Abstract Human T-cell leukemia virus (HTLV-1) infects about 15-20 million individuals worldwide and is the etiological agent of an adult T-cell leukemia/lymphoma (ATLL), and can also result in an inflammatory disease syndrome called HTLV-1-associated myelopathy (HAM)/tropical spastic paraparesis (TSP). HTLV-1 antibody prevalence rates vary among geographical areas, ranging from 0.2 to 10% among adults. This antibody prevalence increases with age, and can affect as much as 20 to 50% of the female population aged 60 and above. HTLV-1 is notorious for being difficult to study in cell culture, which has prohibited a rigorous analysis of how these viruses replicate in cells, including the steps involved in retrovirus assembly. The details for how retrovirus particle assembly occurs are poorly understood even for other more tractable retroviral systems like that of human immunodeficiency virus type 1 (HIV-1). For instance, recent evidence indicates that Gag-Gag interactions differ among retroviruses, which helps explain morphological differences that we have documented among immature retrovirus particles. Furthermore, the role for membrane-bound, non-punctate (np) Gag in the biogenesis of Gag puncta, as well as the nature of Gag puncta biogenesis in the context of cell-to-cell contacts also remain poorly understood aspects of the retrovirus assembly pathway. This is particularly for HTLV-1, for which we have found to have fundamentally distinct differences to that of HIV-1 regarding the role of membrane-bound np Gag in Gag punta biogenesis. In this application, we propose to continue our investigations on HTLV-1 immature and mature particle structure and particle biogenesis through innovative state-of-the-art experimental approaches with appropriate comparative analyses with HIV-1. In particular, we will apply cryo-electron microscopy/tomography (cryo-EM/ET), photoactivated localization microscopy (PALM), total internal reflection fluorescence (TIRF) microscopy and the novel technology of z-scan fluorescence fluctuation spectroscopy (FFS) in living cells to investigate 1) analysis of immature HTLV-1 Gag lattice structure, 2) the importance of membrane-bound, np Gag in HTLV-1 particle biogenesis, and 3) HTLV-1 particle biogenesis in the context of cell-cell contacts. Careful comparisons will be done with HIV-1. These novel studies harness innovative technologies in order to provide new insights into a highly significant and poorly understood aspect of the HTLV-1 particle assembly process, which is fundamentally distinct from that of HIV-1 and other retroviruses. Furthermore, our studies represent some of the most detailed studies conducted on the assembly of virus structural proteins, which ...