Project Summary/Abstract Understanding the nature of the molecular interactions involved in human immunodeficiency virus type 1 (HIV- 1) replication continues to provide important insights into the fundamental nature of retrovirus replication. Beyond the importance of such basic science investigations in addressing crucial knowledge gaps in the field, such studies can inform antiretroviral target identification, and have broad applications towards therapy and cure. One of the key aspects of HIV-1 replication that has remained underexplored has been virus particle assembly. The reasons for this have included the challenges associated with the detailed behavior of Gag translocation to the plasma membrane, the engagement of particle assembly sites, the molecular interactions that drive virus particle assembly, and subsequent particle biogenesis and morphology. Integrative, comparative retrovirology has been particularly informative in gaining a deeper understanding of these steps in retroviral replication. For instance, immature particle morphology and the flexibility of the immature Gag lattice differs among retroviruses, which have been insightful for understanding the role of gaps in the Gag lattice and its relationship to relieving stress of lattice curvature. Furthermore, the recruitment pathways for retroviral Gag punctum formation, as well as the role and nature of the actin cortex on assembly site selection and biogenesis also remain poorly understood aspects of the retrovirus assembly pathway. In this application, we propose to investigate comparative analysis of HIV particle morphology and particle biogenesis through innovative state-of-the-art experimental approaches. In particular, we propose to employ cryo-electron microscopy/tomography (cryo-EM/ET), total internal reflection fluorescence (TIRF) microscopy, photoactivated localization microscopy (PALM), dual-color z-scan imaging and double helix-point spread function (DH-PSF) imaging to perform 3D super-resolution imaging and 3D single particle tracking in living cells to investigate 1) high-resolution comparative structural analysis HIV particle morphology and maturation, 2) investigate Gag puncta biogenesis at particle assembly sites, and 3) investigate the role of the actin cortex as a physical barrier in human retrovirus particle assembly. These novel studies harness innovative technologies in order to provide new insights into a highly significant yet poorly understood aspect of the HIV-1 life cycle, address important knowledge gaps in the field, provide insights into potential targets for therapeutic intervention, and inform efforts toward next-generation HIV therapies.