The defining hallmark of A3-mediated restriction, explaining genomic plus strand G-to-A mutations in patient-derived viral sequences. However, the virus deploys a counteraction strategy that utilizes the virus encoded “virion infectivity factor” (Vif) to polyubiquitinate and degrade the A3s through a cellular E3-ubiquitin ligase complex. Here, I will use robust and unbiased experimental approaches to 1) identify separation-of-function Vif mutants that display differential activity against A3D and A3F restriction enzymes and 2) define the cellular mechanisms that regulate anti-HIV-1 activity of the A3s. My studies will utilize both hypothesis- and technology-driven approaches and a combination of fundamental virology, genetics/genome engineering, cell biology, and biochemistry techniques. I anticipate that a better understanding of the Vif/A3 surfaces and the underlying cellular mechanisms that govern A3 antiviral activity has the potential to lead to novel strategies to boost the anti-HIV-1 activities of these enzymes and contribute to the overall NIAID priority of “supporting innovative strategies for treating or curing HIV infection.”