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• W2079263000 abstract "Adenovirus encodes multiple gene products that regulate proapoptotic cellular responses to viral infection mediated by both the innate and adaptive immune systems. The E3-10.4K and 14.5K gene products are known to modulate the death receptor Fas. In this study, we demonstrate that an additional viral E3 protein, 6.7K, functions in the specific modulation of the two death receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The 6.7K protein is expressed on the cell surface and forms a complex with the 10.4K and 14.5K proteins, and this complex is sufficient to induce down-modulation of TRAIL receptor-1 and -2 from the cell surface and reverse the sensitivity of infected cells to TRAIL-mediated apoptosis. Down-modulation of TRAIL-R2 by the E3 complex is dependent on the cytoplasmic tail of the receptor, but the death domain alone is not sufficient. These results identify a mechanism for viral modulation of TRAIL receptor-mediated apoptosis and suggest the E3 protein complex has evolved to regulate the signaling of selected cytokine receptors. Adenovirus encodes multiple gene products that regulate proapoptotic cellular responses to viral infection mediated by both the innate and adaptive immune systems. The E3-10.4K and 14.5K gene products are known to modulate the death receptor Fas. In this study, we demonstrate that an additional viral E3 protein, 6.7K, functions in the specific modulation of the two death receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The 6.7K protein is expressed on the cell surface and forms a complex with the 10.4K and 14.5K proteins, and this complex is sufficient to induce down-modulation of TRAIL receptor-1 and -2 from the cell surface and reverse the sensitivity of infected cells to TRAIL-mediated apoptosis. Down-modulation of TRAIL-R2 by the E3 complex is dependent on the cytoplasmic tail of the receptor, but the death domain alone is not sufficient. These results identify a mechanism for viral modulation of TRAIL receptor-mediated apoptosis and suggest the E3 protein complex has evolved to regulate the signaling of selected cytokine receptors. epidermal growth factor receptor adenovirus type 2 arabinofuranosylcytosine cytotoxic T lymphocytes Fas-associated death domain-containing protein glycosylphosphatidylinositol lysosome-associated membrane protein-1 nuclear factor-κB natural killer cells phosphate-buffered saline polymerase chain reaction small airway epithelial cells TNF-related apoptosis-inducing ligand TRAIL receptor tumor necrosis factor TNF receptor vesicular stomatitis virus 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide multiplicity of infection fluorescence-activated cell sorter death domain 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. 1Mahr J.A. Gooding L.R. Immunol. Rev. 1999; 1688: 121-130Crossref Scopus (121) Google Scholar and 2Shisler J.L. Gooding L.R. Trends Microbiol. 1998; 6: 337-339Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). 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 (3Sparer T.E. Tripp R.A. Dillehay D.L. Hermiston T.W. Wold W.S. Gooding L.R. J. Virol. 1996; 70: 2431-2439Crossref PubMed Google Scholar). 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 (4Stewart A.R. Tollefson A.E. Krajcsi P. Yei S.P. Wold W.S. J. Virol. 1995; 69: 172-181Crossref PubMed Google Scholar, 5Tollefson A.E. Stewart A.R. Yei S.P. Saha S.K. Wold W.S. J. Virol. 1991; 65: 3095-3105Crossref PubMed Google Scholar, 6Gooding L. Ranheim T. Tollefson A. Aquino L. Duerksen-Hughes P. Horton T. Wold W.S. J. Virol. 1991; 65: 4114-4123Crossref PubMed Google Scholar). 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 (5Tollefson A.E. Stewart A.R. Yei S.P. Saha S.K. Wold W.S. J. Virol. 1991; 65: 3095-3105Crossref PubMed Google Scholar, 7Carlin C.R. Tollefson A.E. Brady H.A. Hoffman B.L. Wold W.S. Cell. 1989; 57: 135-144Abstract Full Text PDF PubMed Scopus (104) Google Scholar). More recently, the E3-10.4K/14.5K complex has been shown to mediate the down-regulation of cell surface Fas (8Shisler J. Yang C. Walter B. Ware C. Gooding L. J. Virol. 1997; 71: 8299-8306Crossref PubMed Google Scholar, 9Elsing A. Burgert H.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10072-10077Crossref PubMed Scopus (93) Google Scholar, 10Tollefson A.E. Hermiston T.W. Lichtenstein D.L. Colle C.F. Tripp R.A. Dimitrov T. Toth K. Wells C.E. Doherty P.C. Wold W.S. Nature. 1998; 392: 726-730Crossref PubMed Scopus (177) Google Scholar), a proapoptotic member of the tumor necrosis factor receptor (TNFR) superfamily. Other members of the TNFR family, such as the LTβR 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. 11Ashkenazi A. Dixit V.M. Curr. Opin. Cell Biol. 1999; 11: 255-260Crossref PubMed Scopus (1151) Google Scholar and 12Griffith T.S. Lynch D.H. Curr. Opin. Immunol. 1998; 10: 559-563Crossref PubMed Scopus (441) Google Scholar). 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 (13Schneider P. Thome M. Burns K. Bodmer J.L. Hofmann K. Kataoka T. Holler N. Tschopp J. Immunity. 1997; 7: 831-836Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar, 14Pan G. O'Rourke K. Chinnaiyan A.M. Gentz R. Ebner R. Ni J. Dixit V.M. Science. 1997; 276: 111-113Crossref PubMed Scopus (1557) Google Scholar, 15Sheridan J.P. Marsters S.A. Pitti R.M. Gurney A. Skubatch M. Baldwin D. Ramakrishnan L. Gray C.L. Baker K. Wood W.I. Goddard A.D. Godowski P. Ashkenazi A. Science. 1997; 277: 818-821Crossref PubMed Scopus (1528) Google Scholar). 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 (15Sheridan J.P. Marsters S.A. Pitti R.M. Gurney A. Skubatch M. Baldwin D. Ramakrishnan L. Gray C.L. Baker K. Wood W.I. Goddard A.D. Godowski P. Ashkenazi A. Science. 1997; 277: 818-821Crossref PubMed Scopus (1528) Google Scholar, 16Schneider P. Bomer J.L. Thome M. Hofmann K. Holler N. Tschopp J. FEBS Lett. 1997; 416: 329-334Crossref PubMed Scopus (248) Google Scholar, 17Pan G. Ni J. Wei Y.F., Yu, G. Gentz R. Dixit V.M. Science. 1997; 277: 815-818Crossref PubMed Scopus (1377) Google Scholar), whereas ligation of TRAIL-R4, which is unable to recruit FADD, can initiate anti-apoptotic signals through the activation of the transcription factor NF-κB (18Degli-Esposti M.A. Dougall W.C. Smolak P.J. Waugh J.Y. Smith C.A. Goodwin R.G. Immunity. 1997; 7: 813-820Abstract Full Text Full Text PDF PubMed Scopus (744) Google Scholar). However, TRAIL-R3 and TRAIL-R4 expression correlates poorly with the sensitivity of tumor cell lines to TRAIL-mediated death (19Griffith T.S. Rauch C.T. Smolak P.J. Waugh J.Y. Boiani N. Lynch D.H. Smith C.A. Goddwin R.G. Kubin M.Z. J. Immunol. 1999; 162: 2597-2605PubMed Google Scholar). These findings, and the fact that TRAIL-R1 and TRAIL-R2 can activate NF-κB in addition to inducing apoptosis (16Schneider P. Bomer J.L. Thome M. Hofmann K. Holler N. Tschopp J. FEBS Lett. 1997; 416: 329-334Crossref PubMed Scopus (248) Google Scholar, 20Chaudhary P.M. Eby M. Jasmin A. Bookwalter A. Murray J. Hood L. Immunity. 1997; 7: 821-830Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar), 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 (21Johnsen A.C. Haux J. Steinkjer B. Nonstad U. Egeberg K. Sundan A. Ashkenazi A. Espevik T. Cytokine. 1999; 9: 664-672Crossref Scopus (74) Google Scholar, 22Kayagaki N. Yamaguchi N. Nakayama M. Kawasaki A. Akiba H. Okumura K. Yagita H. J. Immunol. 1999; 162 (2347): 2639PubMed Google Scholar, 23Thomas W.D. Hersey P. J. Immunol. 1998; 161: 2195-2200PubMed Google Scholar), 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 (24Wilson-Rawls J. Saha S.K. Krajesi P. Tollefson A.E. Gooding L.R. Wold W.S. Virology. 1990; 178: 204-212Crossref PubMed Scopus (26) Google Scholar,25Wilson-Rawls J. Wold W.S. Virology. 1993; 195: 6-15Crossref PubMed Scopus (25) Google Scholar) 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. HT29.14S cells are a human colon adenocarcinoma line that are sensitive to the death inducing activities of TNF-related ligands (26Browning J.L. Miatkowski K. Sizing I. Griffiths D.A. Zafari M. Benjamin C.D. Meier W. Mackay F. J. Exp. Med. 1996; 183: 867-878Crossref PubMed Scopus (136) Google Scholar). 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 mmglutamine and 10% fetal calf serum (HyClone, Logan, UT). All viruses used in these experiments have been described in detail previously (27Wold W.S. Deutscher S.I. Takemori N. Bhat B.M. Virology. 1986; 148: 168-180Crossref PubMed Scopus (41) Google Scholar, 28Brady H.A. Wold W.S. Nucleic Acids Res. 1987; 15 (2416): 9397Crossref PubMed Scopus (19) Google Scholar) 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 (Eco RI-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. The pBluescriptII-Ad2E3 region plasmid (Ad2-E3) has been described previously (9Elsing A. Burgert H.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10072-10077Crossref PubMed Scopus (93) Google Scholar) 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 Sal I and ligating the fragment into pcDNA3.1(−) (Invitrogen, Carlsbad, CA) cut with Xho I. 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 Bam HI and Eco RI, 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 Eco RI 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 withEco RI 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 Eco RI-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 (16Schneider P. Bomer J.L. Thome M. Hofmann K. Holler N. Tschopp J. FEBS Lett. 1997; 416: 329-334Crossref PubMed Scopus (248) Google Scholar) with the addition of a flanking 5′ Bam HI-FLAG sequence and a 3′ Pst I 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-R2ΔC16 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 Bam HI-Pst I fragment in PS089. The TRAIL-R2ΔDD deletion mutant (ΔLeu348–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-R2ΔDD, which was subcloned as a Bam HI-Pst I 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 aSal I-Not I fragment in replacement of the human IgG1 Fc cassette of a TRAIL-R2:Fc construct (described in Ref. 16Schneider P. Bomer J.L. Thome M. Hofmann K. Holler N. Tschopp J. FEBS Lett. 1997; 416: 329-334Crossref PubMed Scopus (248) Google Scholar). 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 Bam HI and Hin dIII 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 Eco RI and ligated into pBABE-puro (29Morgensstern J.P. Land H. Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1898) Google Scholar) to generate pBABE-6.7K. pBABE-FLAG-6.7K was generated by digesting FLAG-6.7K with Sma I and Eco RV to excise the N-terminal FLAG-tagged 6.7K gene, and this fragment was ligated into Sna BI-digested pBABE-puro. The sequences of all constructs were verified unambiguously using an ABI Prism 310 genetic analyzer automatic sequencer (PerkinElmer Life Sciences). Cell viability was determined using an MTT-based assay as described previously (30Rooney I.A. Butrovich K.D. Glass A.A. Borboroglu S. Benedict C.A. Whitbeck J.C. Cohen G.H. Eisenberg R.J. Ware C.F. J. Biol. Chem. 2000; 275: 14307-14315Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) 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-γ, and 20 μg/ml cytosine 1-β-d-arabinofuranosylcytosine (Ara-C) (Sigma) were added (13Schneider P. Thome M. Burns K. Bodmer J.L. Hofmann K. Kataoka T. Holler N. Tschopp J. Immunity. 1997; 7: 831-836Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar). 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. Retroviral vectors were generated as described previously (31Benedict C.A. Tun R.Y.M. Rubinstein D.B. Guillaume T. Cannon P.M. Anderson W.F. Hum. Gene Ther. 1999; 10: 545-557Crossref PubMed Scopus (58) Google Scholar) based on the method of Soneoka et al. (32Soneoka Y. Cannon P.M. Ramsdale E.E. Griffiths J.C. Romano G. Kingsman S.M. Kingsman A.J. Nucleic Acids Res. 1995; 23: 628-633Crossref PubMed Scopus (610) Google Scholar) 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 β-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. 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 CaPO4precipitation method as described previously (33Benedict C. Butrovich K. Lurain N. Corbeil J. Rooney I. Schneider P. Tschopp J. Ware C. J. Immunol. 1999; 126: 6967-6970Google Scholar). 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.Figure 6TRAIL-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7E3 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 thegray 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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. 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 mmNaCl, 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. HT29.14S cells, which have been shown previously to down-regulate cell surface Fas and EGF-R upon infection with adenovirus (8Shisler J. Yang C. Walter B. Ware C. Gooding L. J. Virol. 1997; 71: 8299-8306Crossref PubMed Google Scholar), 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. 1 a). 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 (34Cladras C. Bhat B.M. Wold W.S. Virology. 1985; 140: 44-54Crossref PubMed Scopus (32) Google Scholar)) was similar to the effect of wild-type virus, indicating the E3-14.7K protein is not required for TRAIL receptor down-regulation. 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. 1 b). 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 undetect" @default.
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• W2079263000 date "2001-02-01" @default.
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• W2079263000 title "Three Adenovirus E3 Proteins Cooperate to Evade Apoptosis by Tumor Necrosis Factor-related Apoptosis-inducing Ligand Receptor-1 and -2" @default.
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