Formation of HIV particles is driven by Pr55Gag precursor protein, and Pr55Gag is also the critical determinant to select HIV RNA genome for virion packaging. Full-length recombinant HIV Pr55Gag proteins have not been available in the past 30 years due to technical limitation, making it difficult to precisely define the biophysical and biochemical properties that drive the formation of HIV particles. We have since generated large amount of full-length recombinant HIV Pr55Gag protein for biochemical and biophysical analyses. This presentation comprises of a number of publications from our recent past, detailing how we have defined the protein and RNA requirement on genomic RNA packaging, plus the interplay amongst Gag oligomerization, lipids, RNA, and the thermodynamic properties that drive the formation HIV particles. These discoveries have revealed both novel and practical tools that can be used for the development of antiviral against HIV.