ENDOCYTOSIS AND MEMBRANE FUSION

我们先前的研究表明，塞姆利基森林病毒(SFV)是一种简单的 动物病毒通过包被的吸附内吞进入宿主细胞 囊泡和基因组穿透到组织的细胞质中 通过膜融合反应培养细胞，这种反应可能发生在 溶酶体。我们打算更详细地调查各个步骤 参与SFV进入的遗传、生化、形态和病毒学 技巧。我们的研究将主要集中在两个方面： 进入。第一个是细胞生物学。我们知道内部化 通过一种结构性的细胞内吞作用发生。我们打算 用病毒作为标记来研究细胞表面的附着， 包被坑中的募集、包被囊泡的作用和站 在内化物质的细胞内途径中。我们还将努力 鉴定BHK21细胞上SFV的受体并检测其作用 在病毒基因组的最终穿透和剥离过程中的溶酶体。这个 第二个主要目标是膜学。SFV提供了一种具有生物学相关性的 高度符合生化和遗传学要求的膜融合系统 分析。SFV的低pH依赖膜融合活性非常高 即使是普通的脂质体也是有效的。我们将阐明分子 牵涉到机械师。将开发新的融合分析方法，这些方法一起 对于旧的，应该允许详细描述 反应。修饰的病毒，重组的小泡，亚病毒成分， 分离出的病毒蛋白将用于实验， 电子显微镜和生化技术。我们还将隔离和 缺乏融合的温度敏感型病毒突变体的特征 活动。SFV的融合活性也将被用来开发新的 用于植入的细胞杂交体(多核体和杂核体)的制造方法 外来蛋白质和脂类进入细胞膜，并用于插入水 可溶性分子进入细胞质。SFV对人类没有致病性，但 许多相关病毒是目前无法治愈的重要病原体。 可用。SFV的进入途径可能是共享的，至少在 一部分是被其他病毒感染的。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.