The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC3, A3) family of DNA cytidine deaminases are intrinsic restriction factors against retroviruses. In felids such as the domestic cat (Felis catus, Fca), the APOBEC3 (A3) genes code for the A3Z2s, A3Z3, and A3Z2Z3 antiviral cytidine deaminases. Only A3Z3 and A3Z2Z3 inhibit viral infectivity factor (Vif)-deficient feline immunodeficiency virus (FIV). FIV Vif protein interacts with Cullin (CUL), Elongin B (ELOB), and Elongin C (ELOC) to form an E3 ubiquitination complex to induce the degradation of feline A3s. The functional domains in FIV Vif for interaction with FcaA3s are poorly understood. Here, I have identified several motifs in FIV Vif that are important for selective degradation of different FcaA3s. I initially proposed that FIV Vif would selectively interact with the Z2 and the Z3 A3s. Indeed, I identified two N-terminal Vif motifs (12LF13 and 18GG19) that specifically interacted with the FcaA3Z2 protein but not with A3Z3. In contrast, the exclusive degradation of FcaA3Z3 was regulated by a region of three residues (M24, L25 and I27). Only a FIV Vif carrying a combination of mutations from both interaction sites lost the capacity to degrade and counteract FcaA3Z2Z3. However, alterations in the specific A3s interaction sites did not affect the cellular localization of the FIV Vif protein and binding to feline A3s. Pull-down experiments suggested that the A3 binding region localized to FIV Vif residues 50 to 80, outside the specific A3 interaction domain. Finally, we found that the Vif sites specific to individual A3s are conserved in several FIV lineages of domestic cat and non-domestic cats, while being absent in the FIV Vif of pumas. Our data support a complex model of multiple Vif-A3 interactions in which the specific region for selective A3 counteraction is discrete from a general A3 binding domain.Additionally, the functional domains in FIV Vif for interaction with Cullin are poorly understood. In this study, I found that the expression of dominant-negative CUL5 prevented the degradation of feline A3s by FIV Vif, while dominant-negative CUL2 had no influence on the degradation of A3. In co-immunoprecipitation assays, FIV Vif bound to CUL5 but not CUL2. To identify the CUL5 interaction site in FIV Vif, the conserved amino acids from position 47 to 160 of FIV Vif were mutated, but these mutations did not impair the binding of Vif to CUL5. By focusing on a potential zinc-binding motif (K175 C161 C184 C187) of FIV Vif, I found a conserved hydrophobic region (174IR175) that is important for CUL5 interaction. Mutating this region also impaired the FIV Vif-induced degradation of feline A3s. Based on a structural model of the FIV Vif/CUL5 interaction, residues 52LW53 in CUL5 were identified as mediating the binding to FIV Vif. By comparing our results to the human immunodeficiency virus type 1 (HIV-1) Vif/CUL5 interaction surface (120IR121, a hydrophobic region that is localized in the zinc-binding motif), we suggest that the CUL5 interaction surface in the diverse HIV-1 and FIV Vif is evolutionarily conserved indicating a strong structural constraint. However, the FIV Vif/CUL5 interaction is zinc-independent, which differs from the zinc-dependency of HIV-1 Vif.
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