Molecular Mechanisms of Alphavirus Entry and Exit

甲病毒进入和退出的分子机制

基本信息

批准号：
7922849
负责人：
MARGARET KIELIAN
金额：
$ 6.68万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7922849
关键词：
Address Alanine Alphavirus Amino Acid Substitution Antiviral Therapy Biological Assay Cell membrane Cell surface Cells Cellular Membrane Chimeric Proteins Cholesterol Cysteine DNA Sequence Rearrangement Dengue Virus Dependence Disulfides Drosophila genus E protein Endosomes Eukaryotic Cell Flavivirus Fluorescence Fluorescent Probes Genomics Glycoproteins Goals Grant Growth Half-Life Health Histidine Human Human papillomavirus 16 E1 protein In Situ Infection Label Libraries Lipid Bilayers Mediating Membrane Membrane Fusion Methods Modeling Molecular Molecular Conformation Multivesicular Body Mutagenesis Mutation Nucleocapsid Pathway interactions Peptides Phenotype Positioning Attribute Process Property Proteins RNA Interference RNA Viruses RNA replication Reaction Regulation Role Semliki forest virus Site Staging Structure System Testing Transfection Vacuole Viral Virion Virus Virus Assembly Virus Diseases Virus Replication Work crosslink design env Gene Products insight mutant novel particle pathogen protein E protein degradation protein expression protein transport tool uptake

项目摘要

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.

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) 是一种特征明确的甲病毒，通过低 pH 值触发的膜融合感染细胞，并通过细胞质膜出芽退出。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”，在结构上与黄病毒的融合蛋白相关。在低 pH 值下，E1 将其融合肽环插入含胆固醇的靶膜中，并重新折叠成发夹状三聚体构象以驱动融合反应。在病毒出芽过程中，E1 形成二十面体晶格，组织新病毒颗粒的结构。该资助的总体目标是定义具有 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 系统和黄病毒登革热病毒 (DV)，这是一种 A 类病原体，也是全球重要的人类健康问题。我们之前的研究证明了与 E1 尖端融合环相邻的 ij 环的重要作用。 ij 环涉及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后期阻断了 SFV。我们将使用诱变和融合分析来定义 SFV 和 DV ij 环以及保守组氨酸的功能。 SFV 和 DV 融合蛋白的膜插入将使用位点特异性荧光探针进行表征和比较，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，并且需要质膜上有一定数量的包膜蛋白。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 病毒的萌芽中非常重要，我们将确定其在甲病毒生命周期中的功能。我们还将筛选以确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、潜在的抗病毒疗法以及真核细胞的膜融合和出芽反应提供重要的见解。