Molecular Mechanisms of Alphavirus Entry and Exit

描述（由申请人提供）：所有包膜病毒通过膜融合反应进入细胞，并通过细胞膜出芽产生子代病毒。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触发的膜融合感染细胞，并通过细胞质膜出芽而退出。膜融合由跨膜E1蛋白介导，这是一种与黄病毒融合蛋白在结构上相关的“II类病毒膜融合蛋白”。在低pH下，E1将其融合肽环插入含胆固醇的靶膜，并重新折叠成发夹状三聚体构象，以驱动融合反应。在病毒出芽期间，E1形成一个二十面体晶格，组织新病毒颗粒的结构。这项拨款的总体目标是确定具有II类融合蛋白的病毒进入和退出的分子特征。我们的研究集中在高度发达的SFV系统和黄病毒登革热病毒（DV），一类病原体和一个重要的人类健康问题。

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.