The propagation of all retroviruses is dependent on the proper assembly of infectious viral particles. A key element is the production and correct proteolytic cleavage of polyprotein precursors to virion structural proteins. Cleavage of protein precursors is an ordered process that appears to be driven by a protease that is itself encoded by the retroviral genome. This application proposes to study the nature of the cleavage process and early events in virion assembly of HIV. A major goal is to alter both the dose and timing of protease action and to mutate the protease itself in order to abort the production of infectious viral particles. To study the nature of the cleavage process we will use a cell- free translation system to produce HIV polyproteins and subject them to cleavage using the virion associated protease. Determination of the exact sites of cleavage will then allow us to test a series of synthetic peptides containing the cleavage sites in order to competitively inhibit specific processing. This may provide a rational basis for designing inhibitory peptides for introduction into infected cells. We plan to explore the possibility that proteases from other retroviruses will incorrectly process HIV precursors. In order to interfere with virion assembly in infected cells we plan to take advantage of the fact that HIV appears to regulate the levels, location, and timing of protease action. Inducible protease-expression vectors will be introduced into cells and their effect on HIV multiplication assayed. We also shall use heterologous retroviral proteases, which have been determined to miscleave HIV proteins, as well as randomly mutated proteases in order to develop dominant negative vectors capable of aborting virion production by HIV. To analyse early events in viral assembly we will study the interaction of HIV gag and gag-pol polyproteins with the cell membrane. A key goal will be to ascertain whether specific regions of the viral proteins interact with specific membrane components which may mediate virion assembly.
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