Molinier-Frenkel Valérie, Prévost-Blondel Armelle, Hong Saw-See, Lengagne Renée, Boudaly Sarah, Magnusson Maria K, Boulanger Pierre, Guillet Jean-Gérard
Département d'Immunologie, Institut Cochin, INSERM U567, CNRS UMR 8104, Laboratoire membre de l'Institut Fédératif de Recherche 116, Université R. Descartes, 27 rue du Faubourg Saint Jacques, 75014 Paris, France.
J Biol Chem. 2003 Sep 26;278(39):37175-82. doi: 10.1074/jbc.M303496200. Epub 2003 Jul 10.
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 degrees, AdGFP); (ii) Ad5 vector mutant deleted of the fiber C-terminal knob domain (AdGFPDeltaknob); 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 AdGFPDeltaknob and for fiber protein deletion mutants lacking the beta-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 (FiDeltaHI), 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.
我们利用以下材料研究了5型腺病毒（Ad5）介导的骨髓来源的小鼠树突状细胞（DC）成熟的机制：（i）具有野生型衣壳的Ad5载体（AdE1°，AdGFP）；（ii）缺失纤维C末端球状结构域的Ad5载体突变体（AdGFPΔknob）；以及（iii）从Ad5感染细胞中分离或作为重组蛋白表达的衣壳成分，六邻体、五邻体、五邻体基座、全长纤维、纤维球状结构域和纤维突变体。我们发现，五邻体壳粒（与纤维突起相连的五邻体基座）、全长纤维蛋白及其分离的球状结构域均能够诱导DC成熟，而单独的六邻体或五邻体基座未观察到明显的DC成熟。对于AdGFPΔknob以及缺乏球状结构域β链区域F（亮氨酸-485-苏氨酸-486残基）的纤维蛋白缺失突变体，这种能力严重降低。在缺失HI环（FiΔHI）的重组纤维蛋白中完全保留了DC成熟效应，FiΔHI是一种无法三聚化的纤维（Fi）缺失突变体，这表明纤维球状结构域介导的DC激活不依赖于HI环的完整性和纤维的三聚体状态。有趣的是，用Ad5球状结构域蛋白刺激过的肽脉冲DC在体内诱导了强烈的CD8+T细胞应答。
