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反应进入细胞，并通过细胞膜发芽产生后代病毒。塞姆利基森林病毒是一种特征明确的甲型病毒，它通过低pH触发膜融合感染细胞，并通过细胞膜萌发退出。膜融合是由跨膜E1蛋白介导的，它是一种结构上与黄病毒融合蛋白相关的II类病毒膜融合蛋白。在低pH值下，E1将其融合肽环插入含胆固醇的靶膜，并折叠成发夹状三聚体构象，以驱动融合反应。在病毒萌发期间，E1形成一个二十面体晶格，组织新病毒颗粒的结构。这项赠款的总体目标是确定具有II类融合蛋白的病毒进入和离开的分子特征。我们的研究集中在高度发达的SFV系统和黄病毒登革病毒(DV)，这是一种A类病原体，也是世界范围内一个重要的人类健康问题。 我们以前的研究证明了Ij环在E1末端与融合环相邻的重要作用。Ij环与甲型病毒胆固醇依赖有关，ij环中高度保守的组氨酸的突变在融合的后期阻止了SFV。我们将通过突变和融合分析来确定SFV和DV ij环的功能，并保守组氨酸。将使用位点特异性荧光探针跟踪融合蛋白和ij环的膜相互作用，对SFV和DV融合蛋白的膜插入进行表征和比较。 SFV萌发产生一个高度有组织的颗粒，需要在质膜上有关键数量的包膜蛋白。将确定细胞和病毒过程对细胞表面包膜蛋白水平的调节，并确定它们在萌发过程中的作用。细胞ESCRT机制在许多RNA病毒的萌发过程中是重要的，我们将确定它在甲病毒生命周期中的功能。我们还将筛选与病毒组装和萌发有关的新的细胞蛋白。 了解病毒融合和萌发的分子机制将为了解病毒疾病的机制、潜在的抗病毒治疗以及真核细胞的膜融合和萌发反应提供重要的见解。

DESCRIPTION (provided by applicant): All enveloped viruses enter cells by a membrane fusion reaction and produce progeny virus by budding through a cellular membrane. Semliki Forest virus (SFV) is a well-characterized alphavirus that infects cells by low pH-triggered membrane fusion and exits by budding through the cell plasma membrane. Membrane fusion is mediated by the transmembrane E1 protein, a "class II virus membrane fusion protein" that is structurally related to the fusion proteins of flaviviruses. At low pH E1 inserts its fusion peptide loop into cholesterol-containing target membranes and refolds to a hairpin-like trimer conformation to drive the fusion reaction. During virus budding, E1 forms an icosahedral lattice that organizes the structure of the new virus particle. The overall goal of this grant is to define the molecular features of the entry and exit of viruses having class II fusion proteins. Our studies focus on the highly-developed SFV system and on the flavivirus dengue virus (DV), a class A pathogen and an important human health problem world-wide. Our previous studies demonstrated important roles of the ij loop adjacent to the fusion loop at the tip of E1. The ij loop is involved in alphavirus cholesterol dependence, and mutation of a highly conserved histidine in the ij loop blocked SFV at a late stage in fusion. We will define the functions of the SFV and DV ij loop and conserved histidine using mutagenesis and fusion analysis. Membrane insertion of SFV and DV fusion proteins will be characterized and compared using site-specific fluorescent probes to follow the membrane interactions of the fusion and ij loops. SFV budding produces a highly organized particle and requires a critical number of envelope proteins on the plasma membrane. The regulation of cell surface envelope protein levels by cellular and viral processes will be determined and their role in budding will be defined. The cellular ESCRT machinery is important in the budding of many RNA viruses, and we will determine its functions in the alphavirus lifecycle. We will also screen to identify novel cellular proteins involved in virus assembly and budding. Understanding the molecular mechanisms of virus fusion and budding will provide critical insights into virus disease mechanisms, potential anti-viral therapies, and the membrane fusion and budding reactions of eukaryotic cells.