Vif Proteins from Diverse Human Immunodeficiency Virus/Simian Immunodeficiency Virus Lineages Have Distinct Binding Sites in A3C

Abstract

Lentiviruses have evolved the Vif protein to counteract APOBEC3 (A3) restriction factors by targeting them for proteasomal degradation. Previous studies have identified important residues in the interface of human immunodeficiency virus type 1 (HIV-1) Vif and human APOBEC3C (hA3C) or human APOBEC3F (hA3F). However, the interaction between primate A3C proteins and HIV-1 Vif or natural HIV-1 Vif variants is still poorly understood. Here, we report that HIV-1 Vif is inactive against A3Cs of rhesus macaques (rhA3C), sooty mangabey monkeys (smmA3C), and African green monkeys (agmA3C), while HIV-2, African green monkey simian immunodeficiency virus (SIVagm), and SIVmac Vif proteins efficiently mediate the depletion of all tested A3Cs. We identified that residues N/H130 and Q133 in rhA3C and smmA3C are determinants for this HIV-1 Vif-triggered counteraction. We also found that the HIV-1 Vif interaction sites in helix 4 of hA3C and hA3F differ. Vif alleles from diverse HIV-1 subtypes were tested for degradation activities related to hA3C. The subtype F-1 Vif was identified to be inactive for degradation of hA3C and hA3F. The residues that determined F-1 Vif inactivity in the degradation of A3C/A3F were located in the C-terminal region (K167 and D182). Structural analysis of F-1 Vif revealed that impairing the internal salt bridge of E171-K167 restored reduction capacities to A3C/A3F. Furthermore, we found that D101 could also form an internal interaction with K167. Replacing D101 with glycine and R167 with lysine in NL4-3 Vif impaired its counteractivity to A3F and A3C. This finding indicates that internal interactions outside the A3 binding region in HIV-1 Vif influence the capacity to induce degradation of A3C/A3F.

Importance: The APOBEC3 restriction factors can serve as potential barriers to lentiviral cross-species transmissions. Vif proteins from lentiviruses counteract APOBEC3 by proteasomal degradation. In this study, we found that monkey-derived A3C, rhA3C and smmA3C, were resistant to HIV-1 Vif. This was determined by A3C residues N/H130 and Q133. However, HIV-2, SIVagm, and SIVmac Vif proteins were found to be able to mediate the depletion of all tested primate A3C proteins. In addition, we identified a natural HIV-1 Vif (F-1 Vif) that was inactive in the degradation of hA3C/hA3F. Here, we provide for the first time a model that explains how an internal salt bridge of E171-K167-D101 influences Vif-mediated degradation of hA3C/hA3F. This finding provides a novel way to develop HIV-1 inhibitors by targeting the internal interactions of the Vif protein.

FIG 1

Sensitivity of primate A3C to lentiviral Vifs. (A) Sequence alignment of primate A3C proteins. Black stars represent important residues for HIV-1 Vif interaction. Green stars indicate the dimerization of A3C protein and anti-SIV activity. The red star is the A3C RNA binding site and indicates its viral incorporation. (B) 293T cells were cotransfected with A3C and HIV-1, HIV-2, SIVagm, or SIVmac Vif expression plasmids. Expression of A3Cs, Vifs, and tubulin was detected by immunoblotting using anti-HA, anti-V5, and anti-tubulin antibodies, respectively. h, cpz, gor, agm, smm, and rh represent human, chimpanzee, gorilla, African green monkey, sooty mangabey monkey, and rhesus monkey, respectively.

FIG 2

SIVmac Vif, but not HIV-1 Vif, counteracts primate A3Cs. (A and C) 293T cells were transfected with expression plasmids for SIVmacΔVif-Luc or SIVmac-Luc (A) or SIVmacΔVif-Luc or SIVmacΔVif-Luc plus HIV-1 Vif (C), together with expression plasmids for hA3G and primate A3Cs. pcDNA3.1(+) was used as a control (vector). After normalizing for reverse transcriptase activity, viral infectivity was determined by quantification of luciferase activity in 293T cells. (B and D) Lysates of SIVmac producer cells were used to detect the expression of A3s, SIVmac capsid (p27), or HIV-1 Vif by anti-HA, anti-p27, or anti-V5 antibody, respectively. Tubulin served as a loading control. Encapsidation of A3s into SIVmac was detected by anti-HA antibody. (E) Primate A3Cs and HIV-1 Vif (without tag) expression plasmids were cotransfected into 293T cells. The expression of A3C and HIV-1 Vif was detected by anti-HA and anti-Vif antibodies. (F) Expression plasmids for hA3C and cpzA3C were cotransfected with HIV-1 Vif or SIVcpz Vif into 293T cells. The expression of A3C and Vif was detected by anti-HA and anti-V5 antibodies. Pts, Pan troglodytes schweinfurthii. cps, counts per second. VLP, virus-like particle. Asterisks represent statistically significant differences: ***, P < 0.001; ns, no significance (Dunnett's t test).

FIG 3

Sensitivity of rh/hA3C and h/smmA3C chimeras to HIV-1 Vif-mediated depletion. (A and C) Schematic structure of rh/hA3C and h/smmA3C chimeras. Amino acid positions 120, 150, and 190 in hA3C are indicated. (B and D) Immunoblots of protein lysates of cotransfected cells displaying the A3's sensitivity to HIV-1 Vif-triggered degradation. The expression of A3s and HIV-1 Vif were analyzed by using anti-HA and anti-V5 antibodies. Tubulin served as a loading control. +, with HIV-1 Vif; −, without HIV-1 Vif.

FIG 4

Identification of determinants in rhA3C and smmA3C that confer resistance to HIV-1 Vif. (A) The schematic structure of rhA3C and smmA3C mutants. The numbers represent amino acid positions in A3C. +, sensitive to HIV-1 Vif-induced degradation; −, resistant to HIV-1 Vif-induced degradation. (B and D) hA3C, rhA3C, smmA3C, or A3C mutants were cotransfected with HIV-1 Vif into 293T cells. A3, HIV-1 Vif, and tubulin were detected by using anti-HA, anti-V5, and anti-tubulin antibodies, respectively. (C) Schematic structure of hA3C mutants.

FIG 5

hA3F residues and HIV-1 Vif-induced depletion. (A) The schematic structure of hA3F mutants. +, sensitive to HIV-1 Vif induced degradation; −, resistant to HIV-1 Vif-induced degradation. (B) HIV-1ΔVif luciferase or HIV-1ΔVif luciferase plus HIV-1 Vif were produced in 293T cells in the presence of A3F or mutants. After normalizing for reverse transcriptase activity, viral infectivity was determined by quantification of luciferase activity in 293T cells. (C) A3s in HIV-1 producer cells and HIV-1 viral particles were detected by using anti-HA antibody. HIV-1 Vif and capsid (p24) were detected by anti-Vif and anti-p24 antibodies, respectively. Tubulin served as a loading control. (D) Superimposition of A3C crystal structure (PDB entry 3VOW) (green) and A3F-CTD crystal structure (PDB entry 4J4J) (light blue). Key residues E106, C130, and E133 are shown as orange surfaces in A3C with labels, and the essential residues (E289, Q323, and E324) in A3F are highlighted with gray surfaces and labels. cps, counts per second. Asterisks represent statistically significant differences: ***, P < 0.001; ns, no significance (Dunnett's t test).

FIG 6

HIV-1 Vif binding to A3C and A3F mutants. (A) Coimmunoprecipitation of HIV-1 Vif SLQ/AAA with A3C and A3F mutants. Protein cell lysates (Input) and immunoprecipitated complexes (IP) were analyzed by immunoblotting with anti-Vif for HIV-1 Vif or anti-HA for A3. (B) ImageJ was used to evaluate the band density of A3C and Vif from immunoprecipitation. The ratio of Vif to A3 was calculated as A3-Vif binding activity. WT A3C-Vif and WT A3F-Vif binding activity was set as 100%.

FIG 7

hA3C depletion activities of HIV-1 Vif alleles. (A) The 21 HIV-1 Vif alleles induced depletion of hA3C to various degrees. hA3C and Vif alleles were cotransfected into 293T cells. Protein extracts of transfected cells were used for detecting the expression of hA3C, Vif, and tubulin by anti-HA, anti-Vif, and anti-tubulin antibodies, respectively. Vifs from N and O subtypes were detected by anti-myc antibody. This A3C experiment was repeated at least three times with similar results. (B) ImageJ was used to evaluate the band density of A3C and tubulin. The ratio of A3C to tubulin was calculated as A3C expression efficiency. A3C plasmid cotransfected with empty vector plasmid was set as 100% expression. (C) hA3C, hA3F, hA3G, and hA3H hapII expression plasmids were transfected together with B-NL4-3 expression plasmids and F-1 and F-2 Vif variants. A3 and Vif were detected by using anti-HA and anti-Vif antibodies. Tubulin served as a loading control.

FIG 8

Identification of important residues in subtype F-1 Vif that determine its counteraction of hA3C/F. (A) Schematic structure of F-1 Vif mutants. The N-terminal hA3H hapII box and C-terminal hA3C/F box are shown. (B, C, and D) hA3C and hA3F were transfected into 293T cells together with B-NL4-3 Vif or with F-1 Vif or F-1 Vif mutant. A3 and Vif were detected by using anti-HA and anti-Vif antibodies. Tubulin served as a loading control. (E) The structures of wild-type F-1 Vif and F-1 Vif mutants were modeled by SWISS modeling. The internal interaction between residues 171 and 167 was analyzed by PyMOL and is shown as a red dashed line.

FIG 9

Structural differences between B-NL4-3 Vif and F-1 Vif. (A and C) hA3C and hA3F or hA3G expression plasmids were cotransfected together with B-NL4-3 Vif and its mutants. A3 and Vif were detected by using anti-HA and anti-Vif antibodies. Tubulin served as a loading control. (B) The structures of B-NL4-3, F-1 Vif, and their variants were modeled by SWISS modeling. The internal interaction between residues 171, 167, and 101 were analyzed by PyMOL, and the distances between interacting side chains are between 3.4 and 3.8 Å. The internal interaction is shown as a red dashed line. (D) Structure analysis of HIV-1 Vif (PDB entry 4N9F) for hA3C/hA3F interaction sites. Three discontinuous HIV-1 Vif motifs (F1 box, F2 box, and F3 box) that interact with hA3C/F are shown in red. 167R that contacted the F3 box is shown in magenta.