PROJECT SUMMARY HIV remains a major global health burden, in large part because of its massive evolutionary potential to rapidly evolve escape mutations even from multi-drug therapies. This potential has also allowed HIV to evade the suite of human cell-intrinsic restriction factors that represent the first line of immune defense against infection. In particular, the viral capsid is a critical target of cell-intrinsic immunity, yet human capsid-targeting proteins like TRIM5⍺, MxB, and TRIM34 have almost no ability to inhibit HIV. Although viral sequencing identifies the many capsid mutations sampled by natural isolates, it cannot help to parse the selective pressures that drove this suite of mutations. For example, there is limited information on which capsid mutations allow escape from human restriction factors, and which mutations are disallowed for interaction with virus-required host factors. This knowledge gap leaves us with little insight into the mechanism of capsid targeting by restriction factors, and insufficient information to design capsid-targeting therapies that can evolutionarily “box in” the virus to prevent simultaneous escape from therapies and native restriction factors. This proposal will fill this knowledge gap by comprehensively defining these selective pressures for the HIV-1 capsid, using a high throughput saturating mutagenesis approach. This approach relies on the targeted introduction of all possible single missense mutations at all positions in the HIV-1 capsid, followed by direct measurement of their fitness under different cellular conditions. Using innovative new tools to overcome long-standing technical barriers to the stable production of HIV-1 viral libraries, this proposal will apply saturation mutagenesis to determine the landscape of all deleterious or allowed capsid mutations for completion of the viral life cycle as well as sensitivity to human restriction factors. These data will comprehensively define the capsid surfaces and biochemical moieties recognized by multiple first-line immune defense proteins, which has eluded definition despite decades of study. Moreover, these data will open a new avenue for designing HIV drug therapies with an evolutionary lens; this work will identify Achille's heels of the HIV capsid, which can be targeted by therapies such that viral escape will necessarily sensitize it to immune defense.