Not enough is known about retroviruses to prevent the diseases they cause, and a better understanding of their replication cycle is needed. Some of the least understood steps are those of virion assembly. The Gag protein encoded by each retrovirus plays the central role in assembly. Expression of Gag results in the formation and release of virus-like particles from the plasma membrane, even in the absence of all the other components encoded by the virion. Experiments described in the application continue to focus on Gag-mediated particle assembly. During the previous funding period, the applicant's lab dissected the Gag proteins of Rous sarcoma virus (RSV) and HIV. In each, three small functional domains were found that are essential for budding: the M (membrane-binding), I (interaction), and L (late) domains. Surprisingly, these domains turned out to be fully interchangeable between the two Gag molecules even though they share no sequence homology. Moreover, the order of M, I, and L is different in each case. It is important to learn how the parts of the """"""""particle-making machine"""""""" work to direct the budding process. Towards this end, five Specific Aims are proposed.
The first aim i s focused on the structure and function of the M domain of RSV, shown to be very different from that of HIV. The immediate goal is to determine the three dimensional structure of this domain by NMR and crystallography, and to test the resulting models about how it works. Alternative strategies are proposed toward this end.
The second aim addresses the question of how proteins destined to reside on the cytoplasmic faces of membranes specifically find their targets. For this, a complementation assay will be exploited that was developed in the previous funding period.
Aims 3 and 4 address specific questions regarding the I and L domains. The last aim addresses the determinants that control the rate of budding and the sizes of particles that are produced. A variety of approaches o find host proteins that potentially participate in the budding process are also proposed within several of the specific aims.
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