Imelli Nicola, Meier Oliver, Boucke Karin, Hemmi Silvio, Greber Urs F
Zoologisches Institut. Institut für Molekularbiologie, Universität Zürich, CH-8057 Zürich, Switzerland.
J Virol. 2004 Mar;78(6):3089-98. doi: 10.1128/jvi.78.6.3089-3098.2004.
The species C adenovirus type 2 (Ad2) and Ad5 bind the coxsackievirus B Ad receptor and alphav integrin coreceptors and enter epithelial cells by clathrin-mediated endocytosis. This pathway is rapid and efficient. It leads to cell activation and the cholesterol-dependent formation of macropinosomes. Macropinosomes are triggered to release their contents when incoming Ad2 escapes from endosomes. Here, we show that cholesterol extraction of epithelial cells by methyl-beta-cyclodextrin (mbetaCD) treatment reduced Ad5-mediated luciferase expression approximately 4-fold. The addition of cholesterol to normal cells increased gene expression in a dose-dependent manner up to threefold, but it did not restore gene expression in mbetaCD-treated cells. mbetaCD had no effect in the presence of excess cholesterol, indicating that the inhibition of gene expression was due specifically to cholesterol depletion. Cholesterol depletion inhibited rapid Ad2 endocytosis, endosomal escape, and nuclear targeting, consistent with the notion that clathrin-dependent endocytosis of Ad2 is cholesterol dependent. In cholesterol-reduced cells, Ad2 internalized at a low rate, suggestive of an alternative, clathrin-independent, low-capacity entry pathway. While exogenous cholesterol completely restored rapid Ad2 endocytosis, macropinocytosis, and macropinosome disruption, it did not, surprisingly, restore viral escape from endosomes. Our results indicate that macropinosome disruption and endosomal escape of Ad2 are independent events in cells depleted of and then refilled with cholesterol, suggesting that viral escape from endosomes requires lipid-controlled membrane homeostasis, trafficking, or signaling.
2型腺病毒C种（Ad2）和5型腺病毒（Ad5）与柯萨奇病毒B腺病毒受体和αv整合素共受体结合，并通过网格蛋白介导的内吞作用进入上皮细胞。该途径快速且高效。它导致细胞活化以及依赖胆固醇的巨胞饮体形成。当进入的Ad2从内体逃逸时，巨胞饮体被触发释放其内容物。在此，我们表明，用甲基-β-环糊精（mbetaCD）处理上皮细胞以提取胆固醇，可使Ad5介导的荧光素酶表达降低约4倍。向正常细胞中添加胆固醇可使基因表达以剂量依赖的方式增加至三倍，但它并未恢复mbetaCD处理细胞中的基因表达。在存在过量胆固醇的情况下，mbetaCD没有作用，这表明基因表达的抑制是由于胆固醇的特异性消耗。胆固醇消耗抑制了Ad2的快速内吞作用、内体逃逸和核靶向，这与Ad2依赖网格蛋白的内吞作用依赖胆固醇的观点一致。在胆固醇减少的细胞中，Ad2以低速率内化，提示存在另一种不依赖网格蛋白的低容量进入途径。虽然外源性胆固醇完全恢复了Ad2的快速内吞作用、巨胞饮作用和巨胞饮体破坏，但令人惊讶的是，它并未恢复病毒从内体的逃逸。我们的结果表明，在胆固醇耗尽然后重新填充的细胞中，Ad2的巨胞饮体破坏和内体逃逸是独立事件，这表明病毒从内体的逃逸需要脂质控制的膜稳态、运输或信号传导。
