ABSTRACT, PROJECT 3 The HIV-1 structural protein Gag drives virus assembly resulting in infectious virus. During the assembly process, Gag coordinates the incorporation of essential viral components and interacts with host factors to facilitate virion production. The process of HIV-1 assembly is complex and choreographed, but not well understood mechanistically and dynamically. An essential step in assembling infectious particles is the packaging of the full-length HIV RNA genome (referred to as HIV-1 RNA hereafter). HIV-1 RNA is a minor RNA species in infected cells and yet it is packaged into the vast majority of the viral particles. Gag must anchor in the plasma membrane where it orchestrates particle assembly. However, the intricate interplays between Gag, the lipid membrane components, and the viral RNA genome that govern particle assembly are not understood. HIV-1 usurps host machinery during multiple stages of viral replication. Yet, little is known about cellular (host) factors that are necessary for, and their roles in, Gag trafficking and virus assembly. To study the mechanisms and dynamics of HIV-1 assembly, we have assembled an outstanding team with complementary scientific expertise. We will use multidisciplinary approaches including biophysical methods, structural analyses, biochemical and genetic assays, single-molecule and live-cell imaging techniques to study how infectious viruses are assembled and the dynamics/kinetics of virus assembly. Our studies focus on three knowledge gaps in HIV-1 assembly. In Aim 1 we will define the mechanisms whereby Gag selects HIV-1 RNA during virus assembly by examining the interactions between Gag and viral and nonviral RNAs. We will delineate the impact of nuclear export pathway usage on proteins that complexed with HIV-1 RNA. That Gag and host proteins are required to facilitate viral egress is known; however, less is understood which host factors are necessary for Gag trafficking and virus assembly. Using a novel approach, we have identified multiple host factors that associate with Gag. Furthermore, knocking out some of these identified host genes resulted in alteration of viral production, suggesting that they are dependency factors for assembly and/or trafficking. In Aim 2 we will elucidate how these protein dependency factors assist HIV-1 assembly and promote virus production. In Aim 3 using single-molecule imaging approaches and molecular modeling, we will define the interplays among Gag:lipid:RNA to provide insights into the mechanisms of virus assembly; these studies will be complemented by examining the dynamics and kinetics of cellular HIV-1 assembly using live-cell imaging approaches. Findings from the proposed studies will transform our knowledge of HIV-1 genome packaging mechanisms, advance our understanding of host-viral interactions required for virus assembly, and provide insights into the molecular mechanism of virus assembly and the dynamics of these events in T cells.