A single amino acid of APOBEC3G controls its species-specific interaction with virion infectivity factor (Vif)

Abstract

The virion infectivity factor (Vif) accessory protein of HIV-1 forms a complex with the cellular cytidine deaminase APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) to block its antiviral activity. The antiviral property of APOBEC3G is conserved in several mammalian species, but the ability of Vif to block this activity is species-specific. HIV-1 Vif blocks human APOBEC3G but does not block the mouse or African green monkey (AGM) enzyme. Conversely, SIV(AGM) Vif blocks the antiviral activity of AGM but not human APOBEC3G. We demonstrate that the species specificity is caused by a single amino acid difference in APOBEC3G. Replacement of Asp-128 in human APOBEC3G with the Lys-128 of AGM APOBEC3G caused the enzyme to switch its interaction, becoming sensitive to SIV(AGM) Vif and resistant to HIV-1 Vif. Conversely, the reciprocal Lys to Asp switch in AGM APOBEC3G reversed its specificity for Vif. The reversal of biological activity was accompanied by the corresponding switch in the species specificity with which the enzyme physically associated with Vif and was excluded from virions. The charge of the amino acid at position 128 was a critical determinant of species specificity. Based on the crystal structure of the distantly related Escherichia coli cytidine deaminase, we propose that this amino acid is positioned on a solvent-exposed loop of APOBEC3G on the same face of the protein as the catalytic site.

Fig. 1.

Analysis of human:AGM APOBEC3G chimeras maps the Vif specificity-determining region to residues 1-143. (A) Structure of human:AGM APOBEC3G chimeras. Filled bars denote human sequence; open bars denote AGM sequence. The Zn²⁺-coordination domains are boxed. (B) Luciferase reporter virus assay of chimeric APOBEC3Gs. Wild-type and Δvif luciferase reporter viruses were produced in 293T cells cotransfected with chimeric APOBEC3G expression. vector, Empty vector control. Reporter virus infectivity was measured on HOS.CD4 cells infected with viruses normalized to 1.0 ng of p24. The data are the average of triplicates ± SD. Chimeric APOBEC3G expression in the transfected 293T producer cells detected on immunoblots is shown in Lower.

Fig. 2.

Amino acid 128 of APOBEC3G determines the species specificity of the functional interaction with Vif. (A) Alignment of amino acids 71-143 of human and AGM APOBEC3G. The Zn²⁺-coordination domain is boxed. (B-D) Chimeric and point mutant APOBEC3G activity was determined in the luciferase reporter virus assay as described in Methods. vector, Empty vector control. (E) Amino acid 128 APOBEC3G mutants were tested for their functional interaction with SIV_AGM Vif by using SIV_AGM reporter virus. (F) The role of amino acid 128 in macaque APOBEC3G was tested by using HIV-1 and SIV_Mac luciferase reporter viruses. APOBEC3G in the transfected 293T producer cells was detected on immunoblots (Lower, each histogram). Representative results from one of three repetitions of the experiment are shown.

Fig. 3.

The charge of amino acid 128 determines the specificity of the functional interaction with Vif. The functional interaction of human (A and B) and AGM (C and D) APOBEC3G-containing charge changes at amino acid 128 were measured on HIV-1 (A and C) and SIV_AGM (B and D) luciferase reporter viruses. The experiment was repeated in three experiments with similar results.

Fig. 4.

Amino acid 128 determines the specificity with which Vif excludes APOBEC3G from virions and the specificity of Vif:APOBEC3G complex formation. (A-F) Wild-type and Δvif HIV-1 and SIV_AGM virions produced in 293T cells cotransfected with APOBEC3G expression vector were pelleted and normalized for p24 or p27. The encapsidated APOBEC3G was detected on immunoblots probed with anti-HA mAb (Upper). APOBEC3G expression in the transfected cells was confirmed on immunoblots of the cell lysates (Lower). Human and AGM APOBEC3G amino acid 128 exchange mutants were encapsidated in HIV-1 virions (A) and SIV_AGM virions (B). mock, Control transfection in which the reporter virus plasmid was omitted. Ala-, Glu-, His-, and Arg-substituted human APOBEC3G were encapsidated in HIV-1 (C) and SIV_AGM (D) virions. Ala-, Glu-, His-, and Arg-substituted AGM APOBEC3G was encapsidated in HIV-1 virions (E) or SIV_AGM virions (F). The sensitivity of each virus to Vif as determined in Fig. 3 is summarized above: R, Vif resistant; S, Vif sensitive; s, intermediate. (G-H) Vif:APOBEC3G complexes were immunoprecipitated from 293T cells cotransfected with APOBEC3G expression vector and HIV-1 wild-type or Δvif HIV-1 luciferase reporter virus plasmid. The lysates were immunoprecipitated with anti-HA mAb and protein-A beads, and the complexes were analyzed on immunoblots. The filters were probed with anti-HA mAb to detect APOBEC3G and with rabbit anti-Vif antiserum to detect coimmunoprecipitated Vif. Efficient pull-down of APOBEC3G was confirmed on duplicate blots probed with anti-HA mAb. Equivalent expression of Vif in the transfected cells was confirmed on immunoblots of the cell lysates probed with rabbit anti-Vif antiserum. All of the experiments were repeated three to five times, and representative results are shown.

Fig. 5.

Ribbon representation of E. coli cytidine deaminase. Asp-128 maps to a helical turn (red) situated on a loop (cyan and dotted line) connecting the catalytic and pseudocatalytic domains of the E. coli enzyme. Uridine at the active site is shown in bonds representation. The green sphere represents Zn²⁺.