MOLECULAR MECHANISMS OF ALPHAVIRUS ENTRY AND EXIT

描述(改编自申请者摘要)：所有包膜病毒 使用膜融合来感染细胞，并且必须通过细胞萌发 产生后代病毒的膜。关于这些的分子信息 过程是理解病毒疾病的关键， 新型抗病毒药物的开发，并作为细胞模型 膜融合反应。特征明确的甲型病毒塞姆利基 森林病毒(SFV)通过低pH触发的膜融合进入细胞， 并通过细胞质膜萌发退出。核聚变是 由SFV Spike蛋白介导，它经历了一系列确定的 在酸性pH条件下的构象变化。SFV融合的一个关键特征 它的反应是它对胆固醇和 靶标双层中的鞘磷脂。基利安博士还描述了一种 在SFV退出途径中对胆固醇的新需求。这个 这项资助的总体目标是确定参赛作品的分子特征 甲型病毒从细胞中排出。她的主要假设是 SFV融合和退出涉及病毒尖峰的特定相互作用 细胞膜中含有胆固醇的蛋白质。 她已经分离并鉴定了一种SFV突变体(SRF-3)，它具有 显著更高效的融合和退出胆固醇耗竭 细胞数大于wt SFV。E1峰中的单一氨基酸替代 亚基，一个由脯氨酸226变为丝氨酸的亚基，负责 SRF-3融合和退出的胆固醇非依赖性。离体 将使用该结构域的突变和分离新的SRF突变体 定义赋予甲型病毒胆固醇独立性的序列。 假设SRF-3对关键的E1-3的胆固醇依赖性较低- 融合和退出过程中的膜相互作用。这将由一个 使用纯化的脂类成分的灵敏的新融合分析，通过 E_1构象变化的生化和免疫学检测 膜插入，并通过冷冻电子显微镜观察对照和 类固醇耗尽病毒。 Kielian博士开发了一种生化分析方法，用于 细胞表面刺突蛋白的萌发和释放进入SFV病毒粒子。 这种分析是基于细胞表面的刺激性蛋白的生物素化。 以及使用链霉亲和素结合的磁性颗粒检索病毒。 这个系统将被用来确定胆固醇在 Wt SFV和SRF-3的退出，以表征 完整的细胞，并作为重建细胞的实验基础 SFV在半渗透细胞系统中的表面萌发。

DESCRIPTION (Adapted from Applicant's Summary): All enveloped viruses use membrane fusion to infect a cell, and must bud through a cellular membrane to produce progeny viruses. Molecular information on these processes is critical to the understanding of viral disease, the development of novel anti-viral agents, and as a model for cellular membrane fusion reactions. The well-characterized alphavirus, Semliki Forest virus (SFV), enters cells via low pH-triggered membrane fusion, and exits by budding through the cell plasma membrane. Fusion is mediated by the SFV spike protein, which undergoes a defined series of conformational changes at acid pH. A critical feature of the SFV fusion reaction is its striking dependence on the presence of cholesterol and sphingolipid in the target bilayer. Dr. Kielian has also described a novel requirement for cholesterol in the SFV exit pathway. The overall goal of this grant is to define molecular features of the entry and exit of alphaviruses from cells. Her major hypothesis is that both SFV fusion and exit involve specific interactions of the viral spike protein with cholesterol in the cell membrane. She has isolated and characterized an SFV mutant (srf-3) that has strikingly more efficient fusion and exit from cholesterol-depleted cells than wt SFV. A single amino acid substitution in the E1 spike subunit, a change of proline 226 to serine, is responsible for the cholesterol-independence of both srf-3 fusion and exit. In vitro mutagenesis of this domain and isolation of new srf mutants will be used to define sequences that confer alphavirus cholesterol independence. The hypothesis is that srf-3 is less cholesterol-dependent for key E1- membrane interactions in fusion and exit. This will be tested by a sensitive new fusion assay using purified lipid components, by biochemical and immunological detection of E1 conformational changes and membrane insertion, and by cryo-electron microscopy of control and sterol-depleted virus. Dr. Kielian has developed a biochemical assay for the final steps of budding and release of cell surface spike proteins into SFV virions. This assay is based on cell surface biotinylation of the spike proteins and virus retrieval using streptavidin-conjugated magnetic particles. This system will be used to determine the role of cholesterol in the exit of wt SFV and srf-3, to characterize virus exit requirements in intact cells, and as the basis of experiments to reconstitute the cell surface budding of SFV in a semi-permeabilized cell system.