Summary/Abstract In this Project, we will analyze the structures and molecular transformations of the HIV-1 RNA molecules that become packaged into virions as genomic RNA (gRNA). Beginning with events in the cell, we will elucidate how transcription start site usage defines the structure of the HIV-1 RNA 5’-leader, which, in turn, dictates whether the RNA will dimerize and function as gRNA or remain monomeric and function as messenger RNA. We will study how the dimeric gRNA recruits the viral Gag protein to generate a complex that nucleates assembly of the immature virion. Finally, we will look at processes that occur during virion maturation, in which the gRNA condenses into a viral ribonucleoprotein complex, is re-packaged inside the mature capsid, anneals with the tRNA primer, and prepares for reverse transcription. Our proposed studies will provide deep fundamental understanding of these processes, which represent some of the most critical but incompletely elucidated molecular mechanisms in HIV-1 replication. Importantly, we are now also poised to exploit this understanding towards the identification, characterization and development of novel inhibitors targeted against HIV-1 RNA and RNA complexes. Collectively, our team of investigators have a strong track record and expertise in determining structures of large RNAs and RNA:protein complexes using state-of-the-art techniques; in analyzing their biochemical and biophysical properties in vitro, in virions, and in cells; in visualizing the complexes as they proceed through their programmed processes and pathways in the cell; and in applying genetic and virological tools to identify and characterize host and viral co-factors, probe key mechanistic details, and test small molecule inhibitors.