Retroviruses are important etiological agents for human disease, most notably causing acquired immunodeficiency syndrome (AIDS). Additional human retroviral diseases are likely to be discovered, and these could possess or acquire the ability to spread rapidly through the population. At the present time, there are no cures or suitable vaccines to fight retroviral diseases. Thus, it is necessary to learn as much as possible about these viruses to provide inspiration for novel approaches to control the diseases that they cause. One of the least understood parts of their replication cycle is the process of retrovirion assembly. What is clear is that the Gag proteins they encode play a central role. Expression of the Gag protein results in the formation and release of virus-like particles from the plasma membrane, even in the absence of all the other components of the virion. The focus of this Continuation Application is Gag-mediated particle assembly. We have chosen Rous sarcoma virus (RSV) for our investigations because it is one of the best understood retroviruses and has provided much of what is already known about assembly. At the foundation of our proposed experiments is a rapid and efficient transient expression system, developed during the previous funding period, in which high levels of the RSV Gag protein are made in mammalian cells. Particles are released from the cells with full efficiency by budding from the plasma membrane and contain all of the proper Gag cleavage products. Electron microscopy has shown that the particles have an appearance that is indiscernible from authentic RSV. Using this system, we have recently obtained compelling evidence that suggests that the RSV Gag protein can be diverted to the receptor for pp60(src) (on the cytoplasmic face of the plasma membrane) for particle formation. This finding implies that all of the functions required for particle formation, except for membrane binding which also requires a cell-encoded receptor, are self-contained within Gag itself. Experiments are proposed to explore these ideas by placing appropriate signals on Gag to direct it to other novel membrane sites for particle formation. We have also completed an extensive deletion analysis of the RSV Gag protein and found that surprisingly large sections of this protein can be deleted without impairing the formation and release of particles. Collectively, these non-essential regions amount to 503 of the 701 amino acids in RSV Gag (72%). Moreover, our analysis has suggested the existence of three discrete, movable domains that are essential for particle formation. Defects in two of these domains can be complemented in trans, but deletions in a third cannot, suggesting that it is the region at which Gag-Gag interactions occur. Experiments are proposed to thoroughly analyze the properties and functions of these assembly domains, including their potential role in retroviral protease regulation.
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