Structural determinants of HIV-1 Vif susceptibility and DNA binding in APOBEC3F

Abstract

The human APOBEC3 family of DNA cytosine deaminases serves as a front-line intrinsic immune response to inhibit the replication of diverse retroviruses. APOBEC3F and APOBEC3G are the most potent factors against HIV-1. As a countermeasure, HIV-1 viral infectivity factor (Vif) targets APOBEC3s for proteasomal degradation. Here, we report the crystal structure of the Vif-binding domain in APOBEC3F and a novel assay to assess Vif-APOBEC3 binding. Our results point to an amphipathic surface that is conserved in APOBEC3s as critical for Vif susceptibility in APOBEC3F. Electrostatic interactions likely mediate Vif binding. Moreover, structure-guided mutagenesis reveals a straight ssDNA-binding groove distinct from the Vif-binding site, and a novel ‘aromatic switch’ is proposed to explain DNA substrate specificities across the APOBEC3 family. This study opens new lines of inquiry that will further our understanding of APOBEC3-mediated retroviral restriction and provides an accurate template for structure-guided development of inhibitors targeting the APOBEC3-Vif axis.

Crystal Structure of the DNA Deaminase APOBEC3B Catalytic Domain

“…The single dCMP molecule binds the A3Bctd surface and makes several contacts with the enzyme, but it is also assisted by crystal lattice contacts involving symmetry-related molecules (see PDB entry 5CQH), and thus the significance of this result should be interpreted conservatively. Nonetheless, the general location and orientation of the bound nucleotide appear consistent with previous models of A3 proteins binding ssDNA substrates (30,34,37,38,61,62). A3Gctd and A3A NMR chemical shift perturbation experiments, as well as mutational analyses of A3Fctd and A3Gctd, although differing in details, all suggest ssDNA binding paths spanning the catalytic domain surface and extending from the active site toward the ␣-helix 6, which overlaps with the crystallographically observed dCMP interacting with Arg-372 from the ␣-helix 6 and Tyr-319/Lys-320 from the N terminus of ␣-helix 4 (A3Bctd data in Fig.…”

“…A3Gctd and A3A NMR chemical shift perturbation experiments, as well as mutational analyses of A3Fctd and A3Gctd, although differing in details, all suggest ssDNA binding paths spanning the catalytic domain surface and extending from the active site toward the ␣-helix 6, which overlaps with the crystallographically observed dCMP interacting with Arg-372 from the ␣-helix 6 and Tyr-319/Lys-320 from the N terminus of ␣-helix 4 (A3Bctd data in Fig. 5; prior studies with other family members (30,34,37,38,61,62)). Curiously, this nucleotide-binding site on the A3Bctd surface is accessible even when the active site is in the closed conformation.…”

Identification of a Novel HIV-1 Inhibitor Targeting Vif-dependent Degradation of Human APOBEC3G Protein

“…4A, right panels). A recent study (67) revealed new determinants of Vif binding in the A3F-CTD: Glu-316, Ser-320, and Glu-324. These residues were shown to be part of a negatively charged interface that is directly involved in Vif binding.…”

HIV Genome-Wide Protein Associations: a Review of 30 Years of Research

“…In studies of four subspecies of African green monkey, it has been shown that APOBEC3G is adaptively diversifying within hosts because of the antagonism-driven coevolution between Vif and APOBEC3G (397). In addition to APOBEC3G, Vif binds to other proteins in the human APOBEC3 family (e.g., APOBEC3C [398,399] and APOBEC3F [43,400]). Alanine-scanning analyses revealed six Vif residues (D14, R15, M16, W79, D172, and W174) in three conserved motifs that are essential for the degradation of APOBEC3C and APOBEC3F (401).…”