Human T-cell leukemia virus (HTLV-1) has been estimated to infect 15-20 million individuals worldwide and is known to be the etiological agent of an adult T-cell leukemia/lymphoma (ATLL), an inflammatory disease syndrome known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and pathologies of the lung, skin, eyes, and thyroid gland. HTLV-1 is notorious for being extremely difficult to propagate in cell culture, which has prohibited rigorous analyses of virus replication, including the steps involved in retrovirus assembly. HTLV-1 spread is known to be heavily reliant on virus infection involving cell-to-cell contacts that form what is termed the virological synapse (VS), which represents the primary means for virus spread, including events associated with oral transmission. While HTLV-1 has been previously studied in regard to virus spread via cell-cell contacts, a significant knowledge gap exists regarding the nature of virus particle assembly and transmission via the VS. In general, virus particle spread through cell-cell contacts increases the likelihood of an infection event of virus particles that may possess low particle infectivity in cells without formation of VS. Previous studies have indicated that a low proportion of mature HTLV-1 particles possess an intact capsid core, suggesting that aberrant particle morphology could help to explain the poorly infectious nature of cell-free HTLV- 1. In order to address the current knowledge gap in the field, we propose in this exploratory application to develop workflows for state-of-the-art bioengineering and quantitative imaging technologies that hold high promise in being applied to the efficient study of HTLV-1 particle assembly and spread at the VS. First, we will develop a workflow for the use of cell micropatterning technology in order to reproducibly and efficiently create cell-cell contacts and investigate the role virus budding in virus spread. Second, we propose to establish an efficient workflow in which we can view cell-cell contacts utilizing high-resolution cryo-correlative light and electron microscopy in order to investigate the role of host cell proteins in virus assembly at cell-cell contacts. Development of these workflows will allow for quantitative analysis of virus particle biogenesis at cell-cell contacts. These technologies have broad applicability in virology and the success of this research will be applicable to a variety of questions regarding virus replication and virus-host cell interactions.