The Maturation of Murine Dendritic Cells Induced by Human Adenovirus Is Mediated by the Fiber Knob Domain
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m303496200
ISSN1083-351X
AutoresValérie Molinier‐Frenkel, Armelle Prévost‐Blondel, Saw See Hong, Renée Lengagne, Sarah Boudaly, Maria K. Magnusson, Pierre Boulanger, Jean-Gérard Guillet,
Tópico(s)Immunotherapy and Immune Responses
ResumoWe investigated the mechanism of adenovirus serotype 5 (Ad5)-mediated maturation of bone marrow-derived murine dendritic cells (DC) using (i) Ad5 vectors with wild-type capsid (AdE1°, AdGFP); (ii) Ad5 vector mutant deleted of the fiber C-terminal knob domain (AdGFPΔknob); and (iii) capsid components isolated from Ad5-infected cells or expressed as recombinant proteins, hexon, penton, penton base, full-length fiber, fiber knob, and fiber mutants. We found that penton capsomer (penton base linked to its fiber projection), full-length fiber protein, and its isolated knob domain were all capable of inducing DC maturation, whereas no significant DC maturation was observed for hexon or penton base alone. This capacity was severely reduced for AdGFPΔknob and for fiber protein deletion mutants lacking the β-stranded region F of the knob (residues Leu-485–Thr-486). The DC maturation effect was fully retained in a recombinant fiber protein deleted of the HI loop (FiΔHI), a fiber (Fi) deletion mutant that failed to trimerize, suggesting that the fiber knob-mediated DC activation did not depend on the integrity of the HI loop and on the trimeric status of the fiber. Interestingly, peptide-pulsed DC that had been stimulated with Ad5 knob protein induced a potent CD8+ T cell response in vivo. We investigated the mechanism of adenovirus serotype 5 (Ad5)-mediated maturation of bone marrow-derived murine dendritic cells (DC) using (i) Ad5 vectors with wild-type capsid (AdE1°, AdGFP); (ii) Ad5 vector mutant deleted of the fiber C-terminal knob domain (AdGFPΔknob); and (iii) capsid components isolated from Ad5-infected cells or expressed as recombinant proteins, hexon, penton, penton base, full-length fiber, fiber knob, and fiber mutants. We found that penton capsomer (penton base linked to its fiber projection), full-length fiber protein, and its isolated knob domain were all capable of inducing DC maturation, whereas no significant DC maturation was observed for hexon or penton base alone. This capacity was severely reduced for AdGFPΔknob and for fiber protein deletion mutants lacking the β-stranded region F of the knob (residues Leu-485–Thr-486). The DC maturation effect was fully retained in a recombinant fiber protein deleted of the HI loop (FiΔHI), a fiber (Fi) deletion mutant that failed to trimerize, suggesting that the fiber knob-mediated DC activation did not depend on the integrity of the HI loop and on the trimeric status of the fiber. Interestingly, peptide-pulsed DC that had been stimulated with Ad5 knob protein induced a potent CD8+ T cell response in vivo. Adenovirus (Ad) 1The abbreviations used are: Ad, adenovirus; Hx, hexon; Pn, penton; Fi, fiber; Pb, penton base; DC, dendritic cell(s); LPS, lipopolysaccharide; MLR, mixed lymphocytic reaction; TLR, toll-like receptor(s); CAR, Coxsackievirus adenovirus receptor; WT, wild-type; GFP, green fluorescent protein; TNF, tumor necrosis factor; IFN, interferon; CFSE, 5-6-carboxyfluorescein diacetate succinimidyl ester; MAPK, mitogen-activated protein kinase; NS, non-stimulated; MS, mock-stimulated; Ab, antibody; mAb, monoclonal antibody; m.o.i., multiplicity of infection(s); ELISPOT, enzyme-linked immunoSPOT; FACS, fluorescence-activated cell sorter; MFI, mean fluorescence intensity; Δ, deletion.1The abbreviations used are: Ad, adenovirus; Hx, hexon; Pn, penton; Fi, fiber; Pb, penton base; DC, dendritic cell(s); LPS, lipopolysaccharide; MLR, mixed lymphocytic reaction; TLR, toll-like receptor(s); CAR, Coxsackievirus adenovirus receptor; WT, wild-type; GFP, green fluorescent protein; TNF, tumor necrosis factor; IFN, interferon; CFSE, 5-6-carboxyfluorescein diacetate succinimidyl ester; MAPK, mitogen-activated protein kinase; NS, non-stimulated; MS, mock-stimulated; Ab, antibody; mAb, monoclonal antibody; m.o.i., multiplicity of infection(s); ELISPOT, enzyme-linked immunoSPOT; FACS, fluorescence-activated cell sorter; MFI, mean fluorescence intensity; Δ, deletion. vectors induce a vigorous stimulation of the innate immune system very rapidly after their administration followed by the induction of the cognate immune response mediated by T and B lymphocytes. 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The capsid proteins of the input virus are not only targets for the humoral and T helper responses (5Gahéry-Ségard H. Molinier-Frenkel V. Le Boulaire C. Saulnier P. Opolon P. Lengagne R. Gautier E. Le Cesne A. Zitvogel L. Venet A. Schatz C. Courtney M. Le Chevalier T. Tursz T. Guillet J. Farace F. J. Clin. Invest. 1997; 100: 2218-2226Crossref PubMed Scopus (115) Google Scholar, 6Gahery-Ségard H. Farace F. Godfrin D. Gaston J. Lengagne R. Turz T. Boulanger P. Guillet J. J. Virol. 1998; 72: 2388-2397Crossref PubMed Google Scholar, 7Zabner J. Ramsey B.W. Meeker D.P. Aitken M.L. Balfour R. Gilson R.L. Launspach J. Moscicki R.A. Richards S.M. Standaert T.A. Williams-Warren J. Wadsworth S.C. Smith A. Welsh M.J. J. Clin. Invest. 1996; 97: 1504-1511Crossref PubMed Scopus (240) Google Scholar), but they are also recognized by cytotoxic CD8+ T lymphocytes (8Roth M.D. Cheng Q. Harui A. Basak S.K. Mitani K. Low T.A. Kiertscher S.M. J. 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Gene Ther. 2000; 7: 1112-1120Crossref PubMed Scopus (77) Google Scholar, 16Morelli A.E. Larregina A.T. Ganster R.W. Zahorchak A.F. Plowey J.M. Takayama T. Logar A.J. Robbins P.D. Falo L.D. Thomson A.W. J. Virol. 2000; 74: 9617-9628Crossref PubMed Scopus (206) Google Scholar, 17Réa D. Schagen F.H. Hoeben R.C. Mehtali M. Havenga M.J. Toes R.E. Melief C.J. Offringa R. J. Virol. 1999; 73: 10245-10253Crossref PubMed Google Scholar, 18Rouard H. Leon A. Klonjkowski B. Marquet J. Tenneze L. Plonquet A. Agrawal S.G. Abastado J.P. Eloit M. Farcet J.P. Delfau-Larue M.H. J. Immunol. Methods. 2000; 241: 69-81Crossref PubMed Scopus (68) Google Scholar). We and others have shown that such activated DC are fully competent for initiating protective immune responses (19Mercier S. Gahery-Segard H. Monteil M. Lengagne R. Guillet J.G. Eloit M. Denesvre C. J. Virol. 2002; 76: 2899-2911Crossref PubMed Scopus (36) Google Scholar, 20Ribas A. Butterfield L.H. McBride W.H. Jilani S.M. Bui L.A. Vollmer C.M. 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Virol. 2000; 74: 9617-9628Crossref PubMed Scopus (206) Google Scholar). The purpose of the present study was to identify the viral component(s) of the Ad5 capsid and structural domains thereof, which were responsible for the activation of bone marrow-derived murine DC. By using isolated Ad5 capsid proteins and virions deleted of the fiber terminal domain (knobless Ad5), we provide evidence that the fiber knob was the major viral factor of DC maturation. Experimental data obtained with deletion mutants of recombinant fiber protein mapped this function to region F, one of the 10 β-stranded regions defined by crystallographic analysis of the knob. Ad Virions and Capsid Proteins—AdE1° (kindly provided by Transgene SA, Strasbourg, France) was a replication-defective serotype 5 adenovirus (Ad5) deleted of both early regions E1 and E3 and carrying wild-type (WT) fibers (10Molinier-Frenkel V. Lengagne R. Gaden F. Hong S.S. Choppin J. Gahery-Segard H. Boulanger P. Guillet J.G. J. Virol. 2002; 76: 127-135Crossref PubMed Scopus (142) Google Scholar). AdGFP (AdenoCMV5-GFP, obtained from Quantum Biotechnologies, Inc., Montreal, Canada) was an Ad5 vector carrying WT fibers and encoding the reporter gene for the enhanced green fluorescent protein (GFP). Fiber deletion mutant AdGFPΔknob was derived from the AdGFP vector by insertion of an opale-stop codon in the fiber gene, downstream of an extrinsic trimerization signal introduced into the fiber shaft. This trimerization signal consisted of the neck region peptide from the human lung surfactant protein D, and the fiber construction has been described in detail elsewhere (22Magnusson M.K. Hong S.S. Boulanger P. Lindholm L. J. Virol. 2001; 75: 7280-7289Crossref PubMed Scopus (102) Google Scholar, 23Hong S.S. Magnusson M.K. Henning P. Lindholm L. Boulanger P. Mol. Ther. 2003; 7: 692-699Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The resulting fiber lacked its entire knob domain ("knobless") and contained only the tail domain and the N-terminal seven repeats of the shaft, i.e. residues 1–157. The schematic structure of the fibers of AdE1°, AdGFP, and AdGFPΔknob is depicted in Fig. 1, a and b. AdE1°, AdGFP, and AdGFPΔknob virions were isolated by isopycnic ultracentrifugation in continuous CsCl gradient (10Molinier-Frenkel V. Lengagne R. Gaden F. Hong S.S. Choppin J. Gahery-Segard H. Boulanger P. Guillet J.G. J. Virol. 2002; 76: 127-135Crossref PubMed Scopus (142) Google Scholar). Capsid proteins hexon (abbreviated Hx) and penton capsomers (abbreviated Pn, for penton base + fiber) were isolated from WT Ad5-infected HeLa cells. Recombinant WT Ad5 proteins penton base (Pb), fiber (Fi), and fiber knob domain were isolated from baculovirus-infected Sf9 cells. Four Ad5 fiber mutants carrying deletions of various lengths in the knob domain (24Santis G. Legrand V. Hong S.S. Davison E. Kirby I. Imler J.L. Finberg R.W. Bergelson J.M. Mehtali M. Boulanger P. J. Gen. Virol. 1999; 80: 1519-1527Crossref PubMed Scopus (66) Google Scholar), FiΔ402–481, FiΔHI, FiΔEF, and FiΔ485–486 (Fig. 1c), were produced as recombinant proteins in Sf9 cells. Ad proteins were purified according to a protocol described in earlier studies (10Molinier-Frenkel V. Lengagne R. Gaden F. Hong S.S. Choppin J. Gahery-Segard H. Boulanger P. Guillet J.G. J. Virol. 2002; 76: 127-135Crossref PubMed Scopus (142) Google Scholar, 25Karayan L. Gay B. Gerfaux J. Boulanger P.A. Virology. 1994; 202: 782-795Crossref PubMed Scopus (45) Google Scholar, 26Novelli A. Boulanger P.A. Virology. 1991; 185: 365-376Crossref PubMed Scopus (85) Google Scholar). Briefly, we used a three-step procedure consisting of (i) ammonium sulfate precipitation, (ii) anion exchange high performance liquid chromatography, and (iii) concentration-ultrafiltration through concentrator membranes with a 100-kDa cut-off. Protein samples were analyzed by conventional sodium dodecyl sulfate 12%-polyacrylamide gel electrophoresis (10Molinier-Frenkel V. Lengagne R. Gaden F. Hong S.S. Choppin J. Gahery-Segard H. Boulanger P. Guillet J.G. J. Virol. 2002; 76: 127-135Crossref PubMed Scopus (142) Google Scholar) and immunoblotting (25Karayan L. Gay B. Gerfaux J. Boulanger P.A. Virology. 1994; 202: 782-795Crossref PubMed Scopus (45) Google Scholar). The possible occurrence of endotoxins in Ad virion and protein preparations was assayed using amoebocyte lysate from Limulus polyphemus (E-TOXATE™, Sigma), with Escherichia coli serotype 055:B5 lipopolysaccharide (LPS) as standard. Endotoxin concentration in the different batches of proteins never exceeded 30 pg/ml at the doses used for DC stimulation. At this concentration, Hx protein samples never induced DC maturation above the background of non-stimulated (NS) DC (see Fig. 2). CsCl-purified Ad virion preparations were totally free of detectable endotoxin contamination. Isolation of Dendritic Cells—This method was adapted from Ref. 27Mayordomo J.I. Zorina T. Storkus W.J. Zitvogel L. Celluzzi C. Falo L.D. Melief C.J. Ildstad S.T. Kast W.M. Deleo A.B. Lotze M.T. Nat. Med. 1995; 1: 1297-1302Crossref PubMed Scopus (1061) Google Scholar. Lymphocyte-depleted bone marrows from C57BL/6 (H2b) mice (Harlan, Gannat, France) were cultured overnight in complete medium (RPMI 1640 with 10% fetal calf serum, 2 mml-glutamine, 50 μm β-mercaptoethanol, 100 units/ml penicillin, 100 μg/ml streptomycin). Non-adherent cells were harvested and resuspended in complete medium with 1000 units/ml recombinant granulocyte-macrophage colony-stimulating factor (R&D Systems, Minneapolis, MN) and 100 units/ml rIL4 (R&D Systems). The medium was replaced at day 4. At day 6, aliquots of non-adherent cells were resuspended at 3 × 106/ml in phosphate-buffered saline containing 1% fetal calf serum. Cells were then incubated for 1 h at 37 °C (i) without (non-stimulated DC (NS-DC)), (ii) with Ad5 capsid components, or (iii) with corresponding chromatographic fractions from extracts of Sf9 cells infected with baculoviral empty vector (mock-stimulated DC (MS-DC)), as indicated in the figure legends (expressed as doses/106 cells). After 1-h incubation, the cell concentration was adjusted to 3 × 105/ml with complete medium supplemented with granulocyte-macrophage colony-stimulating factor. Control cells received 1 μg/ml LPS from E. coli (Sigma). At day 8, cells were harvested and analyzed in flow cytometry, mixed lymphocytic reaction, and immunization. Day 8 supernatants were collected. For the experiments described in Fig. 4a, DC were purified by using magnetic beads conjugated to anti-mouse CD11c mAb (Miltenyi Biotec). Flow Cytometry—DC were washed in phosphate-buffered saline with 1% fetal calf serum. After incubation with anti-FcII/IIIR Ab (2.4G2, Pharmingen), cells were incubated with various combinations of the following mAbs (all from Pharmingen): phosphatidylethanolamine-conjugated anti-I-Ab (AF6-120.1) and anti-CD40 (3/23), fluorescein isothiocyanate-conjugated anti-CD11c (HL3) and anti-CD80 (16-10A1), biotinylated anti-H2-Db (28-8-6) and anti-CD86 (GL1) + streptavidin-PerCP (BD Biosciences). Anti-fiber-tail mouse mAb (4D2.5 (28Hong S.S. Karayan L. Tournier J. Curiel D.T. Boulanger P.A. EMBO J. 1997; 16: 2294-2306Crossref PubMed Scopus (303) Google Scholar)) was obtained from Jeff Engler (University of Alabama at Birmingham, Birmingham, AL). For Coxsackievirus-adenovirus receptor (CAR) staining, day 6 DC, Chinese hamster ovary and HeLa cells (obtained from ATCC) were labeled with anti-CAR mAb E1.1 (ascites fluid, kindly provided by Dr. Silvio Hemmi, University of Zurich) or a control irrelevant ascites fluid. Counterstaining was performed using biotinylated rat anti-mouse IgG and streptavidin-phosphatidylethanolamine. Flow cytometric analyses were performed on a FACSCalibur (BD Biosciences). In Vitro Functional Assays (MLR, IL12, and TNFα)—For mixed lymphocyte reaction (MLR), day 8 DC were distributed at increasing doses and co-cultured for 4 days with aliquots of allogeneic (BALB/c, H2d) CD4+ purified splenocytes (2 × 105 cells/well). CD4+ T cell proliferation was measured by [3H]thymidine incorporation (1 μCi/well) during the final 18 h of coculture. For IL12 and TNFα assays, day 8 DC supernatants were tested using mouse IL12p70 and TNFα enzyme-linked immunosorbent assay kits (Pharmingen). Peptides—Immunodominant viral peptides, peptide 33–41 from lymphocytic choriomeningitis virus glycoprotein (KAVYNFATM) and peptide 366–374 from influenza virus nucleoprotein (ASNENMETM), abbreviated GP33 and NP366, respectively, were purchased from Neosystem (Strasbourg, France). Both are H2-Db-restricted. Immunizations and CFSE-labeled Splenocyte Rejection Assays— C57BL/6 mice were immunized subcutaneously into the flank with 3 × 104, 8 × 104, or 2.5 × 105 DC loaded with GP33 or NP366 (10–6m). Ten days later, mice received an intravenous injection of 3 × 107 syngeneic splenocytes labeled with the fluorescent dye 5-6-carboxyfluorescein diacetate succinimidyl ester (CFSE, Molecular Probes) as described in Ref. 29Oehen S. Brduscha-Riem K. Oxenius A. Odermatt B. J. Immunol. Methods. 1997; 207: 33-42Crossref PubMed Scopus (57) Google Scholar. The percentage of CFSE+ donor cells among recipient peripheral blood lymphocytes or splenocytes was determined using FACS analysis. Donor cell rejection was calculated using the formula: (% of CFSE+ cells in immunized mice) (% of CFSE+ cells in naive mice) × 100. ELISPOT-IFNγ—Nitrocellulose microplates (Millipore) were coated with rat anti-mouse IFNγ antibody (R4-6A2, Pharmingen) and then washed and saturated with complete medium. Aliquots (in triplicate) of freshly isolated spleen cells (106 cells/well) or peripheral blood lymphocytes (2 × 105 cells/well) were added to the wells in complete medium containing 30 units/ml human rIL2 (Roche Applied Science) and 10–6m GP33 or NP366. After 20 h at 37 °C, the spot-forming cells were revealed and counted (10Molinier-Frenkel V. Lengagne R. Gaden F. Hong S.S. Choppin J. Gahery-Segard H. Boulanger P. Guillet J.G. J. Virol. 2002; 76: 127-135Crossref PubMed Scopus (142) Google Scholar). The values obtained with NP366 were subtracted from the mean values of triplicate tests obtained with GP33. Cytotoxicity Assay—Splenocytes from DC-immunized mice (effector cells) were cultured for 4 days with 10–6m GP33. Effector cells were then assayed on 51Cr-labeled EL4 target cells (10–4 μCi/cell) pulsed with GP33 or NP366 peptide, in duplicate and at different effector-to-target ratios. After 5 h at 37 °C, supernatants were collected, and radioactivity was measured (Top Count, Packard Instruments). Target cells were incubated with medium alone to determine the level of spontaneous release of 51Cr and with 2% alkyl-trimethyl-ammonium bromide (Sigma) to determine the total 51Cr release. Specific 51Cr release was calculated using the formula: (experimental – spontaneous release)/(total release – spontaneous release) × 100. Effect of Isolated Ad5 Capsid Components on DC Phenotype—Immature DC were incubated with isolated Pn or Hx capsomers, purified from Ad5-infected HeLa cells. The major subset of Hx-treated DC showed a poorly mature phenotype, not significantly different from that shown by NS-DC, with a low expression of major histocompatibility complex class II, CD40, CD80 (B7.1), and CD86 (B7.2) molecules (Fig. 2a). In contrast, Pn-treated DC massively expressed a mature phenotype, similar to the phenotype of LPS-stimulated DC, with a high level of expression of the different markers (Fig. 2a). Since the Ad Pn capsomer is composed of two structural entities, Pb and Fi, the following experiments were designed to determine which of these two constitutive proteins was responsible for the DC maturation. DC were then treated with recombinant Pb or Fi protein isolated from insect cell extracts. The results indicated that isolated Fi protein reproduced the entire stimulatory effect of Pn, whereas Pb protein alone showed no detectable effect on DC maturation (Fig. 2b). Role of the Fiber Knob Domain in Fiber-mediated DC Maturation—The C-terminal domain of Ad fiber has been expressed as recombinant knob protein (26Novelli A. Boulanger P.A. Virology. 1991; 185: 365-376Crossref PubMed Scopus (85) Google Scholar, 28Hong S.S. Karayan L. Tournier J. Curiel D.T. Boulanger P.A. EMBO J. 1997; 16: 2294-2306Crossref PubMed Scopus (303) Google Scholar). DC incubated with the fiber knob protein expressed a mature phenotype similar to that observed in DC stimulated by the full-length Fi (Fig. 3a). Consistent with this, the knob-stimulated DC were able to vigorously induce the proliferation of allogeneic CD4+ T cells in MLR experiments, an effect that occurred in a knob-dependent manner (Fig. 3b). At the higher knob protein dose used (0.5 μg), knob-stimulated DC were much more potent stimulators of allogeneic CD4+ T cells than control NS- or Hx-stimulated DC. However, there was an apparent threshold in the knob-mediated DC activation since a detectable effect was only observed at the dose of 0.2 μg of knob protein, i.e. 5 × 106 molecules of fiber knob per DC. Interestingly, the MLR values for 0.2 μg of knob protein were similar to that of 1 μg of Fi, a result consistent with the fact that the quantity of knob protein present in the 0.2-μg knob sample roughly corresponded to the knob protein content of the 1-μg Fi sample (180 amino acid residues for the knob domain versus 581 amino acid residues for the full-length fiber). The knob protein also induced IL12 and TNFα secretion in a dose-dependent manner (Fig. 3c). We then investigated whether the knob was capable of directly targeting DC and induce their maturation, without the participation of any other intermediate cells. To this aim, CD11c+ cells were purified using anti-CD11c magnetic beads and tested in maturation assays. Although NS-DC showed some up-regulation of expression of co-stimulatory molecules, knob-stimulated DC expressed a significantly more mature phenotype than control cells (Fig. 4a). Moreover, recombinant Fi protein was able to bind to immature DC, with 63% fiber-positive cells at day 6 of culture (Fig. 4b). In this assay, cell-bound Fi was detected using anti-fiber tail mAb, implying that the epitope in the tail was accessible and that the fiber attachment to the cell surface had occurred via the knob domain. These data strongly suggested that DC were direct targets for the fiber knob domain. To assess whether the effect of Ad5 virion on DC activation was also directly mediated by the fiber knob domain, comparative experiments were carried out using virions of AdE1°, carrying WT Fi projections, and of AdGFPΔknob, a knobless Ad5 vector (Fig. 1, a and b). Upon incubation with AdE1°, there was an up-regulation of the expression of maturation markers on DC (Fig. 4c), as expected from previous observations (15Hirschowitz E.A. Weaver J.D. Hidalgo G.E. Doherty D.E. Gene Ther. 2000; 7: 1112-1120Crossref PubMed Scopus (77) Google Scholar, 16Morelli A.E. Larregina A.T. Ganster R.W. Zahorchak A.F. Plowey J.M. Takayama T. Logar A.J. Robbins P.D. Falo L.D. Thomson A.W. J. Virol. 2000; 74: 9617-9628Crossref PubMed Scopus (206) Google Scholar). With the same doses of AdGFPΔknob virions, the DC showed a significantly less mature phenotype. These results confirmed that the fiber knob was the common stimulating factor in all the active viral components assessed, Ad5 virion, Pn capsomer, Fi, and knob proteins, and that the knob domain carried most of the determinants responsible for the stimulation of murine DC. Absence of CAR Expression at the Surface of Murine DC and Low Degree of Permissiveness to Ad5 Infection—The cell entry of Ad5 results from the attachment of the fiber knob to a high affinity primary receptor, identified as the CAR in most Adpermissive cells (24Santis G. Legrand V. Hong S.S. Davison E. Kirby I. Imler J.L. Finberg R.W. Bergelson J.M. Mehtali M. Boulanger P. J. Gen. Virol. 1999; 80: 1519-1527Crossref PubMed Scopus (66) Google Scholar, 30Bewley M.C. Springer K. Zhang Y.B. Freimuth P. Flanagan J.M. Science. 1999; 286: 1579-1583Crossref PubMed Scopus (373) Google Scholar, 31Roelvink P.W. Mi Lee G. Einfeld D.A. Kovesdi I. Wickham T.J. Science. 1999; 286: 1568-1571Crossref PubMed Scopus (335) Google Scholar), followed by the binding of the RGD motifs in Pb to αvβ3/5 integrins, which promotes virus endocytosis (32Wickham T.J. Mathias P. Cheresh D.A. Nemerow G.R. Cell. 1993; 73: 309-319Abstract Full Text PDF PubMed Scopus (1947) Google Scholar). It has already been shown that human and murine DC do not express CAR (17Réa D. Schagen F.H. Hoeben R.C. Mehtali M. Havenga M.J. Toes R.E. Melief C.J. Offringa R. J. Virol. 1999; 73: 10245-10253Crossref PubMed Google Scholar, 33Okada N. Tsukada Y. Nakagawa S. Mizuguchi H. Mori K. Saito T. Fujita T. Yamamoto A. Hayakawa T. Mayumi T. Biochem. Biophys. Res. Commun. 2001; 282: 173-179Crossref PubMed Scopus (72) Google Scholar) but do express αvβ3/5 integrins at their surface, and this probably explains their low permissiveness to Ad. We verified by FACS analysis the level of CAR expression at the surface of DC differentiated in our culture conditions. HeLa cells, which are known to be high CAR expressors, were used as positive control, whereas Chinese hamster ovary cells, which do not express CAR, were used as negative control. As expected, no CAR signal above the background level could be detected on immature DC collected before stimulation (Fig. 5a), confirming previous observations. We thus checked whether knob stimulation of DC was associated with effective cell entry of Ad and Ad-mediated gene expression. AdGFP was used at two different m.o.i., 1 × 104 virions/cell, which was sufficient to induce DC maturation (Fig. 4c), and 3 × 104 virions/cell. At 48 h after infection at an m.o.i. of 104, only 12% of DC expressed GFP, and GFP expression slightly increased (19% of DC) at an m.o.i. of 3 × 104 (Fig. 5b), confirming that murine DC were poorly permissive to Ad5. Mapping of the Fiber Knob Region Involved in DC Maturation—Four Ad5 Fi protein mutants carrying deletions in the knob domain (Fig. 1, c and d) were assayed for DC maturation. The large deletion in recombinant FiΔ402–481 overlapped the AB loop, the β-strands B and C, the CD loop, and the N-terminal moiety of the DG loop, whereas FiΔHI mutant lacked only the HI loop. Two other recombinant Fi proteins, FiΔEF, and FiΔ485–486, carried deletions involving the short double β-stranded region EF, and the two residues Leu-485 and Thr-486, which form the β-sheet F (34Kirby I. Davison E. Beavil A.J. Soh C.P. Wickham T.J. Roelvink P.W. Kovesdi I. Sutton B.J. Santis G. J. Virol. 2000; 74: 2804-2813Crossref PubMed Scopus (146) Google Scholar, 35Xia D. Henry L.J. Gerard R.D. Deisenhofer J. Structure. 1994; 2: 1259-1270Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar), respectively. Both FiΔEF and FiΔ485–486 occur as fiber trimers, whereas Δ402–4
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