ENDOCYTOSIS AND MEMBRANE FUSION

胞吞作用和膜融合

• 批准号: 3294737
• 负责人: ARI H HELENIUS
• 金额: $ 21.19万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-01-01 至 1991-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3294737
• 关键词: HeLa cells; Semliki Forest virus; WI38 cell; electron microscopy; human tissue; hybrid cells; lysosomes; membrane fusion; membrane proteins; membrane reconstitution /synthesis; monoclonal antibody; nucleic acid sequence; pinocytosis; radiotracer; receptor; receptor mediated endocytosis; temperature sensitive mutant; tissue /cell culture; virus cytopathogenic effect; virus genetics; virus infection mechanism; virus protein; viruslike particle

Our previous studies have shown that Semliki Forest Virus (SFV), a simple animal virus enters the host cell by adsorptive endocytosis in coated vesicles and that the genome penetrates into the cytoplasm of tissue culture cells by a membrane fusion reaction, which probably occurs in lysosomes. We intend to investigate in more detail, the individual steps involved in SFV entry genetic biochemical morphological and virological techniques. The main focus of our studies will be on two aspects of entry. The first is cell-biological. We know that the internalization occurs through a constitutive cellular endocytotic activity. We intend to use the virus as a marker to study attachment to the cell surface, recruitment into coated pits, the role of coated vesicles, and the stations in the intracellular pathway of ingetrated material. We will also try to identify the receptors for SFV on BHK21 cells and to examine the role of lysosomes in the final penetration and uncoating of the viral genome. The second major aim is membranological. SFV provides a biologically relevant membrane fusion system highly amenable for biochemical and genetic analysis. The low pH dependent membrane fusion activity of SFV is very potent even with plain liposomes. We will elucidate the molecular mechanics involved. New fusion assays will be developed which, together with the old ones, should allow a detailed characterization of the reaction. Modified viruses, reconstituted vesicles, subviral components, and isolated virus proteins will be used in experiments using electromicroscopic and biochemical techniques. We will also isolate and characterize temperature sensitive virus mutants devoid of fusion activity. The fusion activity of SFV will also be utilized to develop new methods for producing cell hybrids (poly-and heterocaryons), for implanting foreign proteins and lipid into cellular membranes, and for inserting water soluble molecules into the cytoplasm. SFV is nonpathogenic to man, but many related viruses are important pathogens for which no cure is presently available. The pathway of SFV, entry is likely to be shared, at least in part, by other viruses. We hope that our studies will lead to new insights which will help to inhibit virus infection at an early stage.

我们以前的研究表明，Semliki森林病毒（SFV），这是一个简单的 动物病毒通过涂层的吸附性内吞作用进入宿主细胞 囊泡，基因组渗透到组织的细胞质中 通过膜融合反应培养细胞，可能发生在 溶酶体。 我们打算更详细地调查单个步骤 参与SFV进入遗传生化形态和病毒学 技术。 我们研究的主要重点将是 入口。 首先是细胞生物学。 我们知道内在化 通过组成型细胞内吞作用发生。 我们打算 使用病毒作为研究对细胞表面的附着的标记， 招募涂层坑，涂层囊泡的作用和站点 在摄入材料的细胞内途径中。 我们还将尝试 识别BHK21细胞上SFV的受体，并检查 病毒基因组的最终穿透和脱膜中的溶酶体。 这 第二个主要目的是膜学。 SFV提供了与生物学相关的 膜融合系统高度适合生化和遗传 分析。 SFV的低pH依赖性膜融合活性非常 即使有普通的脂质体也有效。 我们将阐明分子 涉及的力学。 将开发新的融合测定法 与旧的有关，应允许详细表征 反应。 修饰的病毒，重建的囊泡，次病毒组件， 和分离的病毒蛋白将用于使用 电显微镜和生化技术。 我们还将隔离和 表征没有融合的温度敏感病毒突变体 活动。 SFV的融合活动也将用于开发新的 产生细胞杂种（聚和异胞菌素）的方法，植入 外蛋白和脂质进入细胞膜，用于插入水 可溶性分子进入细胞质。 SFV对人是非疾病的，但是 许多相关病毒是目前无法治愈的重要病原体 可用的。 SFV的途径，即至少在 部分是其他病毒。 我们希望我们的研究能够带来新的见解 这将有助于在早期抑制病毒感染。