Echovirus 7 entry into polarized intestinal epithelial cells requires clathrin and Rab7.
Echovirus 7 entry into polarized intestinal epithelial cells requires clathrin and Rab7.
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DOI：
10.1128/mbio.00304-11
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发表时间：
2012
期刊：
影响因子：
6.4
通讯作者：
Bergelson JM
中科院分区：
文献类型：
--
作者：
Kim C;Bergelson JM
肠道病毒通过穿过肠粘膜侵入宿主，肠粘膜由极化上皮衬里。许多肠道病毒，包括埃可病毒（EV）和B组柯萨奇病毒（CVB），通过附着在衰变加速因子（decay-accelerating factor，ERF）上启动感染，ERF是一种在极化上皮细胞顶端表面高度表达的分子。我们以前观察到，DAF结合CVB 3进入极化肠上皮细胞发生不寻常的内吞机制，需要小窝蛋白，但不涉及网格蛋白或发动蛋白。在这里，我们检查了结合DAF的埃可病毒EV 7的进入。我们发现，药物，小干扰RNA（siRNA），和显性负突变体，靶向网格蛋白介导的内吞作用所需的因子，包括网格蛋白和发动蛋白，抑制EV 7感染和内化的病毒粒子从细胞表面。一旦病毒进入细胞，它与早期内体（EEA 1）和晚期内体（LAMP-2）的标志物共定位。用siRNA或显性失活突变体靶向Rab 5和Rab 7抑制内体成熟抑制感染并阻止病毒RNA释放到细胞中。这些结果表明EV 7通过网格蛋白介导的内吞作用内化，然后在释放其RNA之前移动到早期和晚期内体。已知通过内体的运输对于依赖于低pH或内体组织蛋白酶蛋白酶来完成进入过程的病毒是重要的。然而，我们发现EV 7感染既不需要低pH值，也不需要组织蛋白酶。结果表明，埃可病毒7（EV 7），结合到细胞表面的衰变加速因子（decay-accelerating factor，ERF）后，进入细胞网格蛋白介导的内吞作用，这种进入机制显着不同的另一种DAF结合肠道病毒，柯萨奇病毒B3（CVB 3）。因此，在附着到相同的细胞表面受体后，这些密切相关的病毒通过不同的机制进入相同的细胞。从肠道病毒衣壳释放病毒RNA（“脱壳”）所需的细胞信号仍然不清楚。我们发现，EV 7移动到晚期内体，RNA的释放依赖于内体成熟;然而，EV 7并不依赖于与其他病毒的脱壳和进入有关的内体因子。结果表明，一种未鉴定的内体因子是EV 7脱壳所必需的，或者通过内体的运输是导致另一个细胞内细胞器的途径中的一个重要步骤，在该细胞器中完成脱壳。
Enteroviruses invade the host by crossing the intestinal mucosa, which is lined by polarized epithelium. A number of enteroviruses, including echoviruses (EV) and group B coxsackieviruses (CVB), initiate infection by attaching to decay-accelerating factor (DAF), a molecule that is highly expressed on the apical surface of polarized epithelial cells. We previously observed that entry of DAF-binding CVB3 into polarized intestinal epithelial cells occurs by an unusual endocytic mechanism that requires caveolin but does not involve clathrin or dynamin. Here we examined the entry of a DAF-binding echovirus, EV7. We found that drugs, small interfering RNAs (siRNAs), and dominant negative mutants that target factors required for clathrin-mediated endocytosis, including clathrin and dynamin, inhibited both EV7 infection and internalization of virions from the cell surface. Once virus had entered the cell, it colocalized with markers of early endosomes (EEA1) and then late endosomes (LAMP-2). Inhibition of endosomal maturation—with siRNAs or dominant negative mutants targeting Rab5 and Rab7—inhibited infection and prevented release of viral RNA into the cell. These results indicate that EV7 is internalized by clathrin-mediated endocytosis and then moves to early and late endosomes before releasing its RNA. Trafficking through endosomes is known to be important for viruses that depend on low pH or endosomal cathepsin proteases to complete the entry process. However, we found that EV7 infection required neither low pH nor cathepsins. The results demonstrate that echovirus 7 (EV7), after binding to decay-accelerating factor (DAF) on the cell surface, enters cells by clathrin-mediated endocytosis; this entry mechanism differs markedly from that of another DAF-binding enterovirus, coxsackievirus B3 (CVB3). Thus, after attachment to the same cell surface receptor, these closely related viruses enter the same cells by different mechanisms. The cellular cues required for release of viral RNA from the enterovirus capsid (“uncoating”) remain poorly defined. We found that EV7 moved to late endosomes and that release of RNA depended on endosomal maturation; nonetheless, EV7 did not depend on the endosomal factors implicated in uncoating and entry by other viruses. The results suggest either that an unidentified endosomal factor is essential for uncoating of EV7 or that trafficking through the endosome is an essential step in a pathway that leads to another intracellular organelle where uncoating is completed.