Baculovirus entry into human hepatoma cells
Baculovirus entry into human hepatoma cells
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DOI：
10.1128/jvi.79.24.15452-15459.2005
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发表时间：
2005-12-01
影响因子：
5.4
通讯作者：
Oker-Blom, C
中科院分区：
文献类型：
--
作者：
Matilainen, H;Rinne, J;Oker-Blom, C
苜蓿银纹夜蛾核型多角体病毒（Autographa californica multiple nucleopolyhedrovirus，AcMNPV）是杆状病毒科的一个原型病毒，作为哺乳动物基因传递的候选载体，已引起越来越多的关注。已知AcMNPV在体外可进入分裂期和非分裂期哺乳动物细胞系，但其进入模式和动力学以及病毒在哺乳动物细胞中的细胞内转运尚不清楚。本研究的总体目标是表征AcMNPV和绿色荧光蛋白展示重组杆状病毒在人肝癌细胞中的进入步骤。发现病毒有效地结合和包裹细胞系，电子显微镜研究显示病毒体位于细胞表面的凹坑中，具有类似网格蛋白的电子致密涂层。此外，在较大的无包膜质膜内陷和类似macropinosome的细胞内囊泡中发现病毒颗粒。在双标记实验中，通过共聚焦显微镜在转导后30分钟的早期内体和在转导后45分钟开始的晚期内体中检测到病毒颗粒。通过纳米金预包埋免疫电子显微镜，在早期内体和晚期内体/溶酶体标记物的特异性结构中也观察到病毒。通过共聚焦显微镜没有观察到病毒进入再循环内体或高尔基复合体的迹象。总之，这些结果表明，AcMNPV进入哺乳动物细胞通过网格蛋白介导的内吞作用，并可能通过巨胞饮。因此，这里提供的数据应该使未来的杆状病毒载体的设计适合于更具体的和增强的遗传物质进入哺乳动物细胞的交付。
Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a prototype member of the Baculoviridae family, has gained increasing interest as a potential vector candidate for mammalian gene delivery applications. AcMNPV is known to enter both dividing and nondividing mammalian cell lines in vitro, but the mode and kinetics of entry as well as the intracellular transport of the virus in mammalian cells is poorly understood. The general objective of this study was to characterize the entry steps of AcMNPV- and green fluorescent protein-displaying recombinant baculoviruses in human hepatoma cells. The viruses were found to bind and transduce the cell line efficiently, and electron microscopy studies revealed that virions were located on the cell surface in pits with an electron-dense coating resembling clathrin. In addition, virus particles were found in larger noncoated plasma membrane invaginations and in intracellular vesicles resembling macropinosomes. In double-labeling experiments, virus particles were detected by confocal microscopy in early endosomes at 30 min and in late endosomes starting at 45 min posttransduction. Viruses were also seen in structures specific for early endosomal as well as late endosomal/lysosomal markers by nanogold preembedding immunoelectron microscopy. No indication of viral entry into recycling endosomes or the Golgi complex was observed by confocal microscopy. In conclusion, these results suggest that AcMNPV enters mammalian cells via clathrin-mediated endocytosis and possibly via macropinocytosis. Thus, the data presented here should enable future design of baculovirus vectors suitable for more specific and enhanced delivery of genetic material into mammalian cells.