Clathrin-Mediated Endocytosis in Living Host Cells Visualized through Quantum Dot Labeling of Infectious Hematopoietic Necrosis Virus
Clathrin-Mediated Endocytosis in Living Host Cells Visualized through Quantum Dot Labeling of Infectious Hematopoietic Necrosis Virus
复制标题
通过传染性造血坏死病毒的量子点标记可视化活宿主细胞中网格蛋白介导的内吞作用
DOI：
10.1128/jvi.00109-11
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发表时间：
2011-07-01
影响因子：
5.4
通讯作者：
Xiao, Gengfu
中科院分区：
文献类型：
--
作者：
Liu, Haibin;Liu, Yi;Xiao, Gengfu
摘要传染性造血细胞坏死病毒（IHNV）是一种重要的鱼类病原体，可感染野生和养殖鲑科鱼类。IHNV作为一种新病毒属病毒，是研究弹状病毒进入宿主机制的重要模式系统。在这项研究中，量子点（QD）被用作荧光标记的敏感，长期跟踪IHNV进入。使用活细胞荧光显微镜，我们发现，IHNV是通过网格蛋白包被的坑后，病毒结合到宿主细胞膜内化。用氯丙嗪（一种阻断网格蛋白介导的内吞作用的药物）和使用RNA干扰的网格蛋白轻链（LCa）耗竭预处理宿主细胞均导致病毒进入显著减少。我们还可视化了病毒通过细胞骨架的运输（即，肌动蛋白丝和微管）。肌动蛋白聚合参与内吞囊泡进入胞质溶胶的运输，而微管是网格蛋白包被的囊泡运输到早期内体、晚期内体和溶酶体所必需的。用细胞松弛素D或诺考达唑破坏宿主细胞骨架显著削弱了IHNV的感染性。此外，通过用巴弗洛霉素A1（一种抑制核内体和溶酶体酸化的化合物）预处理宿主细胞，感染受到显著影响。IHNV与核内体的强共定位表明病毒被内化到这些膜结合区室中。这是第一份报告，其中QD标记是用来可视化病毒和内吞结构之间的动态相互作用，结果表明，IHNV进入宿主细胞通过网格蛋白介导的内吞，依赖于细胞色素依赖性，和低pH值依赖性途径。
ABSTRACT Infectious hematopoietic necrosis virus (IHNV) is an important fish pathogen that infects both wild and cultured salmonids. As a species of the genus Novirhabdovirus, IHNV is a valuable model system for exploring the host entry mechanisms of rhabdoviruses. In this study, quantum dots (QDs) were used as fluorescent labels for sensitive, long-term tracking of IHNV entry. Using live-cell fluorescence microscopy, we found that IHNV is internalized through clathrin-coated pits after the virus binds to host cell membranes. Pretreatment of host cells with chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and clathrin light chain (LCa) depletion using RNA interference both resulted in a marked reduction in viral entry. We also visualized transport of the virus via the cytoskeleton (i.e., actin filaments and microtubules) in real time. Actin polymerization is involved in the transport of endocytic vesicles into the cytosol, whereas microtubules are required for the trafficking of clathrin-coated vesicles to early endosomes, late endosomes, and lysosomes. Disrupting the host cell cytoskeleton with cytochalasin D or nocodazole significantly impaired IHNV infectivity. Furthermore, infection was significantly affected by pretreating the host cells with bafilomycin A1, a compound that inhibits the acidification of endosomes and lysosomes. Strong colocalizations of IHNV with endosomes indicated that the virus is internalized into these membrane-bound compartments. This is the first report in which QD labeling is used to visualize the dynamic interactions between viruses and endocytic structures; the results presented demonstrate that IHNV enters host cells via clathrin-mediated endocytic, cytoskeleton-dependent, and low-pH-dependent pathways.