Structure and Expression of the Paramyxovirus SV5 Genome

副粘病毒 SV5 基因组的结构和表达

• 批准号: 6510345
• 负责人: ROBERT A LAMB
• 金额: $ 25.73万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6510345
• 关键词: Paramyxovirus; gene expression; protein structure function; site directed mutagenesis; tissue /cell culture; virus assembly; virus genetics; virus infection mechanism; virus protein; virus replication

DESCRIPTION (provided by applicant): The paramyxoviruses are the etiological agents of many important diseases of man and lower animals. Collectively, these viruses contribute significantly to worldwide morbidity and mortality. The focus of this study is the mechanism by which a prototype paramyxovirus, simian parainfluenza virus 5 (SV5) enters cells. The entry of enveloped viruses into cells requires the fusion of the viral envelope with a cellular membrane. The mechanism of viral-mediated membrane fusion is a topic of interest to cell biologists and structural biologists as well as those investigating the mechanism of virus entry. This is because membrane fusion is a process central in cell biology as it occurs at many stages of endocytosis and exocytosis. In addition, studies on the mechanism by which paramyxoviruses cause cell fusion are of significance for understanding the pathogenesis of human immunodeficiency virus 1. While our long-term goal is to use X-ray structural analysis to understand the molecular basis for paramyxovirus-mediated membrane fusion, we also need to use other functional approaches to identify determinants of paramyxovirus glycoproteins that are important for viral entry. We will study properties of the SV5 fusion (F) protein in six interrelated specific aims. We will analyze structure/function relationships of the SV5 F protein core trimer, which is composed of the F protein N-1 and C-1 heptad repeat regions, based on the atomic structure that we determined recently at 1.4 A resolution. We will investigate: (a) mutants that are predicted to alter core trimer stability; (b) mutants designed to investigate F protein conformation and (C) mutants designed to test properties of the fusion peptide. We will determine the mechanism of action of peptide inhibitors of SV5 fusion including: (a) determination of IC50 of peptide C-1 containing "guest site" mutations at L447 and I449 which insert into a deep cavity in the N-1 trimer and an IC50/Tm ratio; (b) determination of the mechanism of peptide C-1 inhibition and capture of an early fusion-active conformation of SV5 F protein and (c) determination of the mechanism of peptide N-1 inhibition. We will investigate if core trimer formation brings the bilayers together for fusion and we will endeavor to detect F protein conformational change(s) required for fusion including conformational changes in F on cleavage from F0 to F1 + F2 and conformational changes in membrane bound and soluble forms of F1 + F2. We will investigate the roles of the F cytoplasmic tail and transmembrane domain on fusion activity and lipid 'raft' association. We will elucidate the role of specific mutations in the F protein in the context of a virus infection by using reverse genetics and we will study fusion activity, virus growth and virus assembly of the altered viruses.

描述（由申请人提供）：Paramyxovirus是病因 许多重要疾病的人和下动物的药物。总的来说，这些 病毒对全球发病率和死亡率产生了重大贡献。这 这项研究的重点是原型帕托马病毒，猿猴的机制 Parainfluenza病毒5（SV5）进入细胞。被包膜病毒进入 细胞需要病毒包膜与细胞膜融合。这 病毒介导的膜融合的机制是细胞感兴趣的话题 生物学家和结构生物学家以及研究的生物学家 病毒进入机制。这是因为膜融合是一个中心的过程 在细胞生物学中，它发生在内吞和胞吞作用的许多阶段。在 此外，对帕托病毒引起细胞融合的机制的研究 对于理解人的发病机理具有重要意义 免疫缺陷病毒1。虽然我们的长期目标是使用X射线结构 分析以了解丙糖病毒介导的膜的分子基础 融合，我们还需要使用其他功能方法来识别 对病毒进入至关重要的帕托病毒糖蛋白的决定因素。 我们将研究六个相互关联的SV5融合（F）蛋白的特性 具体目标。我们将分析SV5 F的结构/功能关系 蛋白质核心三聚体，由F蛋白N-1和C-1组成 基于我们最近确定的原子结构的重复区域 1.4分辨率。我们将调查：（a）预计会改变的突变体 核心三聚体稳定性； （b）旨在研究F蛋白的突变体 构象和（c）突变体旨在测试融合肽的性质。 我们将确定SV5融合肽抑制剂的作用机理 包括：（a）确定含有“来宾站点”的肽C-1的IC50 L447和I449的突变，将其插入N-1的深腔中 和IC50/TM比； （b）确定肽C-1机理 抑制和捕获SV5 F蛋白的早期融合活性构象 （c）确定肽N-1抑制的机理。我们将 调查核心三聚体的形成是否将双层组合在一起以进行融合 我们将努力检测F蛋白构象变化所需的F蛋白构象变化 融合包括f0对F1 + f2的裂解的构象变化，并且 F1 + F2的膜结合和可溶形式的构象变化。我们将 研究F细胞质尾部和跨膜结构域在 融合活性和脂质“筏”关联。我们将阐明 在病毒感染的背景下，F蛋白中的特异性突变是 使用反向遗传学，我们将研究融合活性，病毒生长和 病毒的病毒组装。