Human APOBEC3F (huA3F) potently restricts the infectivity of HIV-1 in the absence of the viral accessory protein virion infectivity factor (Vif). Vif functions to preserve viral infectivity by triggering the degradation of huA3F but not rhesus macaque A3F (rhA3F). Here, we use a combination of deletions, chimeras, and systematic mutagenesis between huA3F and rhA3F to identify Glu³²⁴ as a critical determinant of huA3F susceptibility to HIV-1 Vif-mediated degradation. A structural model of the C-terminal deaminase domain of huA3F indicates that Glu³²⁴ is a surface residue within the α4 helix adjacent to residues corresponding to other known Vif susceptibility determinants in APOBEC3G and APOBEC3H. This structural clustering suggests that Vif may bind a conserved surface present in multiple APOBEC3 proteins.

APOBEC3 enzymes form part of the innate immune system by deaminating cytosine to uracil in single-stranded DNA (ssDNA) and thereby preventing the spread of pathogenic genetic information. However, APOBEC mutagenesis is also exploited by viruses and cancer cells to increase rates of evolution, escape adaptive immune responses, and resist drugs. This raises the possibility of APOBEC3 inhibition as a strategy to augment existing antiviral and anticancer therapies. Here we show that, upon incorporation into short single-stranded (ss)DNAs, the cytidine nucleoside analog 2'-deoxy-zebularine (dZ) becomes capable of inhibiting the catalytic activity of selected APOBEC variants derived from APOBEC3A, APOBEC3B, and APOBEC3G supporting a mechanism in which ssDNA delivers dZ to the active site. Multiple experimental approaches, including isothermal titration calorimetry, fluorescence polarization, protein thermal shift, and NMR spectroscopy assays demonstrate nanomolar dissociation constants and low micromolar inhibition constants. These dZ containing ssDNAs constitute the first substrate-like APOBEC3 inhibitors and, together, comprise a platform for developing nucleic acid-based inhibitors with cellular activity.