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 i s 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.
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