Molecular Mechanisms of Alphavirus Entry and Exit

描述(申请人提供)：所有被包膜的病毒通过膜融合的过程进入细胞，并通过细胞膜的萌发产生被包膜的后代病毒。在分子水平上了解病毒膜融合和萌发将为深入了解病毒病的发病机制、抗病毒治疗的发展以及真核细胞的膜融合和萌发反应提供重要的见解。塞姆利基森林病毒(SFV)是一种特征明确的甲型病毒，它通过低pH触发膜融合和通过细胞膜的芽感染细胞。膜融合是由SFV E1糖蛋白通过一系列低pH依赖的构象变化来介导的，并且强烈依赖于靶膜上的胆固醇和鞘磷脂。在萌发过程中，E1形成了一个二十面体的晶格，它组织了新病毒颗粒的结构。有趣的是，寄主细胞膜中的胆固醇也会促进发芽。这笔赠款的总体目标是确定甲型病毒进入和离开细胞的分子特征，以及胆固醇和鞘磷脂在这些过程中的作用。我们已经分离并鉴定了三个SFV突变体(SRF-3、-4和-5)，它们进入和退出胆固醇耗竭细胞的能力都显著增加。每个突变体在E1、SRF-3/P226S、SRF-4/L44F、SRF-5N178A均有单一氨基酸替换。SRF-4和SRF-5也不依赖鞘磷脂进行融合。我们将通过体外突变来定义特定的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这些突变的功能影响，从而表征由这些突变确定的3个不同的E1区域的作用。我们最近的研究表明，在低pH值触发膜插入后，E1融合肽与膜筏有很强的结合。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-RAFT关联的特性以及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检测E1-胆固醇的相互作用。我们已经建立了一种基于细胞表面包膜蛋白的生物素化和链霉亲和素结合的磁性颗粒检索病毒的SFV萌发试验。这个系统将被用来从破碎的细胞中重建SFV的萌芽。我们将专注于定义发芽所需的核苷酸和胞质，并描述胆固醇的作用。

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.