Dissection of the influenza A virus endocytic routes reveals macropinocytosis as an alternative entry pathway.
Dissection of the influenza A virus endocytic routes reveals macropinocytosis as an alternative entry pathway.
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流感A的解剖病毒内吞途径揭示了大型细胞增多症作为替代入口途径。
DOI：
10.1371/journal.ppat.1001329
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发表时间：
2011-03
期刊：
影响因子：
6.7
通讯作者：
de Haan CA
中科院分区：
文献类型：
--
作者：
de Vries E;Tscherne DM;Wienholts MJ;Cobos-Jiménez V;Scholte F;García-Sastre A;Rottier PJ;de Haan CA
甲型流感病毒（IAV）在其血凝素糖蛋白与唾液酸化的宿主细胞受体结合后进入宿主细胞。尽管动力蛋白依赖性网格蛋白介导的内吞作用（CME）通常被认为是IAV感染途径，但一些观察结果表明存在一种尚未表征的替代进入途径。通过操纵入口参数，我们建立了实验条件，允许单独分析的动力依赖和独立的进入IAV。而进入磷酸盐缓冲液中的IAV可以完全抑制dynasore，发动蛋白的特异性抑制剂，dynasore不敏感的进入途径成为胎牛血清的存在下的功能。使用靶向发动蛋白-2的小干扰RNA证实了这一发现。在血清存在的情况下，两种IAV进入途径都是可操作的。在这些条件下，可以通过与dynasore和阿米洛利衍生物EIPA（巨胞饮的标志性抑制剂）联合治疗完全阻断进入，而单独使用任何一种药物都没有效果。非动力蛋白依赖性进入途径对影响肌动球蛋白动力学的抑制剂或显性负突变体以及对生长因子受体酪氨酸激酶及其下游效应物的一些特异性抑制剂的敏感性都指向巨胞饮参与IAV进入。一致地，IAV颗粒和可溶性FITC-葡聚糖显示出在相同囊泡中的细胞中共定位。因此，除了经典的动力蛋白依赖性，网格蛋白介导的内吞途径，IAV进入宿主细胞的动力蛋白不依赖的途径，具有所有的大胞饮的特征。附着和进入宿主细胞是建立成功的病毒感染的第一个关键步骤，也是决定宿主细胞和物种嗜性的关键因素。甲型流感病毒（IAV）通过其主要表面蛋白血凝素与唾液酸结合而附着于宿主细胞，唾液酸在暴露于细胞表面的糖脂和糖蛋白上无所不在。IAV随后通过使用网格蛋白的受体介导的内吞作用以及通过替代的未表征的途径进入鸟类和多种哺乳动物的细胞。IAV的内吞途径的阐明受到阻碍，因为它们在建立生产性感染时明显冗余。通过操纵的入口条件，我们已经建立了实验设置，允许单独的分析动力蛋白依赖（包括网格蛋白介导的内吞作用）和独立的进入IAV。总的来说，我们的研究结果表明，巨胞饮，细胞的非选择性摄取细胞外液的主要途径，作为一种替代IAV进入途径。由于动力素依赖性和非依赖性IAV进入途径是冗余和独立的，因此它们的单独操纵对于识别和表征替代IAV进入途径至关重要。类似的策略可能适用于研究其他病毒的内吞途径。
Influenza A virus (IAV) enters host cells upon binding of its hemagglutinin glycoprotein to sialylated host cell receptors. Whereas dynamin-dependent, clathrin-mediated endocytosis (CME) is generally considered as the IAV infection pathway, some observations suggest the occurrence of an as yet uncharacterized alternative entry route. By manipulating entry parameters we established experimental conditions that allow the separate analysis of dynamin-dependent and -independent entry of IAV. Whereas entry of IAV in phosphate-buffered saline could be completely inhibited by dynasore, a specific inhibitor of dynamin, a dynasore-insensitive entry pathway became functional in the presence of fetal calf serum. This finding was confirmed with the use of small interfering RNAs targeting dynamin-2. In the presence of serum, both IAV entry pathways were operational. Under these conditions entry could be fully blocked by combined treatment with dynasore and the amiloride derivative EIPA, the hallmark inhibitor of macropinocytosis, whereas either drug alone had no effect. The sensitivity of the dynamin-independent entry pathway to inhibitors or dominant-negative mutants affecting actomyosin dynamics as well as to a number of specific inhibitors of growth factor receptor tyrosine kinases and downstream effectors thereof all point to the involvement of macropinocytosis in IAV entry. Consistently, IAV particles and soluble FITC-dextran were shown to co-localize in cells in the same vesicles. Thus, in addition to the classical dynamin-dependent, clathrin-mediated endocytosis pathway, IAV enters host cells by a dynamin-independent route that has all the characteristics of macropinocytosis. Attachment to and entry into a host cell are the first crucial steps in establishing a successful virus infection and critical factors in determining host cell and species tropism. Influenza A virus (IAV) attaches to host cells by binding of its major surface protein, hemagglutinin, to sialic acids that are omnipresent on the glycolipids and glycoproteins exposed on the surfaces of cells. IAV subsequently enters cells of birds and a wide variety of mammals via receptor-mediated endocytosis using clathrin as well as via (an) alternative uncharacterized route(s). The elucidation of the endocytic pathways taken by IAV has been hampered by their apparent redundancy in establishing a productive infection. By manipulating the entry conditions we have established experimental settings that allow the separate analysis of dynamin-dependent (including clathrin-mediated endocytosis) and independent entry of IAV. Collectively, our results indicate macropinocytosis, the main route for the non-selective uptake of extracellular fluid by cells, as an alternative IAV entry route. As the dynamin-dependent and -independent IAV entry routes are redundant and independent, their separate manipulation was crucial for the identification and characterization of the alternative IAV entry route. A similar strategy might be applicable to the study of endocytic pathways taken by other viruses.
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影响因子：
4.8
作者：
Donepudi, Mrudula;Resh, Marilyn D.
通讯作者：
Resh, Marilyn D.
影响因子：
4.8
作者：
Donaldson, Julie G.;Porat-Shliom, Natalie;Cohen, Lee Ann
通讯作者：
Cohen, Lee Ann
DOI：
10.1073/pnas.0405172102
发表时间：
2004-12-28
影响因子：
11.1
作者：
Chu, VC;Whittaker, GR
通讯作者：
Whittaker, GR
DOI：
10.1073/pnas.0803711105
发表时间：
2008-08-19
影响因子：
11.1
作者：
Chen Chen;Zhuang, Xiaowei
通讯作者：
Zhuang, Xiaowei
影响因子：
6.7
作者：
Cureton DK;Massol RH;Saffarian S;Kirchhausen TL;Whelan SP
通讯作者：
Whelan SP