PROJECT SUMMARY During HIV-1 infection, the viral membrane fuses with the host cell membrane to deliver the HIV-1 virus to the host cell cytoplasm. Once within the host cell, HIV-1 must move to the nucleus so that the viral DNA generated by reverse transcription can be incorporated into the host genome. The host microtubule network and the microtubule-associated motor dynein support infection by facilitating HIV-1 transport to the nucleus. This proposal aims to determine how HIV-1 exploits the dynein machinery during transit to the nucleus. Dynein cannot walk on microtubules unless bound to a class of proteins called adaptors. In addition to activating dynein, adaptors also link dynein to cargo. Previously, it was hypothesized that HIV-1 bound to the adaptor BicD2 to hijack the dynein motor. We used single-molecule total internal reflection fluorescence (TIRF) microscopy to reconstitute HIV-1 motility via dynein. Unexpectedly, we found HIV-1 cores bind to dynein directly, unlike host-derived cargoes that require an adaptor to bind to the dynein motor. We also found that HIV-1 cores can “hijack” and move with dynein motors bound to many divergent adaptors. Attaching directly to the dynein motor is likely a viral adaptation that ensures HIV-1 can exploit multiple dynein cargo adaptors for motility in situ. Our long-term goal is to understand the molecular basis of the dynein-mediated trafficking of HIV-1 so that we can eventually develop tools that disrupt the translocation of HIV-1 to the host nucleus without affecting native host trafficking. To facilitate this goal, we have developed the following Aims. In Aim 1, we will use the TIRF-based motility assay we developed to determine how HIV-1 recruits and activates retrograde motility via dynein and associated adaptors. We will also determine if HIV-1 hitchhiking on dynein- cargo complexes affects normal dynein-mediated cargo trafficking. In Aim 2, we will use single particle cryo- EM and cryo-electron tomography of dynein-HIV-1 complexes assembled in vitro to determine the binding site of dynein on the HIV-1 capsid lattice. Biochemical studies in our lab suggest that dynein has two binding sites for HIV-1: the heavy chain dimerization domain and the intermediate/light chains. We will characterize this interaction using individual components bound to HIV-1 capsid in addition to cryo-electron tomography of dynein-HIV-1 complexes on microtubules in vitro. Finally, in Aim 3, we will determine the cargo adaptors that HIV-1 exploits in T-cells and determine if flexible use of dynein cargo adaptors facilitates replication in other cell types. These Aims will show how HIV-1 utilizes a direct interaction with dynein as a flexible platform to navigate the cytoplasm to the host cell nucleus. These experiments will continue to expand our understanding of early steps in HIV-1 replication by providing a framework to understand how HIV-1 reaches the host cell nucleus.