Project Summary/Abstract The deltaretroviruses represent one of seven genera in the Retroviridae and are recognized as being complex retroviruses that encode for gag, pol and env along with several important accessory genes. These viruses have been associated with the development of lymphoid leukemias in humans (i.e., human T-cell leukemia virus, HTLV) and cattle (i.e., bovine leukemia virus, BLV). Deltaretroviruses are notorious for being difficult to propagate in cell culture, which has prohibited rigorous analyses of virus replication, including the steps involved in retrovirus assembly. The detailed steps involved in retrovirus assembly are poorly understood even for the more tractable systems, including human immunodeficiency virus (HIV). Our preliminary studies have revealed that the deltaretroviruses have distinct immature and mature particle morphologies compared to other retroviruses, as well as distinct Gag-Gag and capsid-capsid (CA-CA) lattice structures, which likely impacts virus particle formation. Furthermore, we have made observations which reveal that HTLV Gag puncta biogenesis is distinct among retroviruses, relying heavily on Gag recruitment to particle assembly sites by Gag already at the plasma membrane. This distinctive pathway for particle assembly likely has important implications for establishment of HTLV particle assembly sites and the dynamics of particle assembly. In this application, we propose to advance our research in novel directions through innovative state-of-the-art experimental approaches, making use of comparative retrovirology in order to provide deep insights into the nature of deltaretrovirus particle assembly and maturation. In particular, we will apply cryo-electron microscopy/tomography (cryo-EM/ET), super-resolution microscopy, total internal reflection fluorescence (TIRF) microscopy, and dual-color z-scan microscopy to investigate the 1) structure-function analysis of deltaretrovirus maturation, 2) deltaretrovirus Gag recruitment and puncta biogenesis, and 3) analysis of critical steps in deltaretrovirus particle biogenesis. The proposed studies will extend our preliminary studies and exploit advanced imaging technologies to gain new insights into the deltaretrovirus particle assembly pathway, which we have demonstrated as being distinct from other retroviruses. This research will provide foundationally important knowledge regarding the details of virus assembly at the plasma membrane for enveloped viruses.