Very little is know about the patterns of virus-host protein-protein interactions that influence the pathogenic outcome of infections caused by the classical oncogenic retroviruses of animals and the complex retroviruses of humans and large mammals.
The specific aim of this project is to define the role of the EIAV-encoded proteinase (PR) in viral pathogenesis by identifying its substrate determinants, means of regulation and intracellular targets. EIAV is selected for study because in vitro models for persistent and cytopathic infection are available. We propose to devise an in vitro system in which the virus-specific proteolytic processes that naturally occur in mammalian cells during EIAV replication can be studied at the molecular level. We will analyze in vitro the synthesis of the EIAV-specific proteinase and its activity on a natural substrate. To this end, a DNA sequence specifying PR or gag- pol will be inserted into suitable plasmids just downstream of the promoter for bacteriophage T7 RNA polymerase. The RNA will be isolated in vitro and used to direct the synthesis of virus-specific PR in cell- free translation systems or expression strains of bacteria. The proteolytic activity of PR will be tested by assaying for autoprocessing of gag-pol or for digestion when a recombinant EIAV gag precursor is supplied in trans. cDNA libraries will be constructed from cells in which EIAV mounts cytopathic or persistent infections and screened for cellular proteins that can interact with EIAV gag. If these interactions affect the function of the cellular protein or place potential cellular substrates in the vicinity of activated viral PR, they would become important determinants of the pathogenic potential of both the classical and complex retroviruses.
