Structural Insights into HIV-1 Vif-APOBEC3F Interaction

Abstract

The HIV-1 Vif protein inactivates the cellular antiviral cytidine deaminase APOBEC3F (A3F) in virus-infected cells by specifically targeting it for proteasomal degradation. Several studies identified Vif sequence motifs involved in A3F interaction, whereas a Vif-binding A3F interface was proposed based on our analysis of highly similar APOBEC3C (A3C). However, the structural mechanism of specific Vif-A3F recognition is still poorly understood. Here we report structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Alanine-scanning analysis of Vif revealed that six residues located within the conserved Vif F1-, F2-, and F3-box motifs are essential for both A3C and A3F degradation, and an additional four residues are uniquely required for A3F degradation. Modeling of the Vif structure on an HIV-1 Vif crystal structure revealed that three discontinuous flexible loops of Vif F1-, F2-, and F3-box motifs sterically cluster to form a flexible A3F interaction interface, which represents hydrophobic and positively charged surfaces. We found that the basic Vif interface patch (R17, E171, and R173) involved in the interactions with A3C and A3F differs. Furthermore, our crystal structure determination and extensive mutational analysis of the A3F C-terminal domain demonstrated that the A3F interface includes a unique acidic stretch (L291, A292, R293, and E324) crucial for Vif interaction, suggesting additional electrostatic complementarity to the Vif interface compared with the A3C interface. Taken together, these findings provide structural insights into the A3F-Vif interaction mechanism, which will provide an important basis for development of novel anti-HIV-1 drugs using cellular cytidine deaminases.

Importance: HIV-1 Vif targets cellular antiviral APOBEC3F (A3F) enzyme for degradation. However, the details on the structural mechanism for specific A3F recognition remain unclear. This study reports structural features of interaction interfaces for both HIV-1 Vif and A3F molecules. Three discontinuous sequence motifs of Vif, F1, F2, and F3 boxes, assemble to form an A3F interaction interface. In addition, we determined a crystal structure of the wild-type A3F C-terminal domain responsible for the Vif interaction. These results demonstrated that both electrostatic and hydrophobic interactions are the key force driving Vif-A3F binding and that the Vif-A3F interfaces are larger than the Vif-A3C interfaces. These findings will allow us to determine the configurations of the Vif-A3F complex and to construct a structural model of the complex, which will provide an important basis for inhibitor development.

FIG 1

Critical residues of HIV-1 Vif in the conserved motifs required for A3 interaction. (A) A diagram of HIV-1 Vif functional domains is shown at the top. Three discontinuous motifs, DRMR (F1 box), TGERDW (F2 box), and EDRWN (F3 box), and the YRHHY motif (G box) are important for binding to A3F and A3G, respectively. The FG box, a Zn²⁺ coordination motif, and the BC box are critical for A3F/A3G interaction, CUL5 selection, and ELOC binding, respectively. Alanine-substituted Vif mutants were made in the F1, F2, and F3 boxes. A3C, A3F, or A3G with a C-terminal MH tag was expressed in the presence of WT or mutant Vif in 293T cells. A3 and Vif were detected by Western blotting with anti-HIS MAb and anti-Vif MAb, respectively. The loading control was β-tubulin. −, no Vif expression. Note that four Vif F3-box mutants (E171A, R173A, W174A, and N175A) are not detected by the anti-Vif MAb due to lack of the MAb epitope by the alanine substitution. A representative experiment is shown (n = 3). (B) Reactivity of anti-Vif MAb 319 (Abcam) against a series of C-terminal-truncated Vif proteins and the F3-box mutant with a C-terminal MH tag. 293T cells were transfected with each Vif expression plasmid. The intracellular Vif was immunoblotted with the anti-Vif MAb or anti-HIS MAb. Only WT Vif (WT-MH) and the Vif 1–178 truncation mutant (1–178-MH) were detected by the anti-Vif MAb, whereas all the MH-tagged Vif proteins were detected using the anti-HIS MAb. All four of the mutations (E171A, R173A, W174A, and N175A) within the F3-box motif abolished Vif recognition by the anti-Vif MAb. (C) Effects of the F3-box mutations on A3C and (D) A3F degradation using a C-terminal MH-tagged version of Vif.

FIG 2

Residues involved in each A3 interaction on the Vif structure. (A and B) Based on the crystal structure of Vif (PDB ID no. 4N9F), five residues (RWNKP [residues 173 to 177]) were extended using Modeler. (A) Residues critical only for A3F degradation (R17, E76, E171, and R173) and commonly required for A3C/A3F degradation (D14, R15, M16, W79, D172, and W174) are mapped in magenta and green, respectively. Amino acid residues for which alanine substitution showed no change in A3C/A3F degradation levels are colored in light green. The G box is shown in cyan. The Vif secondary structure is traced in light yellow. Each area of the F1, F2, and F3 boxes is enclosed by the black dotted lines. A gray sphere indicates a zinc atom coordinated in Vif. (B) Electrostatic potential representations of Vif in the same orientation as those in panel A. The surface area is colored according to the calculated potentials from −3.0 kT/e (red) to +3.0 kT/e (blue). The enclosed area with the yellow dotted line indicates the F1, F2, and F3 boxes.

FIG 3

Identification of critical residues in A3F for Vif-mediated degradation. WT A3F and mutants were assayed for intracellular degradation in the presence (+) or absence (–) of HIV-1 Vif in 293T cells. The steady-state levels of A3F were analyzed by Western blotting using an anti-His MAb. (A) Thirteen residues predicted by Bohn et al. (58) were analyzed. (B) Based on three independent analyses for each mutant, the percentage of A3F in the presence of Vif relative to that in the absence of Vif was calculated as the Vif resistance level. (C) Additional A3F residues identified by Siu et al. (59) and the Y314 residue were also analyzed. Vif expression levels were determined by immunoblotting with an anti-Vif MAb. A3F C259K and A3F E289K were used as a control of Vif-resistant A3F as previously reported by our group. The loading control was β-tubulin.

FIG 4

Effects of A3F mutations on the Vif-binding ability of A3F. Shown are the results from coimmunoprecipitation of HIV-1 Vif SLQ/AAA with A3F-MH (WT) and mutants. Total cell lysates (Lysate) and immunoprecipitated complexes (IP) were analyzed by immunoblotting with anti-Vif or anti-His MAbs, and β-tubulin was used as the loading control.

FIG 5

Packaging efficiency and antiviral activity of WT and mutant A3F. (A) 293T cells were cotransfected with pNL4-3ΔVif (HIV-1ΔVif) and each A3F expression plasmid. The amounts of A3F in cells and virions were determined by Western blotting. Virion-associated p24 (CA) levels were detected using anti-p24 antibody. The loading control was β-tubulin. (B) The antiviral effects of WT and mutant A3F on WT HIV-1 or HIV-1ΔVif were assessed in a single-round replication assay using TZM-bl cells. The relative viral infectivity of WT HIV-1 in the absence of A3F (Vector) was set to 100%.

FIG 6

Deaminase activity of WT and mutant A3F. (A) The A3F domain(s) responsible for enzymatic activity are shown. Recombinant GST-A3F proteins were expressed in the baculovirus expression system and purified. The deaminase activity of full-length WT and mutant A3F proteins (E67Q, E251Q, and E67Q E251Q) containing an N-terminal GST tag was assayed using the UDG-dependent assay. The positions of the TTCA-containing DNA oligonucleotide substrate (40 nucleotides [nt]) and the deamination product (18 nt) are indicated. (B) Deaminase activity of the A3F CTD. The recombinant proteins were expressed in the E. coli expression system. The relative amounts of deamination product (percentage) versus the A3F CTD concentration (nanomolar) are shown. Error bars represent the standard deviations from three independent measurements. The A3F CTD E251Q was used as a deaminase-deficient control.

FIG 7

Crystal structure of the A3F CTD and the Vif interaction interface. (A) Ribbon representation of the superimposed structures of the A3F CTD and A3C (PDB ID no. 3VOW). The backbone traces of the A3F CTD and A3C are colored in wheat and gray, respectively. Catalytic zinc ions of A3F and A3C are shown as black and gray spheres, respectively. (B) Ribbon representation of the A3F CTD alone. Critical residues of the Vif interaction interface that are analogous to A3C are shown in green. Four residues (L291, A292, R293, and E324) shown in magenta are uniquely required for the A3F-Vif interaction. (C) Surface representation of A3C with the Vif interaction interface shown in green. (D) Surface representation of A3F CTD with the Vif interaction interface with the same coloring and labeling as in panel B. (E) Electrostatic potentials of A3F CTD. The surface area is colored according to the calculated potentials from −3.0 kT/e (red) to +3.0 kT/e (blue). The enclosed area with the yellow dotted line indicates the Vif-binding interface.

FIG 8

Predicted interaction between the A3F CTD and Vif. (A and B) Two views of their interaction surfaces, from the A3F catalytic groove side (A) and from the A3F CTD α4 helix side (B), are shown. Structures of the A3F CTD and Vif are represented as wheat ribbons and gray surface, respectively. The A3F-unique residues crucial for Vif binding are highlighted with magenta sticks. A zinc ion in the catalytic groove is shown as a black sphere.

FIG 9

Amino acid sequence conservation of A3F-binding Vif motifs and Vif-binding A3F residues. (A) Conservation at each alignment position in the F1, F2, and F3 boxes. A total of 2,935 HIV-1 sequences and 27 SIVcpz Vif sequences were aligned. The conservation rate at each position (residue) was analyzed using the WebLogo 3.4 program and is shown as R_seq values on the y axis. Amino acids are colored according to chemical properties: polar (green), neutral (purple), basic (blue), acidic (red), and hydrophobic (black). (B) Amino acid sequence alignment of human (Homo sapiens) and chimpanzee (Pan troglodytes verus) A3F CTD. Identical residues are shaded in gray. A red arrowhead indicates residue R293 of human A3F.