Su Wen-Chi, Lai Michael M C
China Medical University, Taichung, Taiwan.
Research Center for Emerging Viruses, China Medical University Hospital, Taichung, Taiwan.
Methods Mol Biol. 2018;1836:185-194. doi: 10.1007/978-1-4939-8678-1_9.
Although several virus families are internalized into their host cells by direct fusion of the viral envelope with the plasma membrane, most viruses, for example, influenza virus, make use of endocytic pathways for productive entry and infection. After endocytosis, the influenza virus escapes from the endocytic compartment to the cytosol. The distribution of the incoming influenza virus could be traced by detection of the viral RNA in the distinct cellular compartments, including endosome, cytosol, and nucleus. To accomplish this work, we developed a subcellular fractionation method based on density gradient ultracentrifugation and detected the viral RNA using quantitative reverse transcription-polymerase chain reaction analysis. This chapter is devoted to the practical methods and precautions for studying endocytic traffic of virus as well as host cellular factors affecting viral endocytosis.
尽管有几个病毒家族通过病毒包膜与质膜的直接融合而内化到宿主细胞中，但大多数病毒，例如流感病毒，利用内吞途径进行有效的进入和感染。内吞作用后，流感病毒从内吞区室逃逸到细胞质溶胶中。通过检测不同细胞区室（包括内体、细胞质溶胶和细胞核）中的病毒RNA，可以追踪进入的流感病毒的分布。为了完成这项工作，我们开发了一种基于密度梯度超速离心的亚细胞分级分离方法，并使用定量逆转录-聚合酶链反应分析来检测病毒RNA。本章致力于研究病毒内吞运输以及影响病毒内吞作用的宿主细胞因子的实用方法和注意事项。
