ENDOCYTOSIS AND MEMBRANE FUSION

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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.