Identification of APOBEC3DE as another antiretroviral factor from the human APOBEC family

Abstract

A tandem arrayed gene cluster encoding seven cytidine deaminase genes is present on human chromosome 22. These are APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H. Three of them, APOBEC3G, APOBEC3F, and APOBEC3B, block replication of human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. In addition, APOBEC3A and APOBEC3C block intracellular retrotransposons and simian immunodeficiency virus (SIV), respectively. In opposition to APOBEC genes, HIV-1 and SIV contain a virion infectivity factor (Vif) that targets APOBEC3F and APOBEC3G for polyubiquitylation and proteasomal degradation. Herein, we studied the antiretroviral activities of the human APOBEC3DE and APOBEC3H. We found that only APOBEC3DE had antiretroviral activity for HIV-1 or SIV and that Vif suppressed this antiviral activity. APOBEC3DE was encapsidated and capable of deaminating cytosines to uracils on viral minus-strand DNA, resulting in disruption of the viral life cycle. Other than GG-to-AG and AG-to-AA mutations, it had a novel target site specificity, resulting in introduction of GC-to-AC mutations on viral plus-strand DNA. Such mutations have been detected previously in HIV-1 clinical isolates. In addition, APOBEC3DE was expressed much more extensively than APOBEC3F in various human tissues and it formed heteromultimers with APOBEC3F or APOBEC3G in the cell. From these studies, we concluded that APOBEC3DE is a new contributor to the intracellular defense network, resulting in suppression of retroviral invasion.

FIG. 1.

Amino acid sequence alignment of human A3B, A3DE, A3F, and A3G. Conserved residues are shown against a black background and partially conserved residues are shown against a grey background. Dashes indicate deletions. The key motif for Zn²⁺-binding motif HxE/PCxxC is indicated. D128, which determines the species-specific interaction between A3G and Vif, is indicated by an arrow below the sequences. Eight exon positions are indicated. Intron-exon boundary information for A3B, A3F, and A3G was obtained from a previous report (22), and that for A3DE was obtained from the Ensembl database.

FIG. 2.

Human A3DE blocks HIV-1 replication, and its activity is countered by Vif. (A) A3DE anti-HIV-1 activity. Human A3A, A3B, A3C, A3DE, A3F, A3G, and A3H were cotransfected with HIV-1 reporter provirus pNL-Luc or pNL-LucΔVif in 293T cells, and viruses were collected from cell culture supernatants. After normalization to the levels of p24^Gag, these viruses were used to infect GHOST.X4/R5 cells. Thirty-six hours later, cells were lysed and intracellular luciferase activities were measured. The bottom panel shows protein expression levels of these A3 proteins in viral producer cells (293T) as determined by Western blotting (Ctrl, control vector). (B) Inhibition of Vif-defective HIV-1 by A3DE is dose dependent. The pNL-Luc or pNL-LucΔVif proviral construct was cotransfected into 293T cells with increasing amounts of A3DE, A3F, and A3G expression vector. Viruses were collected and analyzed similarly to the procedure described above. The bottom panel shows A3DE expression levels in viral producer cells as determined by Western blotting. (C) Stable expression of A3DE changes 293 cells from a permissive to a nonpermissive cell type. 293 cells were transfected with linearized A3DE, A3F, or A3G expression vector, and cells were selected by culture medium containing 50 μg/ml G418 to establish stable cell lines. Wild-type (WT) or Vif-defective (ΔVif) HIV-1 was then produced from these cells, and viral infectivity was determined. The bottom panel shows protein expression levels of A3DE, A3F, and A3G proteins in these cells as determined by Western blotting. (D) A previously identified single residue of A3G, D128, does not govern A3DE sensitivity to Vif of HIV-1. The consensus residue in A3DE (D140), A3F (E127), or A3G (D128) was mutated to lysine (K), and anti-HIV activities were determined as described for panel A. The bottom panel shows protein expression levels of these mutants (A3DE_D140K, A3F_E127K, and A3G_D128K mutants) in viral producer cells as determined by Western blotting. Error bars represent the standard deviations from at least three independent experiments.

FIG. 3.

A3DE induces C-to-U hypermutations on viral minus-strand transcripts that are degraded in the target cell. (A) Summary of HIV-1 genome mutations introduced by A3DE and A3G. Wild-type (WT) or Vif-defective (ΔVif) HIV-1 produced in the presence of A3DE or A3G was used to infect GHOST cells. Several hours later, cellular DNAs were extracted and viral DNAs were amplified by PCR. The PCR products were then cloned into the TA cloning vector and sequenced. (B) Summary of A3DE target site sequence specificity. Results from 57 G-to-A mutations in the genome of Vif-deficient HIV-1 introduced by A3DE were used for this analysis. (C) Stability of newly synthesized HIV-1 reverse transcripts. Wild-type and Vif-defective HIV-1 were produced in the presence of A3DE or A3G, and these viruses were used to infect GHOST cells. Four or eight hours later, cellular DNAs were extracted from these infected cells, and newly synthesized viral cDNA was quantified by quantitative real-time PCR with primers specific for the late reverse transcripts (see Materials and Methods). Error bars represent the standard deviations from at least three independent experiments.

FIG. 4.

Vif excludes A3DE from HIV-1 virions by destabilizing A3DE in viral producer cells. (A) Vif excludes A3DE from virions. 293T cells were cotransfected with wild-type (WT) or Vif-defective (ΔVif) HIV-1 proviral clones in the presence of A3DE and A3G. Virions were collected 48 h later and purified. Expression levels of A3, p24^Gag, and Vif in virions or viral producer cells were determined by Western blotting. (B) Vif binds to A3DE. A GST tag was fused to the C termini of A3A (A3A-GST), A3DE (A3DE-GST), and A3G (A3G-GST), followed by another V5 tag. These GST or GST fusion proteins were coexpressed with Vif in 293T cells, and proteins were then pulled down with GSH-Sepharose beads. Both input (I) and pulldown (P) samples were analyzed by Western blotting using antibodies against V5 or Vif. (C) Schematic representation of a reporter system detecting A3 protein stability in the presence of Vif. A firefly luciferase gene was fused to the C termini of A3 proteins. Vif recruits the Cul5-based E3 ubiquitin ligase complex to these A3-luciferase fusions for degradation in the 26S proteasome. The dominant negative Cul5ΔNedd mutant or Cul5ΔRbx mutant, which lacks Nedd8 or the Rbx binding site, respectively, disrupts the assembly of this E3 ligase complex and therefore blocks this degradation pathway. CMV, cytomegalovirus. (D) Vif destabilizes A3DE in cells. A3-luciferase fusions (A3B-Luc, A3DE-Luc, and A3G-Luc) were coexpressed with HIV-1 Vif (pNL-A1) or its control (pNL-A1ΔVif) in the presence of Cul5ΔRbx or Cul5ΔNedd, respectively, in 293T cells. Thirty-six hours later, cells were lysed and intracellular luciferase activity was measured. Results are presented as values relative to those obtained without Vif expression in cells, and error bars represent the standard deviations from at least three independent experiments.

FIG. 5.

Distribution of A3 transcripts in vivo and interactions of A3DE with A3F or A3G. (A) Specificity of the PCR primer pairs for A3DE, A3F, and A3G. One previously reported A3G primer pair (2) and two newly designed A3DE and A3F primer pairs were used to amplify A3DE, A3F, or A3G by use of their expression plasmids as templates. (B) Distribution of A3 transcripts in a variety of human cell lines and PBL. Total RNAs from these cells were subjected to RT-PCR with A3DE-, A3F-, and A3G-specific primers, and amplified PCR products were visualized by agarose gel electrophoresis followed by ethidium bromide staining. GAPDH was used as an internal control to compare each RNA input. (C) Distribution of A3 transcripts in a variety of human tissues. The same primer pairs described above were used to amplify A3DE, A3F, and A3G messengers from a commercially obtained prenormalized human multiple tissue cDNA panel by RT-PCR, and PCR products were analyzed similarly to the procedure described above. (D) A3DE interacts with A3F and A3G to form heteromultimers in cells. This experiment is similar to that described in the legend for Fig. 4B. The GST or A3DE-GST expression vector was cotransfected with the A3F or A3G expression vector into 293T cells. After 48 h, proteins were pulled down by GSH-Sepharose beads and analyzed by Western blotting with a V5 antibody.

FIG. 6.

A3DE blocks the replication of SIVagm and SIVmac but not that of MLV. Wild-type and Vif-defective (ΔVif) SIVagm (A) and SIVmac (B) or wild-type MLV (C) luciferase reporter viruses were produced from 293T cells in the presence or absence of the indicated human A3 proteins. After normalization by p27^Gag enzyme-linked immunosorbent assay or RT activity, viruses were used to infect GHOST cells, and intracellular luciferase activities were quantified after 36 h of infection. Error bars represent the standard deviations from at least three independent experiments. Ctrl, control.