polymerase.
The amplified PCR product was cut with EcoRI and ligated
into PS497 (provided by F. Martinon and J. Tschöpp), an
engineered PCRIII (Invitrogen)-based vector containing the signal
peptide from human IgG fused to an N-terminal FLAG epitope tag.
VSV-6.7K was constructed by amplifying the Ad2 E3-6.7K coding sequence
from isolated adenoviral genomic DNA (primers,
5'-acgtgaattcagcaattcaagtaactctacaagc-3' and
5'-acgtgaattcttatcatcttggatgttgcccccag-3') using Pfu
polymerase. The amplified PCR product was digested with
EcoRI and ligated into PL507, an engineered PCRIII-based
vector containing an N-terminal start codon fused to the VSV epitope
tag. FLAG-6.7K was generated in exactly the same manner as VSV-6.7K;
however, the EcoRI-digested PCR product was ligated into
PL508, an engineered PCRIII-based vector containing an N-terminal start
codon fused to the FLAG epitope tag.
The FLAG-tagged full-length TRAIL-R2 expression construct was generated
by PCR amplification of the mature coding sequence of TRAIL-R2 (amino
acids 52-440) from a PCRIII-TRAIL-R2 plasmid (16) with the addition of
a flanking 5' BamHI-FLAG sequence and a 3' PstI
site allowing subsequent ligation into a PCRIII-derived vector
(Invitrogen, San Diego, CA) containing a signal peptide from the heavy
chain of human IgG (PS089). The TRAIL-R2
C16 construct, lacking the
16 C-terminal amino acids, was derived from the full-length FLAG-TRAIL-R2 construct by PCR amplification using the primers 5-ctgcagctagctcaacaagtggtc-3' and T7. The resulting product was subcloned as a BamHI-PstI fragment in PS089. The
TRAIL-R2
DD deletion mutant (
Leu
348-Ser424) was generated using a dual stage
PCR approach. In the first PCR round, two overlapping fragments
corresponding to the 5' end and 3' end of TRAIL-R2 devoid of the DD
sequence were amplified using the primers
5'-caccaaattgtcctcagcccc-3'and T7 for the 5' fragment and
5'-tttgcagactctggaaagttcatg-3' and sp6 for the 3' end fragment. The two
PCR products obtained were allowed to anneal and were re-amplified
using sp6 and T7 to generate FLAG-TRAIL-R2
DD, which was subcloned as
a BamHI-PstI fragment in PS089. TRAIL-R2:GPI was
obtained by sub-cloning the sequence of the 5 TAPE tandem repeats of
human TRAIL-R3 (amino acids 157-259) as a
SalI-NotI fragment in replacement of the human
IgG1 Fc cassette of a TRAIL-R2:Fc construct (described in Ref. 16).
Retroviral Vector Plasmids--
pBMN-10.4K/14.5K was constructed
by amplification of the E3-10.4K/14.5K coding sequence from Ad2-E3
using Pfu polymerase (Stratagene) and the following primers:
5'-agacggatccgccatgattcctcgagttcttata-3' and 5'-
tcgtaagctttcagtcatctccacctgtcaa-3'. The amplified product was digested
with BamHI and HindIII and ligated into pBMN-LacZ (derived from pBABE series vectors, gift of Garry Nolan, Stanford University), and the resulting retroviral vector expresses both E3
proteins. The pBABE-6.7K plasmid was generated by amplification of the
Ad2 E3-6.7K-coding sequence from isolated adenoviral genomic DNA using
the following primers: 5'-atgagcaattcaagtaactc-3' and 5'-tcatcttggatgttgccc-3. The amplified product was ligated into the
PCRII-Topo vector (Invitrogen). The E3-6.7K-coding sequence was then
excised from PCRII-Topo with EcoRI and ligated into
pBABE-puro (29) to generate pBABE-6.7K. pBABE-FLAG-6.7K was generated
by digesting FLAG-6.7K with SmaI and EcoRV to
excise the N-terminal FLAG-tagged 6.7K gene, and this fragment was
ligated into SnaBI-digested pBABE-puro. The sequences of all
constructs were verified unambiguously using an ABI Prism 310 genetic
analyzer automatic sequencer (PerkinElmer Life Sciences).
Cell Death Assays
Cell viability was determined using an MTT-based assay as
described previously (30) with the following modifications. For adenovirus infection, cells were infected at a m.o.i. of ~30 in 100-µl volume; virus was allowed to preadsorb for 60 min, and then
100 µl of media containing cytokine (FLAG-tagged FasL or TRAIL), 80 units/ml human interferon-
, and 20 µg/ml cytosine 1-
-D-arabinofuranosylcytosine (Ara-C) (Sigma) were added
(13). One µg/ml anti-FLAG M2 antibody was added to wells containing FasL (2 µg/ml for TRAIL-containing wells) to enhance the activity of
the FLAG-tagged cytokines. Fresh Ara-C was added to wells every 12-18
h, and cell viability was determined with MTT 48 h after infection. All cytokine concentrations were performed in triplicate, and error bars represent standard deviations. Retrovirally transduced cells (5 × 103) were assayed for viability with MTT
72 h after addition of cytokines. Fold differences in sensitivity
to TRAIL and FasL mediated killing were determined using
IC50 concentrations of cytokine calculated from cell
viability plots.
Retrovirus Production and Cell Transduction
Retroviral vectors were generated as described previously (31)
based on the method of Soneoka et al. (32) and were
pseudotyped with the vesicular stomatitis virus G protein (VSV-G).
HT29.14S cells (1.5 × 105) were transduced multiple
times in vector supernatant containing 8 µg/ml Polybrene. The volume
of vector supernatant was kept constant for all wells by the addition
of growth medium. Retroviral vector titers were ~1-2 × 107 colony-forming units/ml when determined on HT29.14S
cells, and transduction efficiency was >99% as gauged by resistance
to puromycin (pBABE-6.7K) or staining with 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (X-gal) (pBMN-LacZ virus made in
parallel). Cells were harvested for analysis by FACS or plating into
96-well dishes for killing assays ~48 h after transduction.
Detection of Receptor Surface Levels by Flow Cytometry
HT29.14S and SAEC were infected with adenovirus at a m.o.i.
~30, and cells were detached from plastic with 5 mM EDTA
in PBS 18 h after infection. Cells were resuspended in FACS
binding buffer (PBS + 2% fetal bovine serum + 0.02% sodium azide),
and 1 × 105 cells were used for each staining. Cells
were incubated in 50 µl of FACS buffer plus appropriate antibodies
(all antibodies used at 10 µg/ml) for 1 h, at which time cells
were washed and incubated in either goat anti-rabbit F(ab')2 (for
detection of TRAIL-R1, R2 or R3), goat anti-mouse F(ab')2 (for
detection of Fas and EGF-R), or biotinylated rabbit anti-goat IgG
followed by streptavidin (for TRAIL-R4). All secondary detection
reagents were conjugated to R-phycoerythrin and were from
Southern Biotechnology Associates (Birmingham, AL). Cells were analyzed
on a FACSCalibur (Becton Dickinson, Mountain View, CA), and each
histogram represents 5 × 103 cells gated on forward
and side-angle light scatter. HT29.14S cells transduced with retroviral
vectors were analyzed by the same methods. In all presented figures,
dotted histograms represent either isotype control antibody (Fas and
EGF-R) or goat anti-rabbit F(ab')2 (TRAIL receptors) staining as a
negative control.
293T-Fas and 293T cells were transfected by the CaPO4
precipitation method as described previously (33). Two µg of E3-10.4K and E3-14.5K expression plasmid (4 µg of Ad2 E3 plasmid) were used to
examine the down-regulation of endogenously expressed receptor in
293T-Fas cells (Fig. 4). For analysis of E3-6.7K surface expression
(Fig. 6), 3 µg of VSV-6.7K (or FLAG-6.7K), 1 µg of E3-10.4K, and 1 µg of E3-14.5K or 0.5 µg of FLAG-14.5K were used. For analysis of
TRAIL-R2 mutants (Fig. 7), 0.1 µg of wild-type or mutant receptor
plasmid was used plus or minus 3 µg of Ad2-E3 plasmid. Total DNA
concentration was always kept equivalent by addition of empty vector.
Cells were detached from plastic 36 h after transfection for
analysis by FACS as described above.
Confocal Microscopy
The subcellular distribution of down-modulated receptors was
analyzed by confocal imaging of immunofluorescently labeled HeLa cells.
HeLa cells (3 × 105) were infected with virus at a
m.o.i. of 100 in permanox chamber slides (Nalge Nunc, Naperville, IL).
Twelve hours after infection, the cells were washed with PBS and fixed
in 4% formaldehyde. After washing with 0.2 M glycine, the
cells were permeabilized with 0.1% saponin. The cells were then
incubated in 10% donkey serum for 20 min prior to the addition of the
primary antibodies. Fas was detected with murine anti-Fas (DX2)
followed by incubation with goat anti-mouse Fab (Jackson
ImmunoResearch) and donkey anti-goat fluorescein
isothiocyanate-conjugated antibody (Jackson ImmunoResearch). TRAIL-R2
was detected with rabbit anti-TRAIL-R2 polyclonal antibody followed by
donkey anti-rabbit fluorescein isothiocyanate (Jackson ImmunoResearch).
The biotinylated anti-LAMP1 antibody (PharMingen, San Diego, CA) was
used at a 1:8 dilution according to the manufacturer's instructions
followed by detection with streptavidin-Texas Red. All primary
antibodies were used at 20-25 µg/ml and secondary detection reagents
were used at 2 µg/ml. The immunofluorescently labeled cells were
analyzed with a Bio-Rad MRC 1000 (Emeryville, CA) confocal microscope
using the 60 × objective. Two-color Z-series were collected in
the simultaneous mode, and the overlap of green and red fluorescence
was depicted by a yellow signal. The Z-sections were then projected as
stacks using the Lasersharp image processing software.
Co-immunoprecipitation of 10.4K, 14.5K, and 6.7K
293T cells were transfected in 10-cm dishes as described above
using E3-10.4K (10 µg/dish), FLAG-14.5K (5 µg/dish), and VSV-6.7K (10 µg/dish). Cells were lysed 36 h after transfection (lysis buffer: 1% Nonidet P-40, 50 mM HEPES, 150 mM
NaCl, 20 mM EDTA, 500 µM phenylmethylsulfonyl
fluoride, and 0.018 units of aprotinin), and the lysates were
pre-cleared with mouse IgG (5 µg/ml) and protein G-Sepharose beads
for 3 h at 4 °C. Pre-cleared lysates were then incubated with
anti-FLAG (M2, Sigma, 5 µg/ml) or anti-VSV (for analysis of total
transfected VSV-6.7K) antibody and protein G beads overnight at
4 °C. Samples were then analyzed by SDS-PAGE on 18% Tris glycine
gels and transferred to polyvinylidene difluoride membrane. Membranes
were incubated with anti-VSV antibody (1:5000) followed by rabbit
anti-mouse horseradish peroxidase-conjugated antibody. Protein
(VSV-6.7K) was then visualized by enhanced chemiluminescence using the
Super-Signal detection kit (Pierce). The membranes were then stripped
and reprobed with anti-FLAG or rabbit polyclonal anti-10.4K antibody to
visualize FLAG-14.5K and 10.4K, respectively.
 |
RESULTS |
Adenovirus Infection Results in Down-regulation of Cell Surface
TRAIL-R1 and TRAIL-R2 via an E3-10.4K/14.5K-dependent
Mechanism--
HT29.14S cells, which have been shown previously to
down-regulate cell surface Fas and EGF-R upon infection with adenovirus (8), were infected with either wild-type adenovirus (rec700) or viral
deletion mutants lacking functional E3 region proteins as follows:
10.4K (dl752), 14.5K (dl759), 14.7K (dl762), or both 10.4K/14.5K
(dl799). After infection, cells were analyzed by FACS for surface
levels of TRAIL-R1, -R2, -R3, and -R4 as well as for Fas and EGF-R
(Fig. 1a). Infection with
wild-type virus resulted in significant down-regulation of TRAIL-R1,
TRAIL-R2, Fas, and EGF-R, but surface levels of TRAIL-R3 and TRAIL-R4
were not down-regulated. TRAIL-R1 and TRAIL-R2 surface levels decreased
66 and 60%, respectively, after viral infection (based on peak mean
fluorescence), as compared with a 96% decrease in the levels of Fas
and 88% for EGF-R. Three viral deletion mutants (dl752, dl759, and
dl799) were incapable of down-regulating any receptors analyzed,
indicating that the E3-10.4K/14.5K complex was necessary for TRAIL
receptor down-regulation as has been shown previously for both Fas and
EGF-R. By contrast, infection with a virus deleted for E3-14.7K (14.7K
is encoded by various polycistronic E3-transcripts which also encode
10.4K/14.5K (34)) was similar to the effect of wild-type virus,
indicating the E3-14.7K protein is not required for TRAIL receptor
down-regulation.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 1.
Down-regulation of TRAIL-R1 and TRAIL-R2 by
adenovirus requires E3-10.4K/14.5K. a, HT29.14S cells
were infected with wild-type (rec700) adenovirus or viral mutants
deleted for E3-14.7K (dl762), E3-10.4K (dl752), E3-14.5K (dl759), or
10.4K/14.5K (dl799). b, primary SAEC were infected with
wild-type virus or dl799. Infected cells were analyzed for cell surface
expression of TRAIL receptors, Fas and EGF-R, by flow cytometry.
|
|
Although various studies have analyzed E3-mediated down-regulation of
cell surface receptors, all these experiments have been performed using
transformed human cell lines. To determine whether cell surface levels
of TRAIL receptors decrease in cells that represent the normal target
tissue for adenoviral infection in vivo, primary small
airway epithelial cells (SAEC) were infected with wild-type virus or
dl799 (Fig. 1b). Infection with wild-type virus resulted in
TRAIL-R1 and TRAIL-R2 down-regulation, as well as Fas and EGF-R, from
the cell surface. Notably, TRAIL receptor down-regulation in SAEC was
significantly more pronounced than in HT29.14S cells. TRAIL-R1 was
undetectable, and TRAILR2 was reduced by 93% of normal levels (Fig.
1b). TRAIL-R3 and TRAIL-R4 levels were unaffected by
wild-type virus, and infection with dl799 caused no down-regulation of
the receptors examined. These data indicate that modulation of TRAIL-R1
and TRAIL-R2 by adenovirus occurs in primary cells that are targeted
during host infection. Similar results were seen in infected primary
normal human bronchial epithelial cells, HeLa, 293T, and 293-HEK cells
(data not shown).
Subcellular Localization of Fas and TRAIL Receptors in Infected
Cells--
The steady-state levels of TRAIL-R1 and TRAIL-R2 on the
plasma membrane were reduced within 4 h after viral infection
(data not shown), suggesting that the adenovirus E3 proteins stimulate TRAIL receptor internalization rather than interfering with their biosynthesis, similar to what has been shown for Fas (8, 10). The
intracellular distribution of Fas and TRAIL-R2 was analyzed in HeLa
cells infected with either wild-type virus or dl799. The infected cells
were double-labeled with antibodies directed against Fas or TRAIL-R2
and lysosome-associated membrane protein-1 (LAMP-1), a marker of late
endosomes and lysosomes (35). Confocal imaging (Fig.
2) of cells infected with wild-type virus
revealed that both Fas and TRAIL-R2 were abundant in perinuclear
vesicles (Fig. 2, a and g). In contrast, both Fas
and TRAIL-R2 were absent from these compartments in cells infected with
dl799 (Fig. 2, d and j), and both Fas and
TRAIL-R2 co-localized with LAMP-1 (Fig. 2, c and
i). However, not all internalized Fas and TRAIL-R2 were present in LAMP-1-positive vesicles. Consistent with the presence of
Fas and TRAIL-R2 in late endocytic compartments, these two proteins
extensively co-localized in cells infected with wild-type virus (Fig.
2o) suggesting both receptors have a similar endocytic pathway.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 2.
Fas and TRAIL-R2 co-localize in
intracellular vesicles after adenovirus infection. HeLa cells were
infected with wild-type (rec700) or dl799(-10.4K/14.5K) virus and
stained with antibodies against Fas, TRAIL-R2, and LAMP-1.
a c, g i, and m-o represent cells
infected with wild-type virus, and d-f and j-l
represent cells infected with dl799. The images as follows:
a-f, anti-Fas (green) and anti-LAMP-1
(red); g-l, anti-TRAIL-R2 (green) and
anti-LAMP-1 (red); and m-o: anti-Fas
(green) and anti-TRAIL-R2 (red). The last panel
in each row is a merged image of the previous two panels where
yellow indicates the co-localization of the two analyzed
proteins.
|
|
The E3-10.4K/14.5K Proteins Are Not Sufficient for Down-regulation
of TRAIL Receptors--
To assess whether the E3-10.4K/14.5K proteins
alone were sufficient for down-regulation of TRAIL-R1 and TRAIL-R2 from
the cell surface, expression vectors were transfected into 293T cells that modestly overexpress Fas (293T-Fas), and receptor surface levels
were examined by FACS (Fig.
3a). As expected
co-transfection of E3-10.4K plus E3-14.5K expression vectors resulted
in down-regulation of both Fas and EGF-R from the surface of 293T-Fas
cells (Fig. 3a), whereas transfection of either vector alone
did not affect Fas or EGF-R levels (data not shown). Surprisingly,
transfection with both E3-10.4K and 14.5K did not alter the cell
surface levels of TRAIL-R1 and TRAIL-R2 (Fig. 3a). This
result indicated that an additional viral protein(s) acts in concert
with the E3-10.4K/14.5K complex to down-regulate TRAIL receptors.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 3.
E3-10.4K/14.5K is not sufficient for
down-regulation of TRAIL-R1 and TRAIL-R2. a, 293T-Fas
cells were co-transfected with vectors expressing E3-10.4K and E3-14.5K
or were mock-transfected, and cell surface levels of TRAIL-R1,
TRAIL-R2, Fas, and EGF-R were analyzed by flow cytometry. b,
293T-Fas cells were transfected with a vector containing the entire
adenovirus E3 coding sequence (pBluescript-Ad2E3) or were
mock-transfected and subjected to flow cytometric analysis to determine
receptor surface levels.
|
|
Treatment of adenovirus-infected HT29.14S cells with cytosine
arabinoside (AraC), an inhibitor of viral DNA replication, did not
inhibit down-regulation of TRAIL-R1 and TRAIL-R2 (data not shown), also
suggesting that the additional viral protein(s) needed for TRAIL
receptor down-modulation was expressed as an early gene. To test
whether this gene was contained within the E3 region, a plasmid
containing the entire Ad2 E3 locus (EcoRV C fragment) (9)
was transfected into 293T-Fas cells, and the down-regulation of surface
receptors was analyzed by FACS (Fig. 3b). Decreased levels
of both TRAIL-R1 and TRAIL-R2, as well as Fas, were detected on the
surface of cells transfected with the Ad2-E3 plasmid (Fig. 3b), indicating that the adenoviral protein(s) needed for
down-regulation of TRAIL receptors, in addition to E3-10.4K/14.5K, was
included in the E3 region.
The E3-6.7K Protein Is Required to Down-regulate TRAIL Receptors
from the Cell Surface--
The E3-6.7K protein was identified as a
potential candidate for assisting in TRAIL receptor down-regulation by
its structure as an integral membrane protein (24, 25); however, no
function for the E3-6.7K protein has been previously identified. An
E3-6.7K-expressing retroviral vector (pBABE-6.7K) was generated to test
whether this protein might function in concert with E3-10.4K/14.5K in
the down-regulation of TRAIL-R1 and TRAIL-R2. HT29.14S cells were
transduced with either control retroviral vector expressing LacZ
(Vector), vectors expressing E3-10.4K/14.5K (pBMN-10.4K/14.5K) and/or
E3-6.7K (pBABE-6.7K). Cells transduced with pBMN-10.4K/14.5K or
pBABE-6.7K were then compared with cells transduced with both vectors
for surface receptor levels by flow cytometry (Fig.
4). Transduction of HT29.14S with pBMN-10.4K/14.5K resulted in Fas down-regulation as expected, and
co-transduction with pBABE-6.7K did not show any additional effects on
Fas surface levels (Fig. 4). The identical results were seen for EGF-R
as well (data not shown). TRAIL-R1 was modestly down-regulated by
transduction with pBMN-10.4K/14.5K alone (51% of normal levels), which
was not seen in 293T cells, but co-transduction with pBABE-6.7K
significantly increased TRAIL-R1 down-regulation (28% of normal
levels). Significant TRAIL-R2 down-regulation was only observed when
HT29.14S was co-transduced with pBMN-10.4K/14.5K and pBABE-6.7K (44%
of normal levels). Neither TRAIL-R1 nor TRAIL-R2 surface levels were
affected by transduction with pBABE-6.7K alone (Fig. 4). The surface
levels of TRAIL-R3 and TRAIL-R4 were not affected by transduction with
any combination of E3 protein-expressing vectors. These data indicate
that the E3-6.7K protein is required for the down-modulation of the
death domain-containing TRAIL receptors seen in adenovirus
infection.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 4.
E3-6.7K functions together with
E3-10.4K/14.5K to down-regulate TRAIL-R1 and TRAIL-R2. HT29.14S
cells were transduced with recombinant retroviral vectors expressing
-galactosidase (pBMN-LacZ, serves as "vector" negative control),
E3-10.4K/14.5K (pBMN-10.4K/14.5K), and/or E3-6.7K (pBABE-6.7K) and were
analyzed by flow cytometry to determine receptor cell surface
levels.
|
|
E3-6.7K Is Detectable on the Cell Surface and Forms a Complex with
E3-10.4K/14.5K--
The E3-10.4K and 14.5K proteins have previously
been shown to exist as a heteromeric complex on the cell surface
composed of one 14.5K monomer and two alternatively processed,
disulfide-bridged forms of 10.4K (36, 37). E3-6.7K has been reported to
reside in the endoplasmic reticulum, based upon the sensitivity of this protein to digestion by endoglycosaminidase H (25). Our functional analysis of E3-6.7K led us to hypothesize that the E3-6.7K protein might associate with the E3-10.4K/14.5K complex. Epitope-tagged versions of E3-14.5K (FLAG-14.5K) and E3-6.7K (FLAG-6.7K and VSV-6.7K) were generated to facilitate biochemical analysis of protein-protein interactions and cell surface expression. FLAG-14.5K and FLAG/VSV-6.7K were capable of down-regulating Fas and TRAIL-R equivalent to the
untagged proteins when co-expressed with E3-10.4K, indicating that
epitope tagging of these two E3 proteins did not disrupt their function
(data not shown). 293T cells were transfected with various combinations
of the E3 protein expression vectors and were analyzed for protein cell
surface expression by flow cytometry (Fig.
5, upper panel), or cell
lysates were subjected to immunoprecipitation for analysis of protein
complex formation (Fig. 5, lower panel). Flow cytometry
analysis revealed that the E3-6.7K protein is expressed on the surface
of transfected cells (Fig. 5, a and b) and that co-expression of E3-10.4K/14.5K does not affect the surface levels of
E3-6.7K. Additionally, E3-6.7K cannot supplant the function of E3-10.4K
in helping to transport E3-14.5K to the cell surface (see Fig.
5d). Western blot analysis of immunoprecipitated cell lysates revealed that E3-6.7K co-immunoprecipitates with
E3-10.4K/14.5K. E3-6.7K also co-precipitated with E3-14.5K in the
absence of E3-10.4K, suggesting a direct interaction between these two
proteins. E3-6.7K did not immunoprecipitate with E3-10.4K in the
absence of E3-14.5K (data not shown).

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 5.
E3-10.4K/14.5K/6.7K are expressed on the cell
surface and form a trimolecular complex. Upper
panel, 293T cells were transfected with E3 protein expression
vectors (VSV-6.7K, FLAG-6.7K, E3-10.4K, E3-14.5K, and FLAG-14.5K) and
were subjected to analysis by flow cytometry. Histograms a
and c were samples incubated with anti-VSV antibody to
detect the N-terminal epitope-tagged VSV-6.7K protein. b and
d were samples incubated with anti-FLAG antibody to detect
either FLAG-6.7K or FLAG-14.5K, respectively. Lower panel,
293T cells were transfected with various combinations of E3 protein
expression vectors (FLAG-14.5K, VSV-6.7K, and E3-10.4K). +/ indicates
the presence or absence of the designated expression vectors in the
various transfections. Cell lysates were precipitated using anti-FLAG
antibody followed by SDS-PAGE and Western blot. For Western blots of
immunoprecipitated proteins, anti-FLAG antibody was used to detect
FLAG-14.5K, anti-10.4K polyclonal antiserum for detection of the two
isoforms of 10.4K, and anti-VSV for detection of VSV-6.7K. The
bottom panel indicates the relative levels of VSV-6.7K
protein present in the various transfections and was determined by
immunoprecipitation of cell lysates (approximately half of that used
for analysis of E3 complexes) with anti-VSV antibody followed by
Western blot using anti-VSV.
|
|
E3 Protein-mediated Down-regulation of TRAIL-R2 Is Dependent upon
Sequences in the Cytoplasmic Tail of the Receptor--
In an attempt
to elucidate which sub-domains of TRAIL-R2 are required for
down-regulation by E3-10.4K/14.5K/6.7K, we generated various mutants of
the cytoplasmic domain of TRAIL-R2 that lack either the C-terminal 16 amino acids of the cytoplasmic tail (TRAIL-R2
C16), the entire death
domain (TRAIL-R2
DD), or a chimeric TRAIL-R2/R3 receptor encoding the
extracellular domain of TRAIL-R2 fused to the domain of TRAIL-R3 coding
for the addition of the GPI link to the cell surface (TRAIL-R2:GPI).
The mutant TRAIL-R2 expression plasmids were transfected into 293T
cells either with or without the Ad2-E3 plasmid, and cell surface
receptor levels were analyzed by FACS (Fig.
6). Full-length TRAIL-R2 was included as
a positive control for Ad2-E3 down-regulation. Interestingly, the cell
surface levels of the TRAIL-R2 mutants were unaffected by co-expression of Ad2-E3 (Fig. 6), indicating that the cytoplasmic tail of TRAIL-R2 plays a critical role in the susceptibility of the receptor to down-regulation by E3 proteins. More specifically, the results seen
with the TRAIL-R2
C16 mutant indicate that the C-terminal 16 amino
acids are required for sensitivity of TRAIL-R2 to down-regulation, and
the determinant of specificity does not reside in the death domain.

View larger version (50K):
[in this window]
[in a new window]
|
Fig. 6.
TRAIL-R2 down-regulation by E3 proteins is
dependent upon the cytoplasmic tail of the receptors. 293T cells
were transfected with either wild-type TRAIL-R2 or various TRAIL-R2
mutants in the presence (gray histogram) or absence
(black histogram) of the Ad2-E3 plasmid. All receptor
constructs were detected using anti-FLAG antibody, except TRAIL-R2:GPI
where anti-TRAIL-R2 antibody was used.
|
|
Adenovirus E3 Proteins Are Required to Reduce the Sensitivity of
Infected Cells to Apoptosis by TRAIL and FasL--
To test whether the
down-modulation of death receptors from the cell surface by
E3-6.7K/10.4K/14.5K results in the desensitization to ligand-induced
apoptosis, HT29.14S cells transduced with E3 protein-expressing
retroviral vectors (Fig. 7a)
or infected with wild-type and deletion mutant adenovirus (Fig.
7b) were treated with TRAIL and FasL. Cells transduced with
pBMN-10.4K/14.5K plus pBABE-6.7K showed significant resistance to TRAIL
killing when compared with cells transduced with vector alone
(~8-fold) (Fig. 7a). Transduction with pBMN-10.4K/14.5K
showed some protection against TRAIL killing (~3-fold) and is
probably due to the limited down-regulation of TRAIL-R1 in HT29.14S
cells expressing these two E3 proteins (see Fig. 4). Transduction with
pBABE-6.7K alone showed no protection against TRAIL-mediated apoptosis.
For comparison, transduced cells were also tested for their sensitivity
to FasL. Consistent with the virtual complete down-regulation of Fas in HT29.14S (see Fig. 4), transduction with either pBMN-10.4K/14.5K or
pBMN-10.4K/14.5K plus pBABE-6.7K rendered cells almost entirely resistant to killing by FasL.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 7.
E3 proteins reduce the sensitivity of cells
to FasL and TRAIL apoptosis. a, HT29.14S cells were
infected with wild-type adenovirus (Rec700) or various E3 deletion
mutants, and cells were tested for their sensitivity to TRAIL and
FasL-mediated apoptosis using a MTT-based assay. Adenovirus mutants
lacking E3-10.4K, 14.5K, or both proteins are indicated by the
gray lines. b, HT29.14S cells were transduced
with various retroviral vectors expressing E3 proteins and tested for
sensitivity to FasL and TRAIL using an MTT assay.
|
|
Next, adenovirus-infected cells were tested for sensitivity to TRAIL
and FasL-mediated apoptosis. HT29.14S cells infected with viral mutants
lacking E3-10.4K/14.5K showed an ~6-fold increased sensitivity to
killing by TRAIL when compared with cells infected with wild-type
virus, virus deleted for 14.7K (dl762), or mock-infected cells (Fig.
7b). Similar data were seen when infected cells were analyzed for their sensitivity to FasL-mediated killing, although, analogous to the retroviral transduction experiments, desensitization to FasL (~20-fold) was more dramatic then TRAIL. As was observed for
TRAIL, infection with deletion mutant viruses also increased the
sensitivity of HT29.14S cells to FasL (~2-3-fold). Together, these
data suggest that the E3 protein complex acts to counteract the
increased sensitivity of cells to TRAIL and FasL that occurs during
viral infection.
 |
DISCUSSION |
This report identifies the specific targeting of the proapoptotic
receptors for TRAIL by adenovirus and is the first example of a viral
defense strategy directed toward this group of death receptors. The
E3-6.7K protein is required for the specific down-regulation of TRAIL
receptors seen during adenoviral infection, providing an additional
mechanism that potentially blocks the host innate and immune responses
to viral infection. The adenoviral genome contains several distinct
proteins that regulate cellular apoptosis including E1b-19K, E1b-55K,
E3-14.7K, and E3-10.4K/14.5K. Each viral protein has evolved a
different mechanism to modulate apoptotic signaling pathways. E1b-19K,
a functional homologue of the human BCL-2 protein, is thought to
interfere with apoptosis at the level of the mitochondria (reviewed in
Ref. 38), whereas E1b-55K suppresses p53-mediated apoptosis (39). Both
of these E1b proteins function to block cell death mediated through the
intrinsic cellular apoptotic pathway (reviewed in Ref. 40). Contrary to
the E1b proteins, the E3-encoded proteins appear to act on components
of the extrinsic apoptotic pathways, which are initiated by signaling
through the death domain-containing receptors of the TNFR superfamily.
E3-14.7K is a cytosolic protein that has been shown to inhibit
TNF-mediated apoptosis (41) by a still unknown mechanism, although
recently 14.7K has been implicated in modulating the transcription
factor NF-
B though interactions with IKK
/NEMO (42),
which may induce protection against TNF-mediated death. The commitment of adenoviral genes dedicated to blocking apoptosis signaling by death
receptors indicates an indispensable role for this pathway in immune
control of adenovirus infection in vivo.
The E3-6.7K protein has the predicted structure of a type III integral
membrane protein, a family of transmembrane proteins that encode no
cleavable signal peptide but contain a signal anchor sequence for
targeting and insertion into the membrane of the endoplasmic reticulum,
a compartment that E3-6.7K has previously been reported to reside (25).
In contrast, the E3-10.4K/14.5K proteins are type I membrane proteins
that are thought to localize primarily to the plasma membrane (4),
raising the question of where this trimolecular protein complex resides
in the infected cell. Our data prove that the E3-6.7K protein can be
expressed on the cell surface and interacts directly with the
E3-10.4K/14.5K complex (see Fig. 6), suggesting that the trimolecular
complex exists on the plasma membrane. E3-6.7K is highly expressed on the cell surface in the absence of E3-10.4K/14.5K, indicating there is
no requirement for additional viral proteins to assist in its transport
to the plasma membrane as is the case for E3-10.4K/14.5K (4). However,
there are detectable levels of E3-10.4K and E3-14.5K in both the
endoplasmic reticulum (where E3-6.7K has been described to be localized
previously) and Golgi of adenovirus-infected cells (4), raising the
possibility that the E3 complex may reside in multiple cellular
compartments. We are currently examining the subcellular localization
of the E3-10.4K/14.5K/6.7K complex to understand better the mechanism
for TRAIL receptor down-regulation.
Although the E3-10.4K/14.5K complex was known to down-regulate both Fas
and EGF-R prior to this study, little was known about the mechanism of
action for these proteins. Various deletion mutants were generated in
the cytoplasmic tail of TRAIL-R2 to analyze the role of this receptor
domain in E3 protein-mediated down-regulation, and none of the mutants
were susceptible to down-regulation. Notably, even TRAIL-R2
C16,
which deletes the last 16 amino acids of the receptor cytoplasmic tail,
but still encodes the entire death domain (DD), was unable to be
down-regulated by adenovirus. These data delineate that the DD, which
is highly conserved between Fas and TRAIL receptors, is not the
exclusive target of the E3 complex. Additionally, the
E3-10.4K/14.5K/6.7K complex is unable to down-regulate the DD encoding
receptors DR3/TRAMP and TNFR-1 from the surface of HT29.14S cells (data
not shown), further highlighting the specificity of E3 proteins for Fas
and TRAIL-R. The inability of the TRAIL-R2
DD mutant to be
down-regulated by the E3 complex suggests a positional requirement for
the C-terminal 16 amino acids of TRAIL-R2 in determining sensitivity to
E3-mediated down-regulation. Interestingly, all of the TRAIL-R2 mutants
are expressed on the cell surface of transfected 293T cells at
significantly higher levels than wild-type TRAIL-R2 (see Fig. 7). Since
the total protein levels of the TRAIL-R2 mutants were equivalent to
that of wild-type receptor in 293T cells (data not shown), this result
indicates that the overall steady-state distribution of the mutants is
altered. A significant fraction of TRAIL-R2 is normally present in
intracellular compartments (43), and our data suggest that manipulation
of the TRAIL-R2 cytoplasmic tail can result in a redistribution of TRAIL-R2 to the cell surface, similar to what has been shown for the
LT
R (44). Our continuing efforts to dissect the role trafficking and
subcellular localization of TRAIL-R1 and R2 in E3-mediated down-regulation should help to elucidate further the mechanism of
action of these viral proteins.
Although the trimeric E3 protein complex clearly reduces the
sensitivity of cells to TRAIL killing when expressed in
trans (see Fig. 7a), the role of these proteins
in desensitizing cells to TRAIL in the context of a viral infection
appears a bit more complicated. As has been shown previously for FasL,
infection of cells with viral mutants lacking E3-10.4K and 14.5K
dramatically sensitizes them to TRAIL-induced apoptosis.
Down-regulation of TRAIL-R1 and -R2 by the E3 complex counteracts this
sensitivity, reducing it to the level of mock-infected cells. The
desensitization (when compared with uninfected cells) of
adenovirus-infected HT29.14S cells to FasL killing is likely to be due
to the more pronounced down-regulation of Fas in this cell line when
compared with TRAIL-R1 and -R2. Quite interestingly, down-regulation of
TRAIL-R1 and -R2 is most dramatic in primary small airway epithelial
cells (see Fig. 1b), suggesting that these cells would be
largely resistant to TRAIL-mediated apoptosis if infected in
vivo. However, because most primary cells are not killed by TRAIL
in tissue culture models, we have been prohibited from assaying for
TRAIL sensitivity in this cell line.
Based on our studies in HT29.14S cells, it appears that E3-6.7K is
absolutely required for down-regulation of TRAIL-R2, but that
E3-10.4K/14.5K can function alone to down-regulate TRAIL-R1, albeit at
significantly reduced efficiency when compared with the trimeric E3
complex. If TRAIL is important in mediating innate immune responses
against pathogens in vivo, then perhaps the TRAIL receptor
family has co-evolved to counteract viral strategies directed at
quelling this response, and this is a possible explanation for
differences seen in E3-induced down-regulation of the two proapoptotic
receptors for TRAIL. However, cell-specific issues regarding the extent
of receptor down-regulation are likely to play a role as well, and this
notion is supported by the observation that E3-10.4K/14.5K complex
alone is insufficient to down-regulate TRAIL-R1 in 293T cells in the
absence of E3-6.7K (Fig. 3a).
TRAIL has been implicated in the killing of target cells by both CTL
and NK cells (21-23). Cell lines that were insensitive to killing by
FasL and TNF were susceptible to death mediated by TRAIL (23),
suggesting that the TRAIL signaling pathway is independent of Fas and
TNF. Thus, TRAIL may serve as an additional armament of CTL or NK cells
in the initial defense mounted against viral infection (reviewed in
Ref. 45). The E1A gene product of adenovirus sensitizes infected cells
to NK cell killing (46, 47), further supporting the hypothesis
discussed above that a role for the E3 complex may be to compensate for
the increased sensitivity of infected cells to apoptosis induced by
other viral genes. Taken together, these data indicate that the
inhibition of TRAIL killing by E3-10.4K/14.5K/6.7K may be critical for
adenovirus to evade several host cytotoxic effector mechanisms in
vivo. The apoptotic strategies, together with the E3-gp19K
gene that alters antigen presentation by blocking major
histocompatibility complex transport to the cell surface, may underlie
the success of adenovirus to maintain a persistent infection.