H5N1禽流感病毒以巨胞饮作用侵入细胞的研究

Macropinocytosis: anendocytic pathway for H5N1 subtype avian influenza virus infecting cells in vitro

流感病毒在体外感染细胞的过程中,是分别通过网格蛋白(Clathrin)和小窝蛋白(Caveolae)介导的两种内吞途径进入细胞内。为探索H5N1亚型禽流感病毒(AIV)侵入细胞的其他机制,本实验在胎牛血清存在的情况下,采用Dynasore抑制剂抑制网格蛋白和小窝蛋白介导的两种AIV侵入体外培养细胞途径,然而AIV仍然可以感染A549人源肺癌细胞系。进一步实验结果显示,采用巨胞饮抑制剂5-(N-乙基-N-异丙基)阿米洛利(EIPA)能够完全抑制病毒的感染,确认巨胞饮作用是AIV侵入体外培养的另外一种途径。同时,AIV也可以通过巨胞饮作用侵入不同来源的DF1禽源细胞和MDCK犬源细胞。此外,分别具有与禽源受体、人源受体和禽源-人源双重受体亲和特性的3株流感病毒仍然能够通过巨胞饮作用感染A549细胞。受体特异性试验表明,去除细胞的唾液酸受体后,病毒则不能够感染体外培养的细胞,表明唾液酸受体在巨胞饮作用的病毒侵入方式中仍然具有关键作用。本实验结果证明,巨胞饮作用是流感病毒侵入细胞的另外一种途径。该结果将有助于进一步研究流感病毒的感染机理。

Influenza virus enters cells via clathrin- and caveolae-mediated endocytosis. To verify another pathway of H5N1 subtype avian influenza virus （AIV） entering cells, dynasore was adopted to inhibit the clathrin- and eaveolae-mediated endocytosis, but under the condition of fetal bovine serum （FBS） in presence of the medium, the virus still had the ability to infect A549 ceils. However, the virus infection was blocked by 5-（N-Ethyl-N-Isopropyl） amiloride （EIPA）, the inhibitor of macro- pinocytosis, indicating the macropinocytosis was the mechanism of virus cell entry other than clathrin- and caveolae-mediated endocytosis. While, the AIV was able to infect different cells via macropinocytosis, such as A549, DF-1 and MDCK. Furthermore, all the three strains of the virus, with different receptor affinity of avian-type, human-type and human-avian type receptors, respectively, were capable to infect A549 cells via macropinocytosis. Whereas, the virus failed to infect the cells when the sialic acid receptors were removed from these cells, which suggested that the receptors play a crucial role for the AIV cell entry via macropinocytosis. Therefore, our data provide the evidence that macropinocytosis is another route for virus cell entry which facilitate future study on the infection mechanisms of AIV.