Molecular Mechanisms of Alphavirus Entry and Exit

性状（由申请方提供）：所有包膜病毒均通过膜融合过程进入细胞，并通过细胞膜出芽产生包膜子代病毒。在分子水平上理解病毒膜融合和出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出芽反应提供重要见解。塞姆利基森林病毒（Semliki Forest virus，SFV）是一种特征良好的甲病毒，其通过低pH触发的膜融合感染细胞并通过细胞质膜出芽。膜融合由SFV E1糖蛋白通过一系列确定的低pH依赖性构象变化介导，并且强烈依赖于靶膜中的胆固醇和鞘脂。在出芽过程中，E1形成一个二十面体晶格，组织新病毒颗粒的结构。有趣的是，宿主细胞膜中的胆固醇也能促进出芽。这项资助的总体目标是确定甲病毒进入和离开细胞的分子特征，以及胆固醇和鞘脂在这些过程中的作用。我们已经分离并表征了三种SFV突变体（srf-3、srf-4和srf-5），它们进入和离开胆固醇耗尽的细胞的能力显著增加。每个突变体在E1、srf-3/P226 S、srf-4/L44 F、srf-5 N178 A中具有单个氨基酸取代。srf-4和srf-5两者对于融合也是鞘脂非依赖性的。我们将表征由这些突变确定的3个不同E1区域的作用，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，并测定融合和E1构象变化以确定突变的功能效应。我们最近的研究表明E1融合肽在低pH触发膜插入后与膜筏强烈缔合。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-筏协会的性质和影响的srf和融合肽突变协会。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。我们已经开发了一种基于细胞表面生物素化的包膜蛋白和通过链霉亲和素缀合的磁性颗粒进行病毒检索的SFV出芽测定。该系统将用于从破碎的细胞中重建SFV的出芽。我们将集中在定义的核苷酸和细胞质的要求，出芽，并在表征胆固醇的作用。

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.