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（Semliki Forest virus，SFV）是一种充分表征的甲病毒，其通过低pH触发的膜融合感染细胞，并通过出芽穿过细胞质膜而离开。膜融合由跨膜E1蛋白介导，E1蛋白是一种“II类病毒膜融合蛋白”，其在结构上与黄病毒的融合蛋白相关。在低pH下，E1将其融合肽环插入含胆固醇的靶膜中，并重折叠成发夹样三聚体构象以驱动融合反应。在病毒出芽过程中，E1形成一个二十面体晶格，组织新病毒颗粒的结构。这项资助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。我们的研究重点是高度发达的SFV系统和黄病毒登革病毒（DV），A类病原体和一个重要的人类健康问题的世界范围内。 我们以前的研究表明，在E1尖端的融合环附近的ij环的重要作用。ij环参与甲病毒胆固醇依赖性，并且ij环中高度保守的组氨酸的突变在融合的后期阶段阻断SFV。我们将使用诱变和融合分析来定义SFV和DV ij环和保守组氨酸的功能。将使用位点特异性荧光探针表征和比较SFV和DV融合蛋白的膜插入，以跟踪融合环和ij环的膜相互作用。 SFV出芽产生高度组织化的颗粒，并且需要质膜上的临界数量的包膜蛋白。将确定细胞和病毒过程对细胞表面包膜蛋白水平的调节，并确定它们在出芽中的作用。细胞ESCRT机制在许多RNA病毒的萌芽中很重要，我们将确定其在甲病毒生命周期中的功能。我们还将筛选鉴定参与病毒组装和出芽的新型细胞蛋白。 了解病毒融合和出芽的分子机制将为病毒疾病机制，潜在的抗病毒治疗以及真核细胞的膜融合和出芽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.