The genomes of retroviruses are remarkably small considering the complexity of replicative control which they encode. To attain this level of parsimony, the genomic RNA contains not only coding information for proteins, but also functional signals for transactivation, dimerization, encapsidation, splicing, and poly A addition near the 5' and 3' extremities. Each of these events is vital for productive replication of the virus. In the research proposed here, the structure and function of the cis-element(s) within the genomic RNA that promote dimerization of two copies of the full-length genome will be studied in detail within the context of the HIV- 2 virus, a causative agent for AIDS in humans. Specifically, the signal for dimerization of genomic RNA will be identified and characterized using RNA structure probing, chemical modification interference analysis, mutagenesis, and deletion analysis. Sites identified by these experiments will be further characterized using biochemical methods and a combinatorial in vitro evolution approach to determine the stability and mechanism of the interaction. Detailed secondary/tertiary structure maps of the 5' leader region and parts of the gag coding sequence of the genomic RNA will be constructed to better understand potential interplay between functional motifs in the region. Finally, mutant viruses will be constructed with deletion or mutations in their dimerization domains to assess their effects in vivo. These studies will provide a vital structural basis for developing new anti-retroviral therapies directed toward the functional regions of the genomic RNA.
