Brass Volker, Gosert Rainer, Moradpour Darius
Department of Medicine II, University of Freiburg, Freiburg, Germany.
Methods Mol Biol. 2009;510:195-209. doi: 10.1007/978-1-59745-394-3_15.
Formation of a membrane-associated replication complex, composed of viral proteins, replicating RNA, altered cellular membranes, and other host factors, is a hallmark of all positive-strand RNA viruses. In the case of HCV, RNA replication takes place in a likely endoplasmic reticulum-derived membrane alteration referred to as the "membranous web." In vitro transcription-translation, membrane extraction and flotation analyses, immunofluorescence microscopy, fluorescent in situ hybridization, and RNA metabolic labeling followed by confocal laser scanning microscopy have yielded insights into the structure and function of the HCV replication complex. We describe these techniques and highlight selected results.
由病毒蛋白、正在复制的RNA、改变的细胞膜和其他宿主因子组成的膜相关复制复合体的形成是所有正链RNA病毒的一个标志。就丙型肝炎病毒（HCV）而言，RNA复制发生在一种可能源自内质网的膜改变中，即所谓的“膜性网”。体外转录-翻译、膜提取和浮选分析、免疫荧光显微镜检查、荧光原位杂交以及RNA代谢标记后进行共聚焦激光扫描显微镜检查，已经让我们对HCV复制复合体的结构和功能有了深入了解。我们描述了这些技术并突出了选定的结果。
