Molecular Mechanisms of Alphavirus Entry and Exit

描述（由申请人提供）：所有包膜病毒均通过膜融合进入细胞，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、抗病毒治疗的发展以及真核细胞的膜融合和出芽反应提供重要的见解。塞姆利基森林病毒（SFV）是一种具有良好特征的甲病毒，通过低ph触发的膜融合和通过细胞质膜芽感染细胞。膜融合是由SFV E1糖蛋白通过一系列确定的低ph依赖性构象变化介导的，并且强烈依赖于靶膜中的胆固醇和鞘脂。在出芽期间，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，寄主细胞膜中的胆固醇也会促进出芽。这项资助的总体目标是确定甲病毒从细胞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。我们分离并鉴定了三种SFV突变体（srf-3、-4和-5），它们进入和退出胆固醇耗尽细胞的能力显著增加。每个突变体在E1、srf-3/P226S、srf-4/L44F、srf-5N178A中都有一个氨基酸替换。srf-4和srf-5在融合过程中也不依赖于鞘脂。我们将描述这些突变所鉴定的3个不同的E1区域的作用，使用体外诱变来确定特定残基和所涉及的蛋白质相互作用，并通过融合和E1构象变化的分析来确定突变的功能影响。我们最近的研究表明，E1融合肽在其低pH触发的膜插入后与膜筏密切相关。我们的假设是，这反映了胆固醇与融合肽的关联。我们将描述E1-raft关联的特性以及srf和融合肽突变对该关联的影响。我们将使用光胆固醇和荧光探针直接检测e1 -胆固醇相互作用。我们开发了一种基于细胞表面包膜蛋白的生物素化和链霉亲和素偶联磁颗粒的病毒回收的SFV出芽试验。该系统将用于从破碎的细胞中重建SFV的出芽。我们将着重于定义萌芽所需的核苷酸和细胞质，以及描述胆固醇的作用。

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.