Molecular Mechanisms of Alphavirus Entry and Exit

甲病毒进入和退出的分子机制

• 批准号: 7010380
• 负责人: MARGARET KIELIAN
• 金额: $ 34.25万
• 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 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7010380
• 关键词: Semliki Forest virus; cell membrane; cholesterol; fluorescence resonance energy transfer; gene mutation; laboratory mouse; membrane lipids; sphingolipids; virus genetics; virus infection mechanism; virus protein

DESCRIPTION (provided by applicant): All enveloped viruses enter cells by the process of membrane fusion, and produce enveloped progeny virus by budding through a cellular membrane. Understanding virus membrane fusion and budding at the molecular level will provide critical insights into viral disease mechanisms, the development of anti-viral therapies, and the membrane fusion and budding reactions of eukaryotic cells. Semliki Forest virus (SFV) is a well-characterized alphavirus that infects cells by low pH-triggered membrane fusion and buds through the cell plasma membrane. Membrane fusion is mediated by the SFV E1 glycoprotein through a defined series of low pH-dependent conformational changes, and is strongly dependent on cholesterol and sphingolipid in the target membrane. During budding, E1 forms an icosahedral lattice that organizes the structure of the new virus particle. Interestingly, budding is also promoted by cholesterol in the host cell membrane. The overall goal of this grant is to define the molecular features of the entry and exit of alphaviruses from cells, and the role of cholesterol and sphingolipid in these processes. We have isolated and characterized three SFV mutants (srf-3, -4, & -5) that are strikingly increased in their ability to both enter and exit from cholesterol-depleted cells. Each mutant has a single amino acid substitution in E1, srf-3/P226S, srf-4/L44F, srf-5N178A. Both srf-4 and srf-5 are also sphingolipid-independent for fusion. We will characterize the roles of the 3 distinct E1 regions identified by these mutations, using in vitro mutagenesis to define the specific residues and protein interactions involved, and assays of fusion and E1 conformational changes to determine the functional effects of the mutations. Our recent studies indicate that the E1 fusion peptide strongly associates with membrane rafts following its low pH triggered membrane insertion. Our hypothesis is that this reflects the association of cholesterol with the fusion peptide. We will characterize the properties of E1-raft association and the influence of srf and fusion peptide mutations on association. We will directly assay for E1-cholesterol interactions using photocholesterol and fluorescent probes. We have developed an SFV budding assay based on cell surface biotinylation of the envelope proteins and virus retrieval by streptavidin-conjugated magnetic particles. This system will be used to reconstitute the budding of SFV from broken cells. We will focus on defining the nucleotide and cytosol requirements for budding, and on characterizing the role of cholesterol.

描述（由申请人提供）：所有包络病毒通过膜融合过程进入细胞，并通过细胞膜萌芽而产生包膜的后代病毒。了解分子水平的病毒膜融合和萌芽将为病毒疾病机制，抗病毒疗法的发展以及真核细胞的膜融合和萌芽反应提供关键的见解。 Semliki Forest病毒（SFV）是一种特征良好的α病毒，可通过细胞质膜质膜pH触发的膜融合和芽感染细胞。膜融合是由SFV E1糖蛋白通过定义的一系列低pH依赖性构象变化介导的，并且在靶膜中强烈依赖于胆固醇和鞘脂。在萌芽过程中，E1形成了一个二十面体晶格，该晶格组织了新病毒颗粒的结构。有趣的是，在宿主细胞膜中胆固醇也促进了萌芽。该赠款的总体目的是定义细胞中α病毒进入和退出的分子特征，以及胆固醇和鞘脂在这些过程中的作用。我们已经孤立并表征了三个SFV突变体（SRF -3，-4和-5），它们的进入和退出胆固醇的细胞的能力显着提高。每个突变体在E1，SRF-3/P226S，SRF-4/L44F，SRF-5N178A中具有单个氨基酸取代。 SRF-4和SRF-5均与鞘脂融合无关。我们将使用体外诱变来定义所涉及的特定残基和蛋白质相互作用，以及融合和E1构象变化的测定以确定突变的功能效应。我们最近的研究表明，在其低pH触发膜插入之后，E1融合肽与膜筏密切相关。我们的假设是，这反映了胆固醇与融合肽的关联。我们将表征E1-RAFT关联的性质以及SRF和融合肽突变对关联的影响。我们将使用光胆固醇和荧光探针直接测定E1-胆固醇相互作用。我们基于链霉亲和素偶联的磁颗粒的细胞表面生物素化和病毒检索的细胞表面生物素化开发了一种SFV萌芽测定。该系统将用于重建破碎细胞的SFV的萌芽。我们将专注于定义核苷酸和细胞质的萌芽需求，以及表征胆固醇的作用。