Molecular Mechanisms of Alphavirus Entry and Exit

甲病毒进入和退出的分子机制

• 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
• 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

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，并通过细胞膜萌芽而产生后代病毒。 Semliki Forest病毒（SFV）是一种特征良好的α病毒，可通过低pH触发的膜融合感染细胞，并通过通过细胞质膜萌芽而退出。膜融合是由跨膜E1蛋白（一种“ II类病毒膜融合蛋白”介导的，它与黄叶病毒的融合蛋白在结构上相关。在低pH E1处，将其融合肽环插入含有胆固醇的靶膜中，并重新折叠到类似发夹样的三聚体构象中以驱动融合反应。在病毒萌芽期间，E1形成了一种二十面体晶格，该晶格组织了新病毒颗粒的结构。该赠款的总体目标是定义具有II类融合蛋白的病毒入口和退出的分子特征。我们的研究集中于高度发达的SFV系统和Flavivivure登革热病毒（DV），A类病原体和全球重要的人类健康问题。 我们以前的研究表明，在E1尖端，与融合环相邻的IJ环的重要作用。 IJ环参与α病毒胆固醇依赖性，IJ环中高度保守的组氨酸的突变在融合的后期阻止了SFV。我们将使用诱变和融合分析来定义SFV和DV IJ环和保守的组氨酸的功能。 SFV和DV融合蛋白的膜插入将被表征并使用位点特异性荧光探针进行比较，以遵循融合和IJ环的膜相互作用。 SFV萌芽会产生高度组织的粒子，并且需要质膜上的临界数字包膜蛋白。将确定细胞表面包膜蛋白水平的调节，并确定它们在发芽中的作用。细胞ESCRT机械在许多RNA病毒的萌芽中很重要，我们将确定其在α病毒生命周期中的功能。我们还将筛选以鉴定参与病毒组装和发芽的新型细胞蛋白。 了解病毒融合和萌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萌芽反应提供关键见解。