MOLECULAR MECHANISMS OF ALPHAVIRUS ENTRY AND EXIT

描述（改编自申请人总结）：所有包膜病毒 使用膜融合感染细胞，并且必须通过细胞内的 膜以产生子代病毒。 分子信息 过程对于理解病毒性疾病至关重要， 开发新的抗病毒药物，并作为细胞模型， 膜融合反应 已充分表征的甲病毒Semliki 森林病毒（SFV）通过低pH触发的膜融合进入细胞， 并通过出芽穿过细胞质膜而排出。 融合是 由SFV刺突蛋白介导，该蛋白经历一系列确定的过程 在酸性pH下的构象变化。SFV融合的一个关键特征 反应是其显着依赖于胆固醇的存在， 鞘脂在目标双层。 Kielian博士还描述了一种 SFV出口途径中对胆固醇的新需求。 的 这项资助的总体目标是确定参赛作品的分子特征 以及甲病毒从细胞中退出。 她的主要假设是， SFV融合和退出涉及病毒刺突的特异性相互作用 蛋白质与胆固醇在细胞膜上。 她分离并鉴定了SFV突变体（srf-3）， 显著更有效的融合和退出胆固醇耗尽 细胞比野生型SFV。 E1刺突中的单个氨基酸置换 亚基，脯氨酸226丝氨酸的变化，是负责 SRF-3融合和退出的胆固醇依赖性。 体外 将使用该结构域的诱变和新SRF突变体的分离 以确定赋予甲病毒胆固醇独立性的序列。 假设是srf-3对关键的E1-E2的胆固醇依赖性较低。 融合和退出中的膜相互作用。 这将由一个 使用纯化的脂质组分的灵敏的新融合测定， E1构象变化的生物化学和免疫学检测， 膜插入，并通过冷冻电子显微镜的控制和 甾醇耗尽病毒 博士Kielian开发了一种生化分析方法，用于 出芽和释放细胞表面刺突蛋白进入SFV病毒体。 本试验基于刺突蛋白的细胞表面生物素化 和使用链霉亲和素缀合的磁性颗粒的病毒回收。 该系统将用于确定胆固醇在 野生型SFV和srf-3的退出，以表征病毒退出要求， 完整的细胞，并作为实验的基础， SFV在半透化细胞系统中的表面出芽。

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.