The DNA deaminase activity of human APOBEC3G is required for Ty1, MusD, and human immunodeficiency virus type 1 restriction

Abstract

Human APOBEC3G and several other APOBEC3 proteins have been shown to inhibit the replication of a variety of retrotransposons and retroviruses. All of these enzymes can deaminate cytosines within single-strand DNA, but the overall importance of this conserved activity in retroelement restriction has been questioned by reports of deaminase-independent mechanisms. Here, three distinct retroelements, a yeast retrotransposon, Ty1, a murine endogenous retrovirus, MusD, and a lentivirus, human immunodeficiency virus type 1 (HIV-1), were used to evaluate the relative contributions of deaminase-dependent and -independent mechanisms. Although human APOBEC3G can restrict the replication of all three of these retroelements, APOBEC3G lacking the catalytic glutamate (E259Q) was clearly defective. This phenotype was particularly clear in experiments with low levels of APOBEC3G expression. In contrast, purposeful overexpression of APOBEC3G-E259Q was able to cause modest to severe reductions in the replication of Ty1, MusD, and HIV-1(DeltaVif). The importance of these observations was highlighted by data showing that CEM-SS T-cell lines expressing near-physiologic levels of APOBEC3G-E259Q failed to inhibit the replication of HIV-1(DeltaVif), whereas similar levels of wild-type APOBEC3G fully suppressed virus infectivity. Despite the requirement for DNA deamination, uracil DNA glycosylase did not modulate APOBEC3G-dependent restriction of Ty1 or HIV-1(DeltaVif), further supporting prior studies indicating that the major uracil excision repair system of cells is not involved. In conclusion, the absolute requirement for the catalytic glutamate of APOBEC3G in Ty1, MusD, and HIV-1 restriction strongly indicates that DNA cytosine deamination is an essential part of the mechanism.

FIG. 1.

Model depicting A3G deaminase-dependent and -independent retroelement restriction mechanisms. A3G is packaged along with the RNA genome (gray wavy lines) of a retroelement into assembling virus or virus-like particles. Following packaging, A3G uses either a deaminase-dependent or a deaminase-independent mechanism to inhibit replication at the reverse transcription step. The deaminase-dependent mechanism is depicted on the right. A3G deaminates cytosine to uracil in the first-strand cDNA (black wavy lines), which results in integration of an inactive retroelement due to G-to-A hypermutation or degradation, possibly as a result of uracil excision leaving an abasic site (*). The deaminase-independent mechanism is depicted on the left. A3G causes the degradation of the retroelement genome prior to integration, possibly by preventing reverse transcription. It should be noted that the Ty1, MusD, and HIV-1 life cycles have been oversimplified in order to focus on A3G's deaminase-dependent and -independent activities. Results from these studies indicate that A3G uses predominantly a deaminase-dependent restriction mechanism.

FIG. 2.

Ty1 restriction by A3G and A3G zinc-binding domain mutants. (A) Effect of A3G or A3G mutants on Ty1 retrotransposition under high and low expression conditions as monitored by the frequency of His⁺ colonies. For each condition, the averaged median frequency of Ty1 retrotransposition from five independent experiments, each performed with 8 to 16 independent yeast cultures, was normalized to the vector controls, which had an average frequency of Ty1 retrotransposition of 160 × 10⁻⁸. Error bars, 1 standard errors of the means. P values for the indicated data sets were calculated using the t test. (B) Yeast mutation assay monitoring the frequency of canavanine-resistant (Can^R) colonies for yeast expressing wild-type A3G or A3G mutants for six independent experiments, each performed with 8 to 16 independent cultures. The averaged median mutation frequencies and the corresponding standard errors of the means are shown. P values for the indicated data sets were calculated using the t test. (C) Immunoblots of TCA protein extracts from yeast cells expressing a vector control (V), B42-A3G (wild type [WT]), B42-A3G E67Q (N-terminal mutant [N]), B42-A3G E259Q (C-terminal mutant [C]), or B42-A3G E67Q/E259Q (double mutant incorporating both N- and C-terminal mutations [NC]). Cdc28p is a loading control. It should be noted that five times more cell lysate was loaded for the low-expression immunoblots and A3G could be detected only after an overnight exposure.

FIG. 3.

MusD restriction by A3G and zinc-binding domain mutants. (A) Effects of varying amounts of A3G or mutant derivatives on MusD transposition relative to that with the vector control. Transposition was monitored by the number of G418-resistant colonies. The means and standard errors of the means for four independent experiments are shown; each individual experiment used three cultures per condition. Rel., relative; Freq., frequency. (B) Representative immunoblot of lysates from HeLa cells expressing a vector control (Vect), A3G-HA (wild type [WT]), A3G-E67Q-HA (N-terminal mutant), A3G-E259Q-HA (C-terminal mutant), or A3G-E67Q/E259Q-HA (double mutant with both N- and C-terminal mutations). Tubulin (Tub) is a loading control. Amounts of A3G correspond to ratios as follows: 0.12 μg, 0.1:1 ratio; 0.6 μg, 0.5:1 ratio; 1.2 μg, 1:1 ratio. All of the samples were run on the same gel and cropped to be in the same order as the ratios in panel A.

FIG. 4.

HIV-1 restriction by A3G and A3G zinc-binding domain mutants. (A) Results of a representative HIV-1(ΔVif) single-cycle infectivity assay from four independent experiments, monitoring the effects of different amounts of A3G or A3G mutants on HIV-1(ΔVif) infectivity. Each data point represents the average of two independent cultures relative to the vector control. The standard deviation between the two data sets is shown (error bars); where not visible, it is smaller than the data point. Rel., relative. (B) Immunoblot of lysates from 293T cells (top) expressing a vector control (V), A3G (wild type [WT]), A3G-E67Q (N-terminal mutant), A3G-E259Q (C-terminal mutant), or A3G-E67Q/E259Q (double mutant with both N- and C-terminal mutations) or virion encapsidation of A3G and its variants into viral particles (bottom). Tubulin (Tub) and p24 are loading controls. (C) Quantification of the amounts of A3G and its variants incorporated into viral particles from panel B.

FIG. 5.

Effect of A3G or A3G zinc-binding domain mutants on spreading HIV-1(ΔVif) infections. (A) Representative data showing the replication of HIV-1(ΔVif) on CEM-SS cells stably expressing a vector control (Vect), wild-type A3G, or mutant A3G. (B) Immunoblot of lysates from untransfected CEM-SS cells (−) or CEM-SS cells stably expressing a vector control (V), A3G (wild type [WT]), A3G-E67Q (N-terminal mutant), A3G-E259Q (C-terminal mutant), or A3G-E67Q/E259Q (double mutant with both N- and C-terminal mutations). Tubulin (Tub) is a loading control. CEM cells were used as a control for nonpermissive A3G expression levels.

FIG. 6.

A3G-dependent restriction of Ty1 is independent of Ung1p. (A) Restriction of Ty1 by wild-type A3G. Results are from two independent experiments performed under inducing or noninducing conditions in the presence (WT) or absence (ΔUNG1) of Ung1p. Histograms show the averaged median retrotransposition frequencies (Freq.) relative (Rel.) to those of the vector control (Vect), which had average Ty1 retrotransposition frequencies of 130 × 10⁻⁸ and 110 × 10⁻⁸ for the high- and low-expression experiments, respectively. Error bars, standard errors of the means. (B) Effects of A3G zinc-binding domain mutants on Ty1 retrotransposition in the presence and absence of Ung1p. Histograms show the normalized averaged medians and standard errors of the means from two independent experiments. (C) Assay for uracil DNA glycosylase activity on yeast cell lysates from the wild-type (WT) and Ung1p deletion (ΔUNG1) strains. Lysates were incubated with a fluorescein-labeled 42-mer single stranded (top) or double stranded (bottom) oligonucleotide. A cleaved oligonucleotide results in a 26-mer. The last two lanes show the effects of recombinant Ugi on the wild-type strain and the oligonucleotide alone (Oligo), respectively.

FIG. 7.

A3G-dependent restriction of HIV-1 is independent of UNG. (A and B) Effect of wild-type A3G on infection with wild-type (wt) HIV-1 (A) or HIV-1(ΔVif) (B) in the presence or absence of uracil excision activity. Vect, vector. (C) Oligonucleotide cleavage assay for uracil DNA glycosylase activity on CEM-SS whole-cell extracts (WCE). Lysates were incubated with a fluorescein-labeled 42-mer double-stranded oligonucleotide. Cleavage results in a 26-mer. The last lane shows the effect of recombinant UDG.