Biochemical and structural characterization of the *E1 fusion protein of Semliki Forest virus
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The alphavirus Semliki Forest virus (SFV) contains a single-stranded RNA genome bound to the capsid protein and enveloped by a membrane derived from the host cell. The membrane contains two viral glycoproteins, E1 and E2. E2 is responsible for receptor binding and E1 for a viral-endosomel membrane fusion. The E1 protein responds to the low pH of endosomes by changing conformation to form a homotrimer necessary for the fusion reaction, which is essential for virus infectivity. E1 is believed to be prototypical of a new class of fusion proteins (Class II), including the alpha- and flaviviruses. We therefore chose to study the E1 protein in greater depth to better understand its structure and mechanism of action. The primary results of this work focus on the three parts of the membrane fusion machinery: (1) how the protein associates with target membranes, (2) how the subunits within the homotrimer associate with each other, and (3) the regulation of these events. In studying the relative stabilities of the monomeric and trimeric forms of the protein, the homotrimer was significantly more stable by a number of measures and the monomer appears to exists in a metastable conformation. The change from this metastable monomeric form to an extremely stable homotrimeric form is a tightly regulated event dependent specifically on low pH and the presence of target membranes of the proper composition. By using a monoclonal antibody that maps to the portion of the protein believed to interact with target membranes, termed the fusion peptide, we were able to show that simple exposure of this segment of the protein to solution was not sufficient for stable interaction of the protein with target membranes. However, blockage of this region by binding of the antibody prevented low pH-induced interaction with membranes and all the conformational changes that were assayed. These data emphasize the regulatory interaction between the protein and target membranes, and localize at least the initial interaction to the fusion peptide region. Once formed, the homotrimer is extremely stable to detergents, protein denaturants, heat and proteases. However, selective protease digestion removed the fusion peptide region, but left the protein intact as a soluble homotrimer that dissociated from target membranes. This provides convincing evidence that the two regions of the protein have separable functions, one accounting for protein-membrane interactions and the other for protein-protein interactions. (Abstract shortened by UMI.).
Source: Dissertation Abstracts International, Volume: 65-09, Section: B, page: 4417.;Advisors: Margaret C. Kielian.