A single amino acid difference in human APOBEC3H variants determines HIV-1 Vif sensitivity

Abstract

Several variants of APOBEC3H (A3H) have been identified in different human populations. Certain variants of this protein are particularly potent inhibitors of retrotransposons and retroviruses, including HIV-1. However, it is not clear whether HIV-1 Vif can recognize and suppress the antiviral activity of A3H variants, as it does with other APOBEC3 proteins. We now report that A3H_Haplotype II (HapII), a potent inhibitor of HIV-1 in the absence of Vif, can indeed be degraded by HIV-1 Vif. Vif-induced degradation of A3H_HapII was blocked by the proteasome inhibitor MG132 and a Cullin5 (Cul5) dominant negative mutant. In addition, Vif mutants that were incapable of assembly with the host E3 ligase complex factors Cul5, ElonginB, and ElonginC were also defective for A3H_HapII suppression. Although we found that Vif hijacks the same E3 ligase to degrade A3H_HapII as it does to inactivate APOBEC3G (A3G) and APOBEC3F (A3F), more Vif motifs were involved in A3H_HapII inactivation than in either A3G or A3F suppression. In contrast to A3H_HapII, A3H_Haplotype I (HapI), which differs in only three amino acids from A3H_HapII, was resistant to HIV-1 Vif-mediated degradation. We also found that residue 121 was critical for determining A3H sensitivity and binding to HIV-1 Vif.

FIG. 1.

A3H_HapII has strong anti-HIV-1 activity which is suppressed by HIV-1 Vif. (A) NL4-3 and NL4-3ΔVif viruses were produced in 293T cells cotransfected with either NL4-3 or NL4-3ΔVif plus an APOBEC3H expression vector (A3H_HapI or A3H_HapII) or empty control vector. After 48 h, virus was collected and assayed for virus infectivity using MAGI indicator cells. Relative infectivity was expressed as a percentage of the infectivity obtained in the absence of APOBEC3 (i.e., with the empty vector control, pcDNA3.1). Error bars indicate standard deviations. (B) Virus-producing 293T cells were harvested and analyzed by SDS-PAGE, followed by immunoblotting, to detect intracellular levels of A3H proteins. β-Actin was used as a loading control. (C) Comparison of the antiviral activities of increasing amounts of A3H_HapI with A3H_HapII. (D) Comparison of the intracellular expression and virion packaging of increasing amounts of A3H_HapI with A3H_HapII.

FIG. 2.

Vif hijacks Cul5/ElongBC E3 ligase and suppresses A3H_HapII by inducing its proteasomal degradation. (A) 293T cells were cotransfected with 1 μg of A3H_HapII and 3 μg of wild-type Vif. At 36 h posttransfection, the cells were treated with dimethyl sulfoxide (DMSO) or with MG132 for 16 h. Cell lysates were harvested and analyzed by SDS-PAGE followed by immunoblotting to detect A3H_HapII, with β-actin as the loading control. (B) A dominant negative Cul5 mutant (Cul5ΔNedd8) blocks Vif-induced A3H_HapII degradation. 293T cells were cotransfected with 1 μg of A3H_HapII plus 3 μg of empty vector, wild-type HIV-1 Vif, or wild-type HIV-1 Vif plus Cul5ΔNedd8. Cell were harvested at 48 h posttransfection and analyzed by SDS-PAGE, followed by immunoblotting to detect A3H_HapII, with β-actin as the loading control. (C) 293T cells were cotransfected with 1 μg of A3H_HapII and with 3 μg of empty vector, wild-type HIV-1 Vif, or mutant VifL145A or VifC114S. Cells were harvested at 48 h posttransfection and analyzed by SDS-PAGE, followed by immunoblotting to detect A3H_HapII, with β-actin as the loading control.

FIG. 3.

Characterization of A3H_HapII interaction domains in Vif. (A) Diagram of the functional domains of Vif. (B) 293T cells were transfected with 1 μg of A3H_HapII and with empty vector or 3 μg of one of the Vif mutants VifDR14/15AA, VifK22E, VifRH41/42AA, or VifW79A. Cells were harvested at 48 h posttransfection and analyzed by SDS-PAGE, followed by immunoblotting, to detect A3H_HapII. (C) 293T cells were transfected with 1 μg of A3H_HapII and with empty vector or 3 μg of one of the Vif mutants VifP58A, VifL59S, VifL64S, VifI66S, VifY69A, VifW70A, or VifL72S. Cells were harvested at 48 h posttransfection and analyzed by SDS-PAGE, followed by immunoblotting, to detect A3H_HapII. (D) Coimmunoprecipitation analysis of wild-type Vif, VifDR14/15AA, VifRH41/42AA, or VifY69A with A3H_HapII. Three micrograms of A3H_HapII was cotransfected with 3 μg control plasmid or myc-tagged wild-type Vif, VifDR14/15AA, VifRH41/42AA, or VifY69A. At 36 h posttransfection, the cells were treated with MG132 for 16 h, and the cell lysates were harvested and immunoprecipitated with anti-myc antibody. Cell lysates and immunoprecipitates were analyzed by SDS-PAGE, followed by immunoblotting, to detect intracellular levels of the A3H_HapII and wild-type Vif and mutants and interactions between A3H_HapII and wild-type Vif and Vif mutants.

FIG. 4.

Identification of amino acid changes that are crucial for the antiviral activity of APOBEC3H. (A) Schematic review of A3H constructs generated by site-directed mutagenesis. (B) Antiviral activity of A3H variants compared to A3H_HapII. HIV-1 viruses were produced in 293T cells cotransfected with 2 μg of NL4-3ΔVif and with 2 μg of empty vector or of A3H_HapI, A3H_HapI_G105R, A3H_HapI_K121E, A3H_HapI_G105R/121E, or A3H_HapII. Viral infectivity was assessed by MAGI assay, with viral infectivity in the presence of A3H_HapII set to 100%. Error bars represent the standard deviations from triplicate wells. Cell lysates were harvested at 48 h posttransfection and analyzed by SDS-PAGE, followed by immunoblotting, to detect A3H variants. (C) Cycloheximide (CHX) chase analysis of A3H_HapI and A3H _105R. 293T cells were transfected with 200 ng A3H_HapI or 200 ng A3H_G105R. At 48 h posttransfection, cells were treated with 100 μg/ml CHX (Sigma) and harvested at 0, 1, 3, and 6 h. The cell lysate were analyzed by SDS-PAGE, followed by immunoblotting with anti-V5 antibody, to determine intracellular levels of A3H_HapI and A3H_G105R. (D) Protein structure model for A3H_HapI and HapI_105R, based on homology modeling of the crystal structure of APOBEC2 and the nuclear magnetic resonance (NMR) structure of the C-terminal domain of A3G.

FIG. 5.

Identification of amino acids that are crucial for Vif-mediated degradation and Vif binding. (A) 293T cells were cotransfected with 1 μg of A3H variant and with 3 μg of empty vector or Vif-myc expression plasmid. At 48 h posttransfection, the cells were harvested and analyzed by SDS-PAGE, followed by immunoblotting, to detect A3H variants. (B) 293T cells were cotransfected with 2 μg of A3H variant and 2 μg of Vif-HA expression plasmid or empty vector. At 36 h posttransfection, the cells were treated with MG132 for 16 h, and the cell lysates were harvested and immunoprecipitated with anti-HA antibody. Cell lysates and immunoprecipitates were analyzed by SDS-PAGE, followed by immunoblotting, to detect intracellular levels of the A3H variants and interactions between Vif and the A3H variants. (C) The bar graph shows the relative interactions of the A3H variants with Vif. The intensities of the immunoblot bands of the A3H variants after immunoprecipitation were quantified using ImageJ software and normalized to that of A3H_HapII.

FIG. 6.

Sequence alignment and model structure comparisons between the amino-terminal domains of A3H_HapI, A3H_HapII, and A3G. (A) Amino acid sequence alignment (positions 102 to 127) of the A3H_HapI, HapII, and A3G amino-terminal domains. The DPD motif in A3G is marked by a box. (B) A3H_HapII and A3G N-terminal protein structure models based on homology modeling as described in Materials and Methods. Amino acids 128D to 130D of A3G and 121E of A3H_HapII are marked.