APOBEC3 proteins mediate the clearance of foreign DNA from human cells

Abstract

Bacteria evolved restriction endonucleases to prevent interspecies DNA transmission and bacteriophage infection. Here we show that human cells possess an analogous mechanism. APOBEC3A is induced by interferon following DNA detection, and it deaminates foreign double-stranded DNA cytidines to uridines. These atypical DNA nucleosides are converted by the uracil DNA glycosylase UNG2 to abasic lesions, which lead to foreign DNA degradation. This mechanism is evident in cell lines and primary monocytes, where up to 97% of cytidines in foreign DNA are deaminated. In contrast, cellular genomic DNA appears unaffected. Several other APOBEC3s also restrict foreign gene transfer. Related proteins exist in all vertebrates, indicating that foreign DNA restriction may be a conserved innate immune defense mechanism. The efficiency and fidelity of genetic engineering, gene therapy, and DNA vaccination are likely to be influenced by this anti-DNA defense system.

Figure 1

APOBEC3A is expressed in monocytes and macrophages and induced by interferon and CpG DNA. (a) Relative basal A3 mRNA levels in PBMCs or the specified cell subpopulations, with a value of 1 assigned to the level of A3H mRNA in unsorted PBMCs. Inset: flow cytometry histograms showing the efficiency of CD14+ cell enrichment. (b and c) A3 and IFNβ mRNA levels in PBMCs treated with recombinant IFN or CpG DNA oligonucleotide for the indicated time. mRNA levels are relative to those measured in untreated cells. Inset in c: immunoblot of A3A in PBMCs treated with a CpG DNA oligonucleotide. The same membrane probed with anti-eEF1α is shown as a loading control.

Figure 2

Foreign DNA restriction by APOBEC3A. (a) Schematic of plasmid-based stable gene transfer experiments. (b) Representative plates of Neo^R colonies obtained in a stable gene transfer experiment in HEK-293 cells transfected with a Neo^R plasmid and A3A-expression or control plasmids. (c) Quantification of the data in b and two additional experiments. The amount of A3A-expression plasmid was decreased as indicated. Inset: immunoblot of A3A-GFP and A3A-E72A-GFP and the same membrane stained with Ponceau S. (d) The number of colony forming cells in transfected cell populations in b and c was determined by diluting and plating cells into drug-free medium. (e) The proliferative capacity of transfected cells. (f) The percentage of apoptotic cells in transfected cell populations. Hydrogen peroxide (H₂O₂) was used as a cytotoxic control. (g) Transient expression of a GFP reporter plasmid in HEK-293 cells. (h) pDNA persistence in HeLa cells transfected with A3A or A3A-E72A was determined by quantitative PCR on DNA recovered 48 h post-transfection. For each pDNA, the A3A-E72A data were normalized to one. No t’fection: non-transfected controls. In c, e, g, and h, the mean and s.d. of three replica experiments is shown.

Figure 3

APOBEC3A deaminates transfected plasmid DNA and generates lesions for uracil DNA glycosylase. (a) Agarose gel analysis of 3D-PCR products from stable UGI-expressing HEK-293T cells. Cells were transfected with plasmids encoding A3A or A3A-E72A and a mCherry (pTre2-mCherry) reporter construct. Total DNA was recovered 48 h post-transfection and analyzed by 3D-PCR at the indicated denaturation temperatures (T_d). (b) Agarose gel analysis of 3D-PCR products from HEK-293 cells transiently transfected with increasing amounts of UGI plasmid, a GFP reporter construct (pEGFP-N3), and a plasmid encoding A3A or A3A-E72A. Total DNA was recovered 48 h post-transfection and analyzed by 3D-PCR at the indicated T_d ranges. (c) Summary of the plasmid DNA sequences (nucleotides 1170-1426 of pEGFP-N3) recovered from the experiment in b. C/G-to-T/A hypermutations are indicated as red tics along the consensus sequence, and all other base substitutions as black tics. The control sequences were obtained from cells expressing A3A-E72A. The number of times each sequence was recovered, the number of C-to-T conversions in each sequence (out of 93 total cytidines), and the PCR T_d used to amplify the populations of molecules from which the sequences are derived are indicated.

Figure 4

APOBEC3A mutates foreign DNA in primary human cells. (a) A3A expression by qRT-PCR in CpG-pretreated or untreated monocytes transfected with plasmid DNA (+DNA) or with buffer alone (−DNA) at 8 h post-transfection. Levels are relative to those in untreated, mock-transfected cells. (b) 3D-PCR products from the experiment in a. Total DNA was isolated and a portion of the GFP gene was amplified using a gradient of PCR denaturation temperatures (T_d). DNA extracted from mock-transfected cells and the mock DNA mixed with input plasmid were subjected to the same PCR scheme as controls (labeled “mock” and “pDNA”, respectively). (c) Representative chromatograms of PCR products from DNA transfected into monocytes or control reactions (nucleotides 1289-1314 of pEGFP-N3). Cytidines that have been edited in all or a fraction of molecules are indicated with filled or open arrowheads, respectively. (d) Plasmid DNA recovered from monocytes was subjected to 3D-PCR, cloned, and sequenced. C/G-to-T/A mutations are indicated as red tics; other base substitutions as black tics. The number of times each sequence was recovered and the number of C-to-T conversions in each molecule are indicated. (e) A quantification of the DNA editing site dinucleotide contexts detected by directly sequencing 3D-PCR amplicons (as in c). The editing percentages were grouped by dinucleotide context and the median percentage for each group is indicated. For instance, 5 of the 9 cytidines within 5′-TC dinucleotides were edited in 100% of the molecules amplified (i.e., only a T peak was evident in the chromatogram).

Figure 5

Foreign DNA restriction by multiple human APOBEC3 proteins. (a) Schematic of the DNA transposon-mediated stable gene transfer experiments. (b) A histogram showing the mean and s.d. of three independent transposon-mediated stable gene transfer experiments done in the presence of the indicated A3 expression constructs. Controls included the number of Neo^R colonies resulting from random integration (no transposase; No t’pase) and from no transfection (no foreign DNA; No t’fectection). Flow cytometry confirmed that all of the A3-GFP constructs were expressed similarly (data not shown).

Figure 6

Lack of detectable genomic DNA mutation in cells that restrict foreign DNA. (a) Schematic of the genomic TK mutation assay. (b) Two independent clonal HEK-293 cell lines harboring integrated TK genes were transfected with the indicated expression plasmids and selected with gancyclovir-containing medium. The relative mean number of Gan^R (TK⁻) colonies and s.d. from duplicate or quadruplicate experiments is shown. (c) GFP (or A3-GFP) expression in transfected HEK-293-TK cells (clone #5 in b). (d) 3D-PCR results with primers specific to MDM2 or IFNA2 genomic loci (as described in Fig. 4b). For a positive control (+), genomic DNA was heat denatured and incubated with purified A3A prior to PCR. For a negative control (−), total DNA recovered from untransfected HEK-293 cells was subjected to the same PCR amplification procedures.

Figure 7

A model for foreign DNA restriction. Foreign DNA enters the cell by escaping from an endosomal or phagosomal compartment, by infection, or by other means. TLR-dependent or - independent DNA sensing initiates signaling cascades that result in production of IFN, which in turn induces A3A expression. A3A engages the foreign DNA, deaminating multiple cytidines in a molecule. The resulting uracils are excised by UNG2, creating nuclease-sensitive abasic sites. Cleavage of the backbone by APEX1 or other nucleases results in fragmentation and degradation of the foreign DNA.