ENDOCYTOSIS AND MEMBRANE FUSION

我们以前的研究表明，塞姆利基森林病毒（SFV），一个简单的 动物病毒通过包被细胞内的吸附性内吞作用进入宿主细胞， 囊泡和基因组渗透到细胞质的组织 通过膜融合反应培养细胞，这可能发生在 溶酶体 我们打算更详细地调查， 参与SFV进入的遗传生化形态学和病毒学 技术. 我们的研究主要集中在两个方面， 入境 第一个是细胞生物学。 我们知道内在化 通过组成性细胞内吞活性发生。 我们打算 使用病毒作为标记来研究附着在细胞表面， 招募到被膜小凹，被膜小泡的作用，和车站 进入物质的细胞内途径。 我们还将努力 鉴定SFV在BHK 21细胞上的受体，并检查SFV在BHK 21细胞上的作用。 溶酶体在病毒基因组的最终穿透和脱壳中的作用。 的 第二个主要目标是膜学。 SFV提供了生物学相关的 膜融合系统非常适合生化和遗传 分析. SFV的低pH依赖性膜融合活性非常低， 即使使用普通脂质体也有效。 我们将阐明 机械师参与 将开发新的融合测定法， 与旧的，应该允许一个详细的特点， 反应 修饰病毒、重组囊泡、亚病毒组分， 分离的病毒蛋白将用于实验， 电子显微镜和生物化学技术。 我们还将隔离和 表征缺乏融合的温度敏感病毒突变体 活动 SFV的融合活性也将用于开发新的 生产细胞杂交体（多核和异核体）的方法， 外源蛋白质和脂质进入细胞膜，并将水 可溶性分子进入细胞质。 SFV对人类无致病性，但 许多相关的病毒是重要的病原体， available. SFV进入的途径可能是共享的，至少在 一部分，通过其他病毒。 我们希望我们的研究能带来新的见解 这将有助于在早期阶段抑制病毒感染。

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.