体外研究HIV-1 Gag膜靶向决定因素的方法
Methods to Study Determinants for Membrane Targeting of HIV-1 Gag In Vitro.
作者信息
Todd Gabrielle C, Ono Akira
机构信息
Department of Microbiology and Immunology, University of Michigan Medical School, 5736 Medical Science Building II, 1150 W. Medical Center Drive, Ann Arbor, MI, 48109, USA.
出版信息
Methods Mol Biol. 2016;1354:175-85. doi: 10.1007/978-1-4939-3046-3_12.
Assembly of HIV-1 viral particles is a critical step of the HIV-1 life cycle; yet many details of this complex process are unknown. The Gag polyprotein drives viral particle assembly at the plasma membrane via three different types of interactions: protein-protein, protein-RNA, and protein-membrane interactions. As an approach to tease apart the importance of these interactions during viral particle assembly, in particular at the step of Gag membrane binding, we have developed an in vitro liposome-binding assay. Below we describe how to prepare liposomes, which serve as model membranes, and how to assess their interaction with Gag by liposome flotation centrifugation. Additionally, we outline extensions of this basic assay that can be used to address the role of RNA in regulating Gag-membrane interactions.
HIV-1病毒颗粒的组装是HIV-1生命周期中的关键步骤；然而，这个复杂过程的许多细节仍不清楚。Gag多聚蛋白通过三种不同类型的相互作用在质膜上驱动病毒颗粒的组装：蛋白质-蛋白质相互作用、蛋白质-RNA相互作用和蛋白质-膜相互作用。作为一种剖析这些相互作用在病毒颗粒组装过程中的重要性的方法，特别是在Gag膜结合步骤中，我们开发了一种体外脂质体结合测定法。下面我们描述如何制备用作模型膜的脂质体，以及如何通过脂质体浮选离心法评估它们与Gag的相互作用。此外，我们概述了这种基本测定法的扩展，可用于研究RNA在调节Gag-膜相互作用中的作用。
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