From GenVec, Inc., Rockville, Maryland 20852
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ABSTRACT |
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Adenovirus type 5 encodes a 14.7-kDa protein that
protects infected cells from tumor necrosis factor-induced cytolysis by an unknown mechanism. In this report, we demonstrate that infection of
cells with an adenovirus vector expressing Fas ligand induced rapid
apoptosis that was blocked by coinfection with a virus expressing 14.7K. Moreover, AdFasL/G infection resulted in the rapid activation of
DEVD-specific caspases, and caspase activation was blocked by
coinfection with Ad14.7/G. Cell death induced by the overexpression of
Fas ligand, Fas-associated death domain-containing protein (FADD)/MORT1, or FADD-like interleukin-1-converting enzyme
(FLICE)/caspase-8 in a virus-free system was efficiently blocked by
14.7K expression. Moreover, we demonstrate that 14.7K interacts with
FLICE. These results support the idea that FLICE is a cellular target
for the 14.7-kDa protein.
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INTRODUCTION |
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Apoptosis, or programmed cell death, is a physiological process
that is required for the normal development and homeostasis of
multicellular organisms and involves the destruction of cells that are
no longer required or are potentially dangerous (1). The elimination of
virus-infected cells by apoptosis is an efficient means of limiting
virus production and resolving viral infections (2). This can be
triggered by cytotoxic T lymphocytes (3) or by cytokines such as
TNF1 and interleukin-1
that are released following viral infection (4). However, viruses have
evolved sophisticated mechanisms to counter this host defense.
(5-7).
After ligand binding, Fas (CD-95/APO-1) and TNFR1 induce apoptosis by recruiting the cytosolic adapter protein FADD, also called MORT1, to the activated receptor (8). The TNFR1 does this indirectly through the adapter protein TRADD (9). The signaling complex arranged at these death receptors includes FLICE, also called MACH, Mch5, and caspase-8, a caspase that contains an N-terminal DED (10, 11). FADD recruits FLICE to the receptor complex through interactions with their respective DEDs. Activation of FLICE occurs at this receptor complex, and it is thought that activated FLICE then cleaves and activates downstream DEVD-specific caspases that are involved in executing the death signal (10-13).
The adenovirus type 5 14.7-kDa protein is a highly conserved product of the E3 transcription unit (14). 14.7K functions to inhibit the death of susceptible cells after exposure to TNF (15, 16). Treatment of cells with TNF induces the activation of cPLA2, and in at least some cell types, cPLA2 appears to be required for TNF-induced cytotoxicity (17). The finding that 14.7K blocked arachidonic acid release after TNF treatment (18-20) suggested that it may function by inhibiting the activation or activity of cPLA2. However, the role of the 14.7-kDa protein in the Fas signaling pathway has not been examined, and its precise mechanism of action has remained elusive.
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EXPERIMENTAL PROCEDURES |
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Materials--
The anti-Flag M2 monoclonal antibody was
purchased from Eastman Kodak Co. (Rochester, NY). The
anti-poly(ADP-ribose) polymerase monoclonal antibody was purchased from
Upstate Biotechnology Inc. (Lake Placid, NY). The FasL-specific
antibody was purchased from Transduction Labs (Lexington, KY). The
YAMA-specific antibody was purchased from Santa Cruz (Santa Cruz, CA).
The anti-mouse and anti-rabbit IgG conjugated to horseradish peroxidase
were purchased from Boehringer Mannheim.
Ac-DEVD--(4-methyl-coumaryl-7-amide) and AMC were obtained from
Peptides International, Inc. (Louisville, KY). The enhanced
chemiluminescence Western blot analysis reagents and Promix
35S label were purchased from Amersham Life Sciences, Inc.
Ni2+·nitrilotriacetic acid-agarose resin and the
bacterial expression vector pQE32 were purchased from Qiagen Inc.
(Chatsworth, CA). The pcDNA expression vectors for FLICE, FADD, and
YAMA were generously provided by Vishva M. Dixit, and the cDNA for
CrmA was kindly provided by David Pickup.
Plasmid Construction-- pMT3tagS was constructed by inserting the adapter, R1stopSALs AATTTAGCCCGCC, R1stopSALa TCGAGGCGGGCTA between the EcoRI and SalI sites of pMT3 tag (21). pMT3-14.7HA was constructed by polymerase chain reaction amplification of the Ad5 14.7K gene using oligos A5s30453 (Bam/Pst) CGGGATCCTGCAGCCACCATGACTGACACCCTAGATCTAGAATG and A5a30836 (Pst) AACTGCAGCGTTAAAGGGAATAAGATCTTTG.
The 14.7K polymerase chain reaction product was digested with BamHI and PstI and inserted into the BamHI and PstI sites of pMT3tagS. pMT3F is similar to pMT3tagS, except that it contains the Flag tag in place of the HA tag. pMT3-14.7F expresses a C-terminal Flag-tagged 14.7-kDa protein from the pMT3F vector. pAd14.7K/G and pAdFasL/G are duel expression vectors that drive Ad5 14.7K or the murine FasL, respectively, from the CMV promoter and theCells and Virus-- A549 cells, a human lung carcinoma cell line, and 293 cells, a human embryonic kidney cell line transformed by sheared Ad5 DNA, were obtained from American Type Culture Collection and were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. AdFasL/G, Ad14.7/G, AdNull/G, and AdCrmA express their respective transgenes from the cytomegalovirus promoter inserted at the site of the E1 deletion. All adenovirus vectors were generated using the pAdFasL/G, pAd14.7/G, pAdNull/G, and pAdCrmA shuttle vectors as described previously (22, 23).
14.7K Antibody Production--
The 14.7-kDa protein was purified
by Ni2+·nitrilotriacetic acid chromatography according to
the manufacturer's suggestions. Briefly, 20 ml of a stationary phase
culture of Escherichia coli, DH5F'IQ cells transformed
with the pQE32-14.7K expression plasmid were added to 1 liter of
superbroth containing 100 µg/ml ampicillin. The cells were grown at
37 °C until the absorbance at 600 nm reached 0.6. Isopropyl-
-D-thiogalactopyranoside was added to a final concentration of 1 mM, the temperature was reduced to
28 °C, and the cells were incubated with good aeration for an
additional 20 h. The cells were harvested by centrifugation at
4,000 × g for 10 min at 4 °C, and the pellet was
resuspended in buffer A (6 M guanidine hydrochloride, 0.1 M sodium phosphate, 0.01 M Tris (pH 8)) at 4 ml/gm wet weight and stirred at room temperature for 1 h. The
lysate was centrifuged at 10,000 × g for 20 min. The
supernatant was incubated with 8 ml of a 50% slurry of
Ni2+·nitrilotriacetic acid-agarose resin (previously
equilibrated in buffer A) at room temperature for 45 min and loaded
onto a 1.6-cm diameter column. The column was washed with 10 column
volumes of buffer A and 5 column volumes of buffer B (8 M
urea, 0.1 M sodium phosphate, 0.01 M Tris (pH
8)) and then washed with buffer C (8 M urea, 0.1 M sodium phosphate, 0.01 M Tris (pH 8)) until the optical density at 280 nm reached <0.01. The His-tagged 14.7-kDa protein was eluted with buffer C containing 250 mM
imidazole. The Bradford assay and SDS-polyacrylamide gel
electrophoresis were used to locate the peak fractions. The peak
fractions were pooled and renatured by dialysis for 6 h against
buffer D containing 2 M urea and 1 M NaCl,
followed by dialysis against buffer D (250 mM NaCl, 2 mM MgCl2, 0.1% Nonidet P-40, 25 mM
Tris (pH 8)) for 12 h. The solution was centrifuged at 10,000 × g to remove insoluble materials, and the protein
concentration was determined by the Bradford assay. The
14.7-kDa-specific antiserum was obtained from rabbits immunized with
His-14.7-kDa protein that was generated by electrophoreses of the
renatured, partially purified protein on a preparative, 15%
SDS-polyacrylamide gel stained with Coomassie Blue.
AMC Assay--
293 cells were infected with viruses at a
multiplicity of infection of 5 plaque-forming units/cell. At 4 h
post-infection, cells were lysed in a buffer containing 1% Triton
X-100, 50 mM Tris (pH 8) and 150 mM NaCl.
Aliquots (300 µg) of cell lysate were incubated with the
Ac-DEVD-(4-methyl-coumaryl-7-amide) fluorogenic substrate for the
indicated time periods (min), and AMC release was measured by
spectrofluorometry. AMC concentration was determined from a standard
curve.
Coimmunoprecipitation Assay-- 293 cells were cotransfected by calcium phosphate precipitation with expression vectors encoding 14.7F (10 µg), HA-tagged FLICE (5 µg) or control vectors along with a CrmA expression vector (5 µg) to ensure that apoptosis induced by the overexpression of FLICE was efficiently blocked. 12-16 h after transfection, cells were labeled with 200 µCi/ml Promix 35S label (Amersham Corp.) for 2-4 h at 37 °C. Labeled cells were washed and lysed in 1 ml of ice-cold Nonidet P-40 lysis buffer (20 mM Tris (pH 8), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40) containing 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin. Immunoprecipitated products were analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography.
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RESULTS |
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14.7K Blocks Apoptosis and DEVD-specific Caspase Activity Induced by FasL-- To determine whether 14.7K blocks apoptosis induced by Fas oligomerization, we used an adenovirus vector that expresses FasL to induce apoptosis in 293 cells. FasL expression was detectable at 3 h post-infection and increased significantly at 4 h post-infection with AdFasL/G but not with a control virus (Fig. 1A). Cell surface-associated FasL was rapidly proteolytically cleaved, liberating soluble FasL and leaving behind the intracellular domain anchored to the membrane. The time course of FasL expression correlated well with apoptosis induction as evidenced by poly(ADP-ribose) polymerase cleavage (Fig. 1B), morphology, and the induction of DEVD-specific caspase activity (data not shown). Elevation in YVAD-specific caspase activity was not observed between 1 and 5 h post-infection (data not shown). Coinfection with virus that expressed either 14.7K or CrmA efficiently blocked Fas ligand-triggered apoptosis (Fig. 1C) and prevented the induction of DEVD-specific caspase activity (Fig. 1D). These results suggest that 14.7K functions to impede FasL-induced apoptosis by blocking the activation or the activity of one or several caspases.
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14.7K Blocks Cell Death Induced by the Overexpression of FasL, FADD, or FLICE-- To confirm the results obtained with the adenovirus constructs in a virus-free system and to further explore the mechanism of 14.7K activity, the ability of 14.7K to block apoptosis induced by overexpressing FasL, FADD, or FLICE was assessed. 293 cells were transiently transfected with plasmids that express GFP and either FasL, FADD, or FLICE together with either 14.7K or CrmA. In this assay, GFP was used to tag transfected cells. Like CrmA, 14.7K expression efficiently protected cells from apoptosis induced by overexpression of each of these death inducing proteins (Fig. 2). Inhibition of cell death by the 14.7K product was not cell type-specific, as we observed similar effects in A549 cells (Fig. 2D). This analysis indicated that 14.7K interferes with FasL-induced apoptosis at the level of or downstream of FLICE and that 14.7K function does not require any additional adenovirus products. Since 14.7K blocked the generation of DEVD-specific caspase activity induced by FasL expression (Fig. 1D) and FLICE can directly activate DEVD-specific caspases (13, 24, 25), these results suggest that FLICE or DEVD-specific caspases such as YAMA/CPP32/caspase-3 may be cellular targets for 14.7K.
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14.7K Interacts with FLICE-- To directly test whether 14.7K interacts with FLICE in vivo, 293 cells were transiently transfected with plasmids that direct the synthesis of Flag epitope-tagged 14.7K (14.7F) and HA epitope-tagged FLICE. The 14.7K- or Flag-specific antibodies coprecipitated a 55-kDa protein that corresponds to FLICE and a 26-kDa protein from 35S-labeled cell lysates (Fig. 3). Control antibodies did not coprecipitate FLICE or the 26-kDa protein nor were they efficiently coprecipitated from lysates that contained FLICE but not 14.7F (Fig. 3). It is likely that the 26-kDa product corresponds to the N-terminal-processed DED of FLICE, since the molecular mass of this proteolytic fragment was reported to be 26 kDa (12).
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DISCUSSION |
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FLICE is the first caspase to be activated during apoptosis induced by Fas and TNFR1 oligomerization. FLICE becomes processed and activated within the death receptor signaling complex, possibly by an autocatalytic mechanism involving FLICE oligomerization (12, 13). After activation, FLICE is thought to trigger a caspase cascade that involves the activation of downstream DEVD-specific caspases (13, 25, 27).
Interruption of the caspase cascade at the level of the apex protease appears to be an effective point of intervention for DNA viruses. Recently, Muzio et al. (13) demonstrated that CrmA formed a complex with and inhibited the activity of FLICE. In addition, several anti-apoptotic DED-containing viral products, termed vFLIPs, have recently been identified by data base searches (5-7). These vFLIPs include the molluscum contagiosum virus proteins MC159 and MC160 and the equine herpes virus 2 protein E8. MC159 was shown to bind to FADD and inhibit apoptosis induced by TNFR1 and Fas (5-7). E8 interacted with FLICE (5, 7) and inhibited apoptosis induced by the overexpression of Fas, TRADD and FADD but not by FLICE (5). On the other hand, 14.7K lacks a discernible DED, interacted with FLICE, and attenuated FLICE-induced apoptosis. This suggests that the mechanism of action of 14.7K may be different from that of the vFLIPs.
Since FLICE is a shared effector caspase for both TNFR1 and Fas, it is likely that 14.7K interferes with TNF-induced apoptosis through its interaction with FLICE and through its inhibition of DEVD-specific caspase activity. However, previous studies have suggested that 14.7K may inhibit TNF-induced apoptosis by blocking the activation or activity of cPLA2 (18-20). The role of 14.7K in apoptosis appears to be further clarified by the recent finding that TNF-induced apoptosis and arachidonic acid release can occur by a mechanism involving the activation of cPLA2 by caspase cleavage (28). This finding suggests that attenuation of DEVD-specific caspase activation by 14.7K may prevent the subsequent activation of cPLA2 and thus inhibit arachidonic acid release and apoptosis.
The ability of 14.7K to protect cells from TNF and FasL-induced apoptosis may be involved in the establishment and maintenance of latent or persistent adenovirus infections in lymphocytes. During lytic infection, the protective effect of the 14.7K protein might be essential for optimal viral replication. Considering the lack of persistence of adenoviruses used for gene therapy applications (26) and the functional exclusion of 14.7K from these vectors, the expression of 14.7K in future adenovirus constructs may be beneficial in a clinical setting.
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ACKNOWLEDGEMENTS |
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We thank Doug Brough, Damodar Reddy, Lou Cantolupo, Duncan McVey, Chris Keller, Alena Lizonova, Angela Appiah, Faith Beams, and Lu Qin for their support with virus production and cell culture, Doug Brough and Tom Wickham for comments on the manuscript, and Rena Cohen for preparation of the manuscript. We also thank Vishva M. Dixit and David Pickup for providing expression vectors and cDNAs.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: GenVec, Inc., 12111 Parklawn Dr., Rockville, MD 20852. Tel.: 301-816-5541; Fax: 301-816-0440; E-mail: bruder{at}genvec.com.
1
The abbreviations used are: TNF, tumor necrosis
factor; TNFR1, TNF receptor 1; Ad5, adenovirus type 5; AMC,
Ac-DEVD-amino-4-methylcoumarin; caspase, cysteine aspartic
acid-specific protease; CMV, cytomegalovirus; cPLA2,
cytosolic phospholipase A2; DED, death effector domain; FADD, Fas-associated death domain-containing protein; FasL, Fas ligand;
FLICE, FADD-like interleukin-1-converting enzyme; GFP, green
fluorescent protein; HA, hemagglutinin; vFLIPs, viral FLICE inhibitory
proteins.
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REFERENCES |
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