Visualization of intracellular pathways of engineered baculovirus in mammalian cells.
Visualization of intracellular pathways of engineered baculovirus in mammalian cells.
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DOI：
10.1016/j.virusres.2014.01.006
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发表时间：
2014-03-06
期刊：
影响因子：
5
通讯作者：
Wang P
中科院分区：
文献类型：
--
作者：
Liu Y;Joo KI;Lei Y;Wang P
杆状病毒是一种很有前途的基因传递载体。它们有能力在哺乳动物细胞中表达大量的转基因，而不会对人类产生致病性；然而，人们对它们在靶细胞中的转导机制知之甚少。在这项研究中，我们使用共定位和活细胞成像研究，通过直接显示VP39-GFP标记的病毒颗粒和靶细胞内的各种内吞结构来阐明杆状病毒的内化和细胞内传播途径。药物抑制和共聚焦显微镜结果表明，杆状病毒以动力蛋白依赖的方式通过网状蛋白介导的内吞作用进入细胞。病毒颗粒被显示通过早期的内体运输，触发了病毒的低pH依赖的内体融合过程。自噬活性的抑制增强了病毒的转导，而自噬小体的过度表达抑制了病毒的转导，提示自噬参与了病毒颗粒的降解过程。肌动蛋白细丝参与了病毒的转导，而微管通过促进自噬小体与溶酶体的融合形成自溶酶体，从而对病毒转导进行负向调控，从而发生病毒颗粒的降解。这些结果揭示了限制病毒转导的基本细胞因素，这些因素可用于改进杆状病毒载体在细胞和基因治疗中的使用。
Baculoviruses are a promising gene delivery vector. They have the ability to express large transgenes in mammalian cells without displaying pathogenicity in humans; however, little is known about their transduction mechanisms in target cells. In this study, we use colocalization and live-cell imaging studies to elucidate the internalization and intracellular trafficking pathways of baculoviruses through direct visualization of VP39-GFP-labeled viral particles and various endocytic structures within target cells. Drug inhibition and confocal microscopy results suggested that baculoviruses enter the cells via clathrin-mediated endocytosis in a dynamin-dependent manner. Viral particles were shown to traffic through early endosomes, triggering a low-pH-dependent endosomal fusion process of viruses. Suppressed autophagy activity enhanced viral transduction and overexpression of autophagosomes reduced viral transduction, suggesting that autophagy is involved in degradation process of viral particles. Actin filaments were involved in the viral transduction, while microtubules negatively regulated viral transduction by facilitating the fusion of autophagosomes with lysosomes to form autolysosomes, where degradation of viral particles occurs. These results shed some light on the essential cellular factors limiting viral transduction, which can be used to improve the use of baculoviral vectors in cell and gene therapy.
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