MOLECULAR MECHANISMS OF ALPHAVIRUS ENTRY AND EXIT

甲病毒进入和退出的分子机制

• 批准号: 6351246
• 负责人: MARGARET KIELIAN
• 金额: $ 28.52万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2003-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6351246
• 关键词: Semliki Forest virus; acid base balance; biotin; cell membrane; cholesterol; cryoelectron microscopy; gene mutation; immunomagnetic separation; membrane lipids; membrane permeability; protein structure; virus infection mechanism; virus protein

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.

描述（根据申请人的摘要改编）：所有包围病毒 使用膜融合感染细胞，必须通过细胞芽 膜产生后代病毒。 有关这些的分子信息 过程对于理解病毒疾病至关重要 新型抗病毒药物的发展，作为细胞模型 膜融合反应。 特征良好的α病毒，semliki 森林病毒（SFV），通过低pH触发的膜融合进入细胞， 并通过萌芽穿过细胞质膜而退出。 融合是 由SFV Spike蛋白介导的，该蛋白经历了一系列定义的一系列 酸性pH的构象变化。 SFV融合的关键特征 反应是其对胆固醇的存在的惊人依赖性 目标双层中的鞘脂。 Kielian博士还描述了 SFV出口途径中胆固醇的新颖需求。 这 该赠款的总体目标是定义条目的分子特征 并从细胞出口出口。 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 蛋白质在细胞膜中胆固醇。 她已经孤立并表征了具有的SFV突变体（SRF-3） 更有效的融合并从胆固醇耗尽的融合中退出 细胞比WT SFV。 E1尖峰中的单个氨基酸取代 亚基是脯氨酸226丝氨酸的变化，负责 SRF-3融合和退出的胆固醇独立性。 体外 将使用该域的诱变和新的SRF突变体的隔离 定义赋予α病毒胆固醇独立性的序列。 假设是SRF-3对关键E1-的胆固醇依赖性较小。 融合和退出中的膜相互作用。 这将通过 使用纯化的脂质成分的敏感新融合测定，通过 E1构象变化的生化和免疫学检测以及 膜插入，以及对照和对照的冷冻电子显微镜和 缺乏固醇的病毒。 Kielian博士为最后一步开发了生化测定 细胞表面峰值蛋白的萌芽和释放到SFV病毒体中。 该测定基于峰值蛋白的细胞表面生物素化 并使用链霉亲和素偶联的磁颗粒检索病毒。 该系统将用于确定胆固醇在 WT SFV和SRF-3的退出，以表征病毒退出要求 完整的细胞，也是重建细胞的实验的基础 SFV在半渗透的细胞系统中的表面萌芽。