Retroviruses are single-stranded, positive-sense RNA viruses that cause many diseases in a variety of species. During the replication cycle, the retroviral genome generates three species of viral RNA (vRNA): the first is an unspliced vRNA that serves as the template for the translation of the Gag and Gag-Pol proteins, the second is an unspliced vRNA that is packaged into newly formed virions, and the third is a spliced vRNA that serves as the template for translation of Env and other viral proteins. Studies in our laboratory of the avian oncoretrovirus Rous sarcoma virus (RSV) demonstrated that the Gag transiently traffics through the nucleus, and this step is linked to efficient packaging of the genome into virions.Retroviruses are unique in that they package their genomes into newly formed virions as a non-covalently linked genome dimer. Disruption of genome dimer formation in any manner reduces the infectivity of any newly formed virions, which has motivated researchers to further characterize this step in the retrovirus replication cycle. Genome dimerization has been characterized to involve two cis-acting sequences in the 5’ UTR of the genome. The first is a dimer linkage sequence (DLS), a sequence initially identified in RSV via electron microscopy images of genome dimers isolated from virions demonstrating a physical linkage between two strands of viral RNA. The second is a dimer initiation site (DIS), a sequence identified during deletion analysis experiments of the 5’ UTR of retroviruses. The DIS has been characterized to be a stem-loop containing a palindromic sequence across the top of the stem-loop allowing for intermolecular interactions.In this study, we sought to answer questions about genome dimerization in RSV using a combination of single molecule microscopy techniques including RNA stem loops and fluorescent-tagged coat proteins and RNA FISH. These techniques allowed us to observe single molecules of RNA in the context of a whole cell. We determined the subcellular location of RSV genome dimerization, the composition of genome dimers in cells and in virions, and whether Gag played a role in genome dimerization. Additionally, we used microscopy techniques to visualize genome splicing in RSV. Live-cell imaging techniques were used to observe genome dimerization and genome splicing in real-time and measure the kinetics of these processes.Taken together, we propose a model in which a threshold of nuclear Gag is functional in facilitating genome dimerization and possibly genome splicing. The expression of additional nuclear-trapped Gag in trans does not increase the number of genome dimers formed in the cell. We propose that nuclear Gag serves to regulate efficient dimerization and packaging of genomes. Further study of these functions in RSV, and in other retroviruses, will increase our understanding of the role of nuclear Gag in retrovirus replication and infectivity.