Distinct Domains within APOBEC3G and APOBEC3F Interact with Separate Regions of Human Immunodeficiency Virus Type 1 Vif

Abstract

Human APOBEC3G (A3G) and APOBEC3F (A3F) inhibit the replication of Vif-deficient human immunodeficiency virus type 1 (HIV-1). HIV-1 Vif overcomes these host restriction factors by binding to them and inducing their degradation. Thus, the Vif-A3G and Vif-A3F interactions are attractive targets for antiviral drug development, as inhibiting these interactions could allow the host defense mechanism to control HIV-1 replication. Recently, it has been reported that amino acids 105 to 156 of A3G are involved in the interaction with Vif; however, to date, the region of A3F involved in Vif binding has not been identified. Using our previously reported Vif mutants that are capable of binding to only A3G (3G binder) or only A3F (3F binder), in conjunction with a series of A3G-A3F chimeras, we have now mapped the APOBEC3-Vif interaction domains. We found that the A3G domain that interacts with the Vif YRHHY region is located between amino acids 126 and 132 of A3G, which is consistent with the conclusions reported in previous studies. The A3F domain that interacts with the Vif DRMR region did not occur in the homologous domain but instead was located between amino acids 283 and 300 of A3F. These studies are the first to identify the A3F domain that interacts with the Vif DRMR region and show that distinct domains of A3G and A3F interact with different Vif regions. Pharmacological inhibition of either or both of these Vif-A3 interactions should prevent the degradation of the APOBEC3 proteins and could be used as a therapy against HIV-1.

HIV-1 Vif-mediated ubiquitination/degradation of APOBEC3G involves four critical lysine residues in its C-terminal domain

“…In the present A3G model, the distance between the 128 DPD 130 cluster and the critical Lys residues ranges from 45Å to 60Å, very similar to the distance between the E2 active-site Cys and the substratebinding site within the Cul 5-EloB/C-Vif-Rbx2-E2 complex. Interestingly, in a recent study by Russell et al on A3F, the Vif binding region was identified as amino acids 283 to 300 (25). Reference to the A3G structural model suggests that the A3F Vif-binding site corresponds to the A3G ubiquitination region where Lys-297, 301, and 303 are located.…”

“…In contrast to the residues that are involved in ubiquitination of A3G, amino acids implicated in Vif binding are located in the NTD (43) within the 128 DPD 130 motif (31,43) that is embedded in the 126 FWDPDYQ 132 (25) sequence in a loop preceding helix 4, which protrudes out from the overall structure (Fig. S2).…”

“…Interestingly, it has been reported that Vif contains three sequence motifs for binding to A3G: 12 (24)(25)(26). The region in A3G responsible for binding to HIV-1 Vif was initially identified by comparative studies of the species specificity of A3G degradation by Vif.…”

Nuclear import of APOBEC3F-labeled HIV-1 preintegration complexes

“…Transfections were carried out using polyethylenimine transfection reagent (Sigma), as previously described (50,51). Briefly, 3.5 × 10 6 293T cells were seeded onto 10-cm-diameter dishes and cotransfected the next day with an HDV-EGFP (10 μg), S15-mCherry (5 μg), HCMV-G (VSV-G; 2 μg), and either A3F-YFP (1.25 μg), A3F-E251Q-YFP (5 μg; referred to as A3F*-YFP), A3G-YFP (10 μg), A3G-E259Q-YFP (5 μg; referred to as A3G*-YFP), A3C-YFP (10 μg), or P22-YFP (5 μg).…”

HIV-1 and HIV-2 Vif Interact with Human APOBEC3 Proteins Using Completely Different Determinants

“…Further, Vif1 has several domains through which it binds APOBEC3 proteins, with some regions being specific for certain APOBEC3s (34)(35)(36)(37)(38)(39). For example, the 40 YRHHY 44 motif of Vif1 is known to play a critical role in inducing the degradation of A3G, while the 14 DRMR 17 region is known to be critical for inducing the degradation of A3C, A3D, and A3F (34).…”