LAUSR

APOBEC3 enzymes are single-stranded DNA cytosine-to-uracil deaminases with beneficial roles in antiviral immunity and detrimental roles in cancer mutagenesis. Regarding viral infection, all seven human APOBEC3 enzymes have overlapping roles in restricting virus types that require DNA for replication, including EBV, HIV, human papillomavirus (HPV), and human T-cell leukemia virus (HTLV). ABSTRACT APOBEC3B is an innate immune effector enzyme capable of introducing mutations in viral genomes through DNA cytosine-to-uracil editing. Recent studies have shown that gamma-herpesviruses, such as Epstein-Barr virus (EBV), have evolved a potent APOBEC3B neutralization mechanism to protect lytic viral DNA replication intermediates in the nuclear compartment. APOBEC3B is additionally unique as the only human DNA deaminase family member that is constitutively nuclear. Nuclear localization has therefore been inferred to be essential for innate antiviral function. Here, we combine evolutionary, molecular, and cell biology approaches to address whether nuclear localization is a conserved feature of APOBEC3B in primates. Despite the relatively recent emergence of APOBEC3B approximately 30 to 40 million years ago (MYA) in Old World primates by genetic recombination (after the split from the New World monkey lineage 40 to 50 MYA), we find that the hallmark nuclear localization of APOBEC3B shows variability. For instance, although human and several nonhuman primate APOBEC3B enzymes are predominantly nuclear, rhesus macaque and other Old World primate APOBEC3B proteins are clearly cytoplasmic or cell wide. 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. Ancestral APOBEC3B reconstructed from present-day primate species also shows strong nuclear localization. Together, these results indicate that the ancestral nuclear localization of APOBEC3B is maintained in present-day human and ape proteins, but nuclear localization is not conserved in all Old World monkey species despite a need for antiviral functions in the nuclear compartment. IMPORTANCE APOBEC3 enzymes are single-stranded DNA cytosine-to-uracil deaminases with beneficial roles in antiviral immunity and detrimental roles in cancer mutagenesis. Regarding viral infection, all seven human APOBEC3 enzymes have overlapping roles in restricting virus types that require DNA for replication, including EBV, HIV, human papillomavirus (HPV), and human T-cell leukemia virus (HTLV). Regarding cancer, at least two APOBEC3 enzymes, APOBEC3B and APOBEC3A, are prominent sources of mutation capable of influencing clinical outcomes. Here, we combine evolutionary, molecular, and cell biology approaches to characterize primate APOBEC3B enzymes. We show that nuclear localization is an ancestral property of APOBEC3B that is maintained in present-day human and ape enzymes, but not conserved in other nonhuman primates. This partial mechanistic conservation indicates that APOBEC3B is important for limiting the replication of DNA-based viruses in the nuclear compartment. Understanding these pathogen-host interactions may contribute to the development of future antiviral and antitumor therapies.