Little is known about the process through which viral proteins and RNA assemble on the plasma membranes of host cells. The investigators have been studying this process in vitro and find that these complexes can be reconstituted to give particles that resemble immature virion assemblies morphologically and functionally, and thus provide a way to study, at the molecular level, the assembly pathway leading to infectious particle formation. In this proposal, they will test a model for viral assembly that involves initial membrane interactions involving several domains of Gag, that are replaced with protein-protein and protein-RNA interactions as assembly proceeds. They propose that myristylation plays a role in orienting viral Gag on the membrane surface. Further, they propose that a highly conserved region in the capsid domain transduces the conformational changes that occur upon membrane association and find that mutations in this region severely impair assembly. Processing of Gag releases MA causing altered protein contacts mediated through a particular portion of the protein. This serves to weaken MA interaction with the membrane surface, thus allow it to be transported to the host cell nucleus during a new round of infection. To identify properties of the nucleoprotein complexes that might be targeted for design of anti-viral agents, Dr. Scarlata proposes using Gag, the viral protease, and RNA containing the encapsidation signal in the 5' end of the viral genome to define the macromolecular interactions of functional reconstituted nucleocapsids. The applicant will first model by characterizing the in vitro assembly of wild type Gag in terms of conformation, orientation, and role of myristylation using fluorescence energy transfer; nucleoprotein assembly will be monitored by gel-shift, centrifugation, and protection from nuclease degradation; functional competence will be assessed by PR assay; and limited proteolysis and hydrolysis with nucleases will localize altered regions in Gag and RNA, respectively. Second, the applicant will compare the assembly of Gag proteins with mutations in this highly conserved region. Third, the applicant will examine the role of a region of MA in weakening membrane interactions. The proposed study makes it possible to identify critical interactions required for assembly and to design assembly inhibitors.
