The goal of this research is to define biochemical mechanisms of avian retrovirus replication and the role of viral proteins in this process. These are multidisciplinary model studies that have direct application to development of anti-HIV agents. The first specific aim of this proposal is to understand the role of the matrix (MA) protein in viral replication. Experiments are proposed to further analyze the biological effect of point mutations in MA. Initial results suggest that the RSV MA has at least two additional biological functions in replication besides structural and transport roles. One of these, may be related to virus entry into the cell and/or uncoating of the virus particle. The other may be related to RNA packaging during assembly. Experiments are proposed to examine specific packaging of viral RNA by the RSV Gag protein in vitro and in vivo and directly evaluate a role for MA in this process. These studies are particularly important since they analyze the first phosphorylation site protein mutants in retroviral replication that alter growth in vivo. RNA mapping studies are also proposed to extend our knowledge of what constitute an RNA packaging signal for avian retroviruses. The second specific aim is to understand the mechanism of initiation of reverse transcription. These studies will continue to address the role of secondary structure in the U5 region of viral RNA in initiation of reverse transcription through the analysis of mutations in U5 RNA. Chemical and enzymatic mapping techniques will also be employed, both to confirm U5 structures already genetically defined and to probe the nature of initiation complexes formed with RT. Furthermore, efforts will be made to establish if there are sequence specific contacts between RT, U5 RNA, and primer in forming the initiation complex. In a separate line of investigation, we will characterize second site revertants which spontaneously arose from viruses containing deletions in RNA structures required for initiation of this process. We have also note that U5 sequences required for initiation of reverse transcription are homologous to known leader RNA packaging signal. We will therefore determine if US sequences contain overlapping functions for reverse transcription and RNA packaging.
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