DESCRIPTION: The overall goal of this proposal is to improve our understanding of the molecular mechanisms involved in retroviral RNA packaging, particularly for HIV-1, by investigating the nature and specificity of RNA and protein sequences that participate in packaging. During assembly, retroviruses incorporate two full-length viral RNA genomes into the budding particle. This process depends upon specific sequences in the viral RNA and in the Gag protein, and may involve additional viral and cellular proteins. The specific aims of this proposal are to precisely define in vivo the functional secondary structure of retroviral RNA packaging sites by using a newly developed iterative in vivo genetic selection technique and to identify the complete set of viral proteins that contribute to the specificity of RNA packaging through the construction and analysis of viral chimeras. The results of the studies proposed here should make important contributions to both our understanding of retroviral RNA packaging and our knowledge of protein-RNA interactions in general. The characterization of the precise elements involved in HIV-1 packaging will provide the background to future X-ray crystallographic investigations of HIV-1 RNA-protein complexes and should lay the foundation for identifying and optimizing chemotherapeutic agents that inhibit this highly specific and essential aspect of the virus life cycle.