The objectives of this proposal are to learn about the principles that govern the assembly of retrovirus particles at the plasma membrane of the infected cell. The formation of virus particles requires protein-membrane interactions, protein-protein interactions, and protein-RNA interactions. In addition, proteolytic cleavages take place in the maturation of virus particles. Using as a model system the avian sarcoma and leukemia viruses, we will continue to investigate each of these aspects of assembly. 1. Point mutations that render the viral protease defective will be introduced into the viral genome, and quail cell lines will be derived that produce the defective particles. The biochemical properties of these presumably immature virus particles will be characterized. Also, the regulation of protease activity will be studied in vitro, from partially purified protease fusion proteins obtained from an expression vector in E. coli. 2. The site of interaction of gag protein with lipids in the viral membrane will be studied, using different cross-linking agents. Both normal virus and protease-defective virus containing the uncleaved gag precursor protein Pr76 will be analyzed. Further, the interaction of Pr76 made in vitro with chicken membranes will be characterized to learn if this is a biologically relevant model. Studies will be continued to reconstitute phospholipids around delipidated immature virus cores. 3. Detailed deletion and linker scanning mutagenesis of defined 5' and 3' regions on the RNA will be carried out, in order to learn exactly what RNA sequences are required for an RNA to be recognized and efficiently packaged into virus particles. The interaction of Pr76 with viral RNA will be studied in vitro, and in vivo using protease-defective particles and cross-linking techniques. The intent of these experiments is to learn what RNA sequences Pr76 interacts specifically with, and what domains on Pr76 are involved in this interaction. Also, a series of experiments will be carried out to learn what gag proteins interact with newly synthesized viral DNA. 4. The minimum portion of the gag gene needed for formation of a virus particle will be determined using a series of nested deletion mutants. The mechanism by which gag protein inside the virus particle interacts with env protein on the surface will be investigated using several different crosslinking agents. Finally, an attempt will be made to clone a chicken gene that when introduced into mammalian cells, allows these normally non- permissive cells to assemble avian retrovirus particles.
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