MOLECULAR MECHANISMS OF ALPHAVIRUS ENTRY AND EXIT

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DESCRIPTION (Adapted from Applicant's Summary): All enveloped viruses use membrane fusion to infect a cell, and must bud through a cellular membrane to produce progeny viruses. Molecular information on these processes is critical to the understanding of viral disease, the development of novel anti-viral agents, and as a model for cellular membrane fusion reactions. The well-characterized alphavirus, Semliki Forest virus (SFV), enters cells via low pH-triggered membrane fusion, and exits by budding through the cell plasma membrane. Fusion is mediated by the SFV spike protein, which undergoes a defined series of conformational changes at acid pH. A critical feature of the SFV fusion reaction is its striking dependence on the presence of cholesterol and sphingolipid in the target bilayer. Dr. Kielian has also described a novel requirement for cholesterol in the SFV exit pathway. The overall goal of this grant is to define molecular features of the entry and exit of alphaviruses from cells. Her major hypothesis is that both SFV fusion and exit involve specific interactions of the viral spike protein with cholesterol in the cell membrane. She has isolated and characterized an SFV mutant (srf-3) that has strikingly more efficient fusion and exit from cholesterol-depleted cells than wt SFV. A single amino acid substitution in the E1 spike subunit, a change of proline 226 to serine, is responsible for the cholesterol-independence of both srf-3 fusion and exit. In vitro mutagenesis of this domain and isolation of new srf mutants will be used to define sequences that confer alphavirus cholesterol independence. The hypothesis is that srf-3 is less cholesterol-dependent for key E1- membrane interactions in fusion and exit. This will be tested by a sensitive new fusion assay using purified lipid components, by biochemical and immunological detection of E1 conformational changes and membrane insertion, and by cryo-electron microscopy of control and sterol-depleted virus. Dr. Kielian has developed a biochemical assay for the final steps of budding and release of cell surface spike proteins into SFV virions. This assay is based on cell surface biotinylation of the spike proteins and virus retrieval using streptavidin-conjugated magnetic particles. This system will be used to determine the role of cholesterol in the exit of wt SFV and srf-3, to characterize virus exit requirements in intact cells, and as the basis of experiments to reconstitute the cell surface budding of SFV in a semi-permeabilized cell system.