CELL BIOLOGY OF VIRUS ENTRY

我们建议阐明包膜形成的途径和机制。 无包膜的动物病毒将它们的基因组送入宿主细胞 复制，并表征促进细胞功能的 他们的入场券。我们对塞姆利基森林病毒(SFV)和甲型病毒的研究， 已经表明，病毒颗粒首先是由 包被囊泡中的吸附内吞作用，以及穿透 基因组进入细胞质是通过低pH依赖的细胞内进行的 融合反应，可能发生在溶酶体中。在这 应用程序，我们将研究扩展到其他三种病毒：流感 水疱性口炎病毒和脊髓灰质炎病毒。这些是 特性良好的模型病毒属于三个病毒科(Myxo-， Rhabdo和Picornia病毒科)，它们是世界性疾病的罪魁祸首。 我们的目标是检查这些病毒是否遵循类似的进入途径 并对所涉及的细胞功能进行了表征。 将特别关注脊髓灰质炎病毒，它是无包膜的， 必须利用一种不同的、到目前为止完全没有特征的机制 在穿过细胞膜屏障时。我们会研究 病毒附着在细胞表面，内化的过程， 细胞内途径和溶酶体在穿透中的可能作用 并揭开基因组的外衣。以水疱性口炎病毒为例 和流感病毒，我们将研究低pH依赖的膜 聚变活动(我们最近演示过的)，并建立 它在感染中是否重要。融合缺陷的ts-突变体 流感病毒将被分离和鉴定。在脊髓灰质炎的情况下 病毒，我们还将检查低pH的影响，并测试 以脂质体为靶膜，可在体外诱导透皮吸收。 溶酶体亲和胺，如氯喹和 金刚烷胺，抑制病毒进入将被研究与 它们可能只是通过提高溶酶体的pH值来起作用。这个 使用的方法包括电子显微镜、细胞提取、 荧光显微镜、生化分析、单克隆和多克隆 抗体、放射性标记病毒和遗传方法。

We propose to elucidate the pathway and the mechanisms by which enveloped and nonenveloped animal viruses deliver their genome into host cells for replication, and to characterize the cellular functions that facilitate their entry. Our studies with Semliki Forest Virus (SFV), and alphavirus, have indicated that the virus particles are first internatlized by adsorptive endocytosis in coated vesicles, and that the penetration of the genome into the cytoplasm occurs intracellularly by a low pH-dependent fusion reaction, which probably takes place in lysosomes. In this application, we extend our studies to three other viruses: influenza virus, vesicular stomatitis virus and polio virus. These are well-characterized model viruses belonging to three virus families (myxo-, rhabdo- and picornia viridae) which are responsible for world-diseases. Our aim is to examine whether these viruses follow an entry pathway similar to that of SFV and to characterize the cellular functions involved. Special attention will be given to polio virus, which being nonenveloped, must utilize a different, and so far, completely uncharacterized mechanism in passing through the cellular membrane barriers. We will study the attachment of the viruses to cell surface, the process of internalization, the intracellular pathway and the possible role of lysosomes in penetration and uncoating of the genomes. In the case of vesicular stomatitis virus and influenza virus, we will investigate the low pH-dependent membrane fusion activity (which we have recently demonstrated), and establish whether it is important in infection. Fusions deficient ts-mutants of influenza virus will be isolated and characterized. In the case of polio virus, we will also examine the effects of low pH, and test whether penetration can be induced in vitro using liposomes as target membranes. The mechanism by which lysosomotropic amines such as chloroquine and amantadine, inhibit virus entry will be studied in relation to the possibility that they act simply by elevating the lysosomal pH. The methods to be used include electronmicroscopy, cell tractionation, fluorescence microscopy, biochemical assays, mono- and polyclonal antibodies, radioactively labelled viruses and genetic approaches.