ABSTRACT The highly conserved 5' untranslated region (UTR) of the HIV-1 genome plays a central role in regulating viral replication. Groundbreaking NMR experiments, along with significant biochemical data support a model in which the 5'UTR can transition between at least two conformational states: in one state the genome remains a monomer, leading to translation of the viral genes; the second state is competent for dimerization and packaging into assembling virions. Therefore, according to this model the conformation of the 5'UTR determines the fate of each genome molecule. How the virus maintains an appropriate balance of genomes fated to packaging and translation remains unknown. But significant evidence indicates that viral proteins, as well as, potentially, host factors, facilitate switching between these functional states of the 5'UTR. The viral Gag protein in particular contains two RNA-binding domains, the nucleocapsid (NC) and matrix (MA) domains. Efficient recruitment of the dimerized genome into assembling virions occurs by way of specific interactions between the NC domain of Gag and the 5'UTR. As a nucleic acid chaperone, NC facilitates folding of the genome into thermodynamically favorable conformations that likely favor dimerization and packaging. In contrast, the MA domain counteracts the activity of NC either through interaction with NC or by modulating the structure of the 5'UTR. We have established a single-molecule Frster resonance energy transfer (smFRET) imaging approach to visualize the conformational dynamics of the 5'UTR. In our approach individual UTR molecules carrying donor and acceptor fluorophores are surface immobilized and imaged with total internal reflection fluorescence (TIRF) microscopy. Here, we will further develop this approach to elucidate the order and timing of 5'UTR conformational changes, dimerization, and Gag binding events. We will test the prevailing model that the 5'UTR adopts a distinct conformation prior to dimerization and packing. We will generate a more complete understanding of how Gag modulates the structure of the 5'UTR, thereby regulating viral replication.