Investigating the relevance of lipid microdomains for (HI) virus protein assembly and virus budding in an artifical cell environment

研究人工细胞环境中脂质微结构域与 (HI) 病毒蛋白组装和病毒出芽的相关性

• 批准号: 13204897
• 负责人: Professorin Dr. Petra Schwille
• 金额: --
• 依托单位: Forschungsgruppe Zelluläre und molekulare Biophysik
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2005
• 资助国家: 德国
• 起止时间: 2004-12-31 至 2012-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/13204897?language=en
• 关键词: Investigating; relevance; lipid; microdomains; HI

During the HIV replication cycle, the immature viral core is assembled at the plasma membrane and subsequently released from the host cell via a budding process. This process is primarily driven by its cytoplasmic core-precursor protein Gag. In addition, constituents of the plasma membrane become incorporated into the viral envelope. These are, e.g., GPI (glycosylphosphatidyl inositol)-anchored proteins, but also the ganglioside GM1, cholesterol and Sphingomyelin, which are supposed to be enriched in so-called lipid rafts , putative platforms for protein sorting and signaling events in the cell membrane which exhibit a condensed lipid order. Consequently, lipid rafts seem to play a crucial role in virus infection of a cell, and their control could be an essential step in antiviral therapies. However, to date very little is known about the dynamics of the protein-lipid interactions during virus assembly and budding, due to the complexity of the cellular system being infected by viruses. To investigate the role of lipid microdomains or rafts on virus proliferation independent from other factors such as membrane proteins or the cellular secretion machinery, we propose to study the assembly, membrane-association and budding of viral particles, after in vitro expression of Gag protein, in an artificial cell system of defined membrane composition. To this end, a generally applicable scheme of cell-free protein expression in Giant Unilamellar Vesicles (GUVs) will be established. Of particular relevance will further be the control of cholesterol levels in the GUV, i.e., artificial cell membrane. Using fluorescent lipids and Gag protein chimeras fused to various fluorescent proteins, the different steps such as Gag protein aggregation, membrane association and budding of viral particles will be investigated by various kinds of single molecule-based fluorescence correlation spectroscopy and imaging, techniques specifically developed and tailored in our laboratory.

在HIV复制周期中，未成熟的病毒核心在质膜上组装，随后通过出芽过程从宿主细胞中释放。该过程主要由其细胞质核心前体蛋白 Gag 驱动。此外，质膜的成分并入病毒包膜中。例如，这些是 GPI（糖基磷脂酰肌醇）锚定蛋白，还有神经节苷脂 GM1、胆固醇和鞘磷脂，它们被认为富含所谓的脂筏，是细胞膜中蛋白质分选和信号传导事件的推定平台。表现出稠密的脂质顺序。因此，脂筏似乎在细胞的病毒感染中发挥着至关重要的作用，并且它们的控制可能是抗病毒治疗中的重要步骤。然而，迄今为止，由于被病毒感染的细胞系统的复杂性，人们对病毒组装和出芽过程中蛋白质-脂质相互作用的动态知之甚少。为了研究脂质微结构域或筏对独立于膜蛋白或细胞分泌机制等其他因素的病毒增殖的作用，我们建议在体外表达 Gag 蛋白后研究病毒颗粒的组装、膜缔合和出芽，在具有确定的膜组成的人造细胞系统中。为此，将建立一种普遍适用的巨单层囊泡（GUV）中无细胞蛋白表达方案。特别相关的是 GUV（即人造细胞膜）中胆固醇水平的控制。使用荧光脂质和与各种荧光蛋白融合的 Gag 蛋白嵌合体，通过各种基于单分子的荧光相关光谱和成像、专门开发的技术和技术来研究病毒颗粒的不同步骤，例如 Gag 蛋白聚集、膜缔合和出芽。在我们的实验室量身定制。