The central hypothesis of this proposal is that HIV-1 Gag assembles into immature capsids through a stepwise, energy dependent pathway of intermediates that contain cellular factors important for Gag targeting and assembly, as well as HIV-1 genomic RNA encapsidation. Using an antibody to the ATP-binding protein ABCE1, which functions during assembly and is present in these intermediates, we can track and isolate these intermediates. Here we propose to use the assembly pathway as a temporal and spatial framework for understanding the role of a variety of cellular factors in human cells in Gag targeting and assembly as well as in genomic RNA packaging. In aim 1 we propose to further test the hypothesis that the ABCE1 pathway is universally used during Gag assembly in cells by examining whether Gag constructs that contain only the minimum necessary domains for assembly utilize this pathway. In aim 2, we will examine at what point during the assembly pathway HIV-1 genomic RNA becomes specifically encapsidated. In aims 3 and 4, we will follow up our recent identification of the assembly intermediate responsible for relocating Gag from the cytosol to membranes. We will examine the role of AP-3delta, PIP2 in membrane targeting of this ABCE1-containing intermediate. A proteomics approach is proposed for identification of other factors in this intermediate that may be important for directing membrane targeting. Immunogold EM will be utilized to demonstrate colocalization of cellular factors in ABCE1-containing assembly intermediates. Finally, in aim 4, we will further test the hypothesis that a block at this intermediate is responsible for the failure of Gag to assemble in murine cells. This hypothesis is suggested by preliminary data indicating that Gag in murine cells is arrested in the form of this membrane targeting intermediate. Mass spectrometry will be used to identify cellular factors in this intermediate that overcome the targeting and assembly block in murine cells. Together these biochemical and ultrastructural approaches will give us an integrated view of how a variety of cellular factors act sequentially to promote targeting and assembly of HIV-1 Gag in human cells.