Elucidating HIV-1 nuclear trafficking to integration sites - Project Summary The nuclear entry and transport of HIV-1 to genes for viral (v)DNA integration is determined by viral capsid and its interaction with the host cellular polyadenylation specific factor CPSF61-3. In the absence of CPSF6 interactions, HIV-1 remains tethered to nuclear pore complexes (NPCs) and integrates outside of genes in lamin associated genomic domains or LADs3, 4. We have made a seminal discovery2 that CPSF6 interactions drive HIV-1 replication complexes to accumulate in phase-separated membraneless compartments called nuclear speckles, in multiple cell-types including primary CD4+ T-cells and macrophages. Notably, nuclear speckles are formed at sites of transcription and are known to sequester actively transcribing genes5. As such our finding indicated that the speckle associated genomic domains or SPADs are the primary targets for HIV-1 vDNA integration. The scientific premise of this proposal is to delineate the mechanisms of how? HIV-1 navigates the nucleoplasm to reach speckles and integrate into SPADs, a key step in viral replication that remains under- explored and poorly understood. We hypothesize that the intrinsically disordered low-complexity regions (LCRs)6 and mixed charged RSLD-domains of CPSF6 cooperatively recruit host-factors including nuclear filaments to capsids for its release into- and transport to nuclear speckle compartments for integration. We will use novel technologies developed in our lab and, (1) Determine a role for CPSF6-RSLD domain phase-separation in releasing HIV-1 from NPCs, and in recruiting new host-proteins including speckles for virus nuclear transport, (2) apply live-cell imaging to visualize HIV-1 nuclear co-trafficking with CPSF6, nuclear actin and nuclear speckles and determine the contribution of individual CPSF6 LCRs and RSLD domains to this process, and (3) determine the role of nuclear speckles, and proximal genomic architecture in biasing vDNA integration into genes. We expect these studies to provide key mechanistic insights into CPSF6-capsid interactions that mediate nuclear HIV-1 trafficking to integration sites, identify novel SPADs that respond to virus infection and highlight its implication on global genome architecture, including virus-cell biology.
