Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
PLoS Pathog. 2011 Mar;7(3):e1001329. doi: 10.1371/journal.ppat.1001329. Epub 2011 Mar 31.
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.
甲型流感病毒(IAV)通过其血凝素糖蛋白与唾液酸化的宿主细胞受体结合进入宿主细胞。虽然依赖于胞质动力蛋白的网格蛋白介导内吞作用(CME)通常被认为是 IAV 的感染途径,但一些观察结果表明存在一种尚未确定的替代进入途径。通过操纵进入参数,我们建立了实验条件,允许单独分析 IAV 的依赖于胞质动力蛋白和不依赖于胞质动力蛋白的进入。虽然 IAV 在磷酸盐缓冲液中的进入可以被胞质动力蛋白的特异性抑制剂 dynasore 完全抑制,但在胎牛血清存在的情况下,出现了一种不依赖于 dynasore 的进入途径。使用针对 dynamin-2 的小干扰 RNA 进一步证实了这一发现。在血清存在的情况下,两种 IAV 进入途径均起作用。在这些条件下,通过 dynasore 和 amiloride 衍生物 EIPA(吞噬作用的标志性抑制剂)的联合处理可以完全阻断 IAV 的进入,而单独使用任何一种药物都没有效果。不依赖于胞质动力蛋白的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许多特定抑制剂的敏感性均表明吞噬作用参与了 IAV 的进入。一致地,已显示 IAV 颗粒和可溶性 FITC-葡聚糖在同一囊泡中在细胞内共定位。因此,除了经典的依赖于胞质动力蛋白的网格蛋白介导内吞途径外,IAV 还通过一种不依赖于胞质动力蛋白的途径进入宿主细胞,该途径具有吞噬作用的所有特征。