ABSTRACT APOBEC3B (A3B) is an innate immune enzyme capable of introducing mutations in viral genomes and has been implicated in the restriction of several viruses. In addition, A3B is unique as the only human DNA deaminase family member that is constitutively nuclear. Recent studies have shown that -herpesviruses, such as Epstein-Barr virus (EBV), have evolved potent mechanisms for A3B neutralization and re-localization. The ribonucleotide reductase of EBV, BORF2, is capable of binding and relocalizing A3B from the nuclear compartment to cytoplasmic aggregates. Therefore, nuclear localization has been inferred to be essential for innate antiviral function. Regarding A3B subcellular localization, we have previously shown that A3B nuclear localization requires the presence of three residues within the N-terminal half of the protein and that nuclear localization is not a conserved feature of A3B across non-human primates, despite a need for antiviral function in the nuclear compartment. For instance, although human and several non-human primate A3B enzymes are predominantly nuclear, rhesus macaque and other Old World monkey A3B enzymes are clearly cytoplasmic. A series of human/rhesus macaque chimeras and mutants combined to map localization determinants to the N- terminal half of the protein with residues 15, 19, and 24 proving critical. Despite advances in our knowledge of A3B, the mechanism of nuclear localization and the broad role of subcellular localization in virus restriction activities remain uncharacterized. Taken together, we hypothesize that A3B uses a non-canonical mechanism of nuclear import and that nuclear localization is a key feature of A3B’s role as a restriction factor. This hypothesis will be tested through two complementary Specific Aims. The first will identify the molecular mechanism of A3B nuclear import through evolutionary and proteomics approaches, using the human/rhesus macaque chimeras described above. The second will address whether nuclear localization is required for A3B-mediated antiviral activities by testing A3B and select mutants against a panel of viruses in cell culture and in vivo. These studies will elucidate the mechanism of A3B nuclear import and the role of subcellular localization in DNA virus restriction, thus enhancing our understanding of these important host-pathogen interactions.