Assembly and maturation are essential steps in the replication of orthoretroviruses such as HIV-1 and are proven therapeutic targets. These processes require the coordinated functioning of the viral Gag protein’s multiple biochemical activities. We describe here the development of an experimental system that allows an integrative analysis of how Gag’s multiple functionalities cooperate to generate a retrovirus particle. Our current studies help to illuminate how Gag synergizes the formation of the virus compartment with RNA binding and how these activities are modulated by the small molecule IP6. Further development and use of this system should lead to a more comprehensive understanding of the molecular mechanisms of HIV-1 assembly and maturation and may provide new insights for the development of antiretroviral drugs.