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（改编自申请人的摘要）：所有包膜病毒 使用膜融合来感染细胞，并且必须通过细胞出芽 膜以产生子代病毒。 有关这些的分子信息 过程对于理解病毒性疾病至关重要 开发新型抗病毒药物，并作为细胞模型 膜融合反应。 充分表征的甲病毒，Semliki 森林病毒 (SFV) 通过低 pH 值触发的膜融合进入细胞， 并通过细胞质膜出芽退出。 融合是 由 SFV 刺突蛋白介导，该蛋白经历一系列确定的过程 酸性pH下构象变化。 SFV 融合的一个关键特征 反应是其对胆固醇的存在的显着依赖， 目标双层中的鞘脂。 Kielian 博士还描述了 SFV 退出途径对胆固醇的新要求。 这 这笔赠款的总体目标是定义条目的分子特征 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定相互作用 细胞膜上含有胆固醇的蛋白质。 她分离并鉴定了一种 SFV 突变体 (srf-3)，该突变体具有 明显更有效的融合和退出胆固醇耗尽 细胞比wt SFV。 E1 刺突中的单个氨基酸取代 亚基，将脯氨酸 226 变为丝氨酸，负责 srf-3融合和退出均与胆固醇无关。 体外 将使用该结构域的诱变和新 srf 突变体的分离 定义赋予甲病毒胆固醇独立性的序列。 假设 srf-3 对关键 E1- 的胆固醇依赖性较低 融合和退出时的膜相互作用。 这将由一个测试 使用纯化的脂质成分进行灵敏的新融合测定，通过 E1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控制和 甾醇耗尽的病毒。 Kielian 博士开发了一种生化检测方法，用于最后步骤 细胞表面刺突蛋白出芽并释放到 SFV 病毒体中。 该测定基于刺突蛋白的细胞表面生物素化 以及使用链霉亲和素结合的磁性颗粒进行病毒检索。 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，并作为重建细胞的实验基础 半透化细胞系统中 SFV 的表面出芽。