Project Summary/Abstract It is therapeutically advantageous to inhibit HIV at early stages of its replication cycle owing to the permanence of the integrated provirus and the persistence of viral latency in vivo. To rationally design new inhibitors of early replication steps, a detailed molecular understanding is required. We propose integrated biochemical, biophysical, and cellular approaches to probe critical steps in early viral replication. Recent studies found that only a minority of capsids are transported intact through the nuclear pore and uncoat in the nucleus successfully integrate. The HIV capsid is a promising target for therapeutic intervention owing to the critical timing of its dissolution, but it remains unknown how uncoating after viral DNA synthesis is initiated and what molecular interactions control its timing. That successful capsid uncoating occurs inside the nucleus explains why human host APOBEC3 (A3) proteins must be packaged into the virus in producer cells to successfully inhibit reverse transcription. Because so few A3 proteins are packaged, these proteins must be extremely efficient inhibitors. Modified A3 super-restrictors have been developed but how they inhibit retroviral replication remains a critical knowledge gap. Viral genome transcription after successful integration is regulated by many proteins, including HIV-1 Vpr. Although Vpr alters cellular and viral genome transcription, the interactions that enhance HIV transcription and alter cellular gene expression are unknown. To address these questions, three aims are proposed: (1) Determine the roles of HIV-1 nucleocapsid, IN and Vpr proteins in regulating the timing of capsid uncoating during viral reverse transcription. To test the hypothesis that NC compacts double- stranded DNA to regulate capsid uncoating, we will measure the DNA condensing activity of wild type and mutant HIV-1 NC. We will correlate the results with live cell studies of capsid uncoating and reverse transcription, as well as overall viral replication, in the presence of the same NC mutations. (2) Determine how APOBEC3 (A3) super-restrictors optimize inhibition of HIV-1 reverse transcription. To test the hypothesis that different DNA-bound forms of A3 super-restrictors have different enzyme activities and mechanisms of HIV-1 replication inhibition, we will measure the DNA binding characteristics of effective super-restrictors. We will compare our results with measurements of A3 packaging, reverse transcription inhibition, deaminase-dependent and deaminase-independent replication inhibition, and the resistance of the super-restrictors to HIV-1 Vif countermeasures. (3) Determine how HIV-1 Vpr regulates nucleosome accessibility and transcription. To test the hypothesis that Vpr binds DNA and alters nucleosomes to facilitate transcription of HIV-1 and cellular genes, we will measure how Vpr and its mutants alter nucleosome stability as a function of DNA sequence. Results will be directly compared...