CELL BIOLOGY OF VIRUS ENTRY

我们建议阐明的途径和机制，包膜 无包膜的动物病毒将它们的基因组递送到宿主细胞中， 复制，并表征细胞功能，促进 他们的入境。 我们对塞姆利基森林病毒（SFV）和甲病毒的研究， 已经表明，病毒颗粒首先是通过 在包被的囊泡中的吸附性内吞作用， 基因组进入细胞质发生在细胞内的低pH依赖性 融合反应，这可能发生在溶酶体中。 在这 应用，我们将我们的研究扩展到其他三种病毒： 病毒、水泡性口炎病毒和脊髓灰质炎病毒。 这些是 属于三个病毒家族（myxo-， rhabdo-和picornia viridae），它们是世界性疾病的罪魁祸首。 我们的目的是检查这些病毒是否遵循类似于 SFV和表征所涉及的细胞功能。 将特别注意脊髓灰质炎病毒，因为它没有包膜， 必须利用一种不同的，迄今为止，完全未知的机制， 通过细胞膜屏障的能力。 我们会研究 病毒附着在细胞表面，内化过程， 细胞内途径和溶酶体在渗透中的可能作用 和基因组的脱壳。 在水泡性口炎病毒的情况下， 和流感病毒，我们将研究低pH依赖性膜 融合活性（我们最近已经证明），并建立 它在感染中是否重要。 融合缺陷型TS突变体 流感病毒将被分离和鉴定。 在小儿麻痹症的案例中， 病毒，我们还将研究低pH值的影响，并测试是否 可以使用脂质体作为靶膜在体外诱导渗透。 促溶酶体胺如氯喹和 金刚烷胺，抑制病毒进入将进行研究， 它们的作用可能仅仅是通过升高溶酶体的pH值。 使用的方法包括电子显微镜，细胞牵引， 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.