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Author:
Date:
2015-04-05
[Abstract] Many postitive-stranded RNA viruses, such as Hepatitis C virus (HCV), highjack cellular membranes, including the Golgi, ER, mitchondria, lipid droplets, and utilize them for replication of their RNA genome or assembly of new virions. By investigating how viral proteins associate with cellular membranes we will better understand the roles of cellular membranes in the viral life cycle. Our lab has focused specifically on the role of lipid droplets and lipid-rich membranes in the life cycle of HCV. To analyze the role of lipid-rich membranes in HCV RNA replication, we utilized a membrane flotation assay based on an 10-20-30% iodixanol density gradient developed by Yeaman et al. (2001). This gradient results in a linear increase in density over almost the entire length of the ...
[摘要] 许多假性绞线的RNA病毒，例如丙型肝炎病毒（HCV），高压细胞膜，包括高尔基体，ER，马钱子树，脂滴，并利用它们复制其RNA基因组或组装新的病毒粒子。通过调查病毒蛋白如何与细胞膜结合，我们将更好地了解细胞膜在病毒生命周期中的作用。我们的实验室特别关注脂质滴和富含脂质的膜在HCV的生命周期中的作用。为了分析富含脂质的膜在HCV RNA复制中的作用，我们使用基于由Yeaman等人（2001）开发的10-20-30％碘克沙醇密度梯度的膜浮选测定。该梯度导致在梯度的几乎整个长度上密度的线性增加，并且膜颗粒基于其浮力特性在梯度中分离。为了保存裂解物中的膜，在缺乏洗涤剂的缓冲液中机械破碎细胞。将细胞裂解物加载在梯度的底部，用梯度覆盖，并且膜随着碘克沙醇梯度自我产生而浮起。膜的脂质含量和相关蛋白的浓度将决定梯度内不同膜的分离。离心后，可以从梯度的顶部取样级分，并使用标准SDS-PAGE和蛋白质印迹分析来分析感兴趣的蛋白质。
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