Project Summary Immature virus particle structure can provide valuable clues to the nature of virus particle assembly in cells. In this proposal, we sought to decipher key structural details of immature retroviral Gag assemblies by obtaining high-resolution structures using cryo-electron microscopy (cryo-EM). Reconstructions from the immature Gag structure of distinct types of virus-like particles reveals distinct similarities among Gag lattice organization of the viruses studied to date. Structural insights have enhanced from a comparative analysis human immunodeficiency virus type 1 (HIV-1) cryo-EM reconstructions regarding the importance of the capsid (CA) carboxy-terminal domain (CTD) and spacer peptide 1 (SP1) regions in forming hexameric assemblies of CA in the intermolecular contacts of the overall lattice structure. In order to gain a fundamental understanding of retrovirus assembly and maturation, atomic resolution maps of immature particles for other members of Orthoretrovirinae are required. Here, I propose a strategy, i.e., cryo-electron tomography guided single particle reconstruction (Cryo-ET guided SPR), to achieve atomic resolution reconstruction maps of immature retrovirus particles. Initial efforts at streamlining the EM methodology to resolve immature Gag lattices using the strengths from each imaging processing technique combined into a new approach. I will apply this methodology to study HIV type 2 (HIV-2) and human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 has an immature Gag lattice that forms a uniquely flattened capsid that is not observed in other retroviruses morphologies, such as with HIV-1. My central hypothesis is that HTLV-1 Gag CA protein mediates unique intermolecular contacts in the immature Gag lattice that allows hexameric Gag conformations to generate a flattened morphology that does not exist within immature HIV viral particles. By studying the CA structures of HIV-2 and HTLV-1, I expect to generate biophysical and structural information to provide fundamental aspects of virus particle assembly and Gag architecture. To expand the biological significance of this proposed research morphological aspects of authentic HIV-2 and HTLV-1 immature virus particle capsid lattices will be analyzed and compaired to the more ordered viral-like immature particles used for high-resolution efforts. A single particle and tomography cryo-EM hybrid approach of both capsid lattices will provide novel insights into this flattened CA morphology as well as the conformational flexibility of Gag in forming immature capsid structures. Identifying structural differences in the Gag lattice organization among closely related human retroviruses sheds new details on virus particle assembly that will ultimately identify novel drug targets for antiretroviral therapies targeting Gag capsid lattice formation.