Analysis of human APOBEC3H haplotypes and anti-human immunodeficiency virus type 1 activity

Abstract

Human APOBEC3H (A3H) has one cytidine deaminase domain (CDD) and inhibits the replication of retrotransposons and human immunodeficiency virus type 1 (HIV-1) in a Vif-resistant manner. Human A3H has five single amino acid polymorphisms (N15Δ, R18L, G105R, K121D, and E178D), and four haplotypes (I to IV) have previously been identified in various human populations. Haplotype II was primarily found in African-derived populations, and it was the only one that could be stably expressed. Here, we identified three new haplotypes from six human population samples, which we have named V, VI, and VII. Haplotypes V and VII are stably expressed and inhibit HIV-1 replication. Notably, haplotype V was identified in samples from all African-, Asian-, and Caucasian-derived populations studied. Using haplotype VII, we investigated the A3H anti-HIV-1 mechanism. We found that A3H virion packaging is independent of its CDD but dependent on a (112)YYXW(115) motif. This motif binds HIV-1 nucleocapsid in an RNA-dependent manner, and a single Y112A mutation completely disrupts A3H virion incorporation. We further studied the mechanism of A3H resistance to Vif. Although the previously identified APOBEC3G Vif-responsive motif (128)DPDY(131) is not conserved in A3H, placement of this motif into A3H does not make it become less resistant to HIV-1 Vif. We conclude that stably expressed A3H haplotypes may be more broadly distributed in humans than previously realized, and A3H protein is resistant to Vif. These results have important implications for the role of A3H in retrotransposon and HIV-1 inhibition.

FIG. 1.

Expression of human A3H genes in 293T cells. A3H genes containing various mutations at the six polymorphic sites N15Δ, R18L, G105R, K121D, E178D, and E140K were inserted into the pcDNA3.1 vector with a V5 tag and were transiently expressed in 293T cells. Protein expression was detected by Western blotting with the indicated antibodies. Expression of A3H hap II and its mutants is shown in panel A, expression of the seven A3H haplotypes is shown in panel B, expression of A3H hap VII and AGM A3H bearing an E140K mutation is shown in panel C, and expression of A3H-L and its mutants is shown in panel D. In addition, the insert of the seven A3H gene fragments was sliced out from the pcDNA3.1 vector by HindIII/XhoI digestion and analyzed by agrose gel electrophoresis (B).

FIG. 2.

Anti-HIV-1 activity of the seven A3H haplotypes. 293T cells were transfected with ΔVif HIV-1 expression vector pNL4-3Δvif and A3H- or A3G-expressing pcDNA3.1 vector, or a control (Ctrl). Virions were collected from cell culture, and their infectivity was analyzed in the HIV-1 luciferase reporter cell line TZM-bl. In addition, virions were further purified by ultracentrifugation, and proteins in virions and viral producer cells were analyzed by Western blotting with the indicated antibodies. The standard errors of the means (SEMs) were calculated from three independent experiments.

FIG. 3.

Anti-HIV-1 activity of A3H CDD mutants. (A) Expression of A3H hap VII CDD mutants from the pcDNA3.1 vector. Indicated CDD mutants with a V5 tag were created from the A3H hap VII-expressing pcDNA3.1 vector and were transfected into 293T cells. Protein expression was determined by Western blotting with the indicated antibodies. WT, wild type. (B) Expression of A3H CDD mutants from the VR vector and their incorporation into virions. Indicated CDD mutants with an HA tag were created from the A3H hap I-expressing VR vector (VR-A3H-L). 293T cells were transfected with the indicated A3H expression constructs plus the HIV-1 proviral vector pNL4-3. Virions were purified by ultracentrifugation, and the presence of A3H protein in cells and in virions was determined by Western blotting with the indicated antibodies. (C) The anti-HIV-1 activity of A3H CDD mutants. HIV-1 reporter viruses were produced from 293T cells by transfection of pNL-Luc with VR vector expressing the indicated A3H hap I CDD mutants. Viral infectivity was analyzed in GHOST cells. The SEMs were calculated for three independent experiments.

FIG. 4.

Mechanism of A3H virion packaging. Mapping of the interaction between A3H and HIV-1 Gag. 293T cells were transfected with the pcDNA3.1-A3H Hap VII-FLAG-HA vector and another pcDNA3.1 vector expressing the indicated HIV-1 Gag proteins with a C-terminal GST-V5 tag into 293T cells. The Gag and A3H interaction was then determined by GST pulldown in the absence (A) or presence (B) of RNase A. (C) Virion incorporation of the A3H ¹¹²YYHW¹¹⁵ mutants. 293T cells were transfected with pNL4-3 and the indicated pcDNA3.1-A3H Hap VII-V5-expressing vectors. Virions were purified by ultracentrifugation. The levels of A3H in cells and virions were determined by Western blotting with the indicated antibodies. (D) Interaction of the A3H ¹¹²YYHW¹¹⁵ mutants with HIV-1 NC. 293T cells were transfected with the pcDNA3.1-NC-GST-V5 vector and indicated pcDNA3.1-A3H Hap VII-FLAG-HA vectors. The interaction between A3H mutants and HIV-1 NC was determined by GST pulldown as in panel A. (E) Anti-HIV-1 activity of the A3H ¹¹²YYHW¹¹⁵ mutants. HIV-1 reporter viruses (pNL-Luc) were produced in the presence of each A3H hap VII CDD mutant, and viral infectivity was determined as in Fig. 3C. The SEMs were calculated from three independent experiments.

FIG. 5.

Analysis of human A3H sensitivity to HIV-1 and SIVmac Vif. (A) Amino acid sequence alignment of the A3G virion packaging and Vif-responsive domains with the corresponding A3H region. The conserved residues are shown in red, and residues in the Vif-responsive domain are shown in blue. Mut I, II, and III were created from A3H hap VII. (B) The sensitivity of A3H to Vif was measured by a single-round HIV-1 replication assay. 293T cells were transfected with the HIV-1 reporter construct pNL-LucΔenvΔvif, pNL-A1 expressing HIV-1 Vif-HA or SIVmac Vif-HA, a VSV-G expression vector, and a pcDNA3.1 vector expressing the indicated V5-tagged A3G or A3H proteins. The infectivity of the virus produced from the transfected cells was determined by infection of GHOST cells. The SEMs were calculated from three independent experiments. (C) The sensitivity of A3H to Vif was measured by Western blotting in viral producer cells. 293T cells were transfected with the indicated A3 expression vector plus pNL-A1ΔVif, pNL-A1 HIV-1 Vif-HA, or pNL-A1 SIVmac Vif-HA. Levels of A3G, A3H, Vif, and actin expression were determined by Western blotting with the indicated antibodies.

FIG. 6.

(A) Expression of untagged (ΔV5) or V5-tagged A3H Hap II and Hap VII from pcDNA3.1 vector in 293T cells as determined by Western blotting using the indicated antibodies. (B) Sensitivity of A3H Hap II (ΔV5) and Hap VII (ΔV5) to HIV-1 Vif as measured by a single-round HIV-1 replication assay as in Fig. 5B. The SEMs were calculated from three independent experiments.