Cellular inhibitors of long interspersed element 1 and Alu retrotransposition

Abstract

Long interspersed element (LINE) 1 retrotransposons are major genomic parasites that represent approximately 17% of the human genome. The LINE-1 ORF2 protein is also responsible for the mobility of Alu elements, which constitute a further approximately 11% of genomic DNA. Representative members of each element class remain mobile, and deleterious retrotransposition events can induce spontaneous genetic diseases. Here, we demonstrate that APOBEC3A and APOBEC3B, two members of the APOBEC3 family of human innate antiretroviral resistance factors, can enter the nucleus, where LINE-1 and Alu reverse transcription occurs, and specifically inhibit both LINE-1 and Alu retrotransposition. These data suggest that the APOBEC3 protein family may have evolved, at least in part, to defend the integrity of the human genome against endogenous retrotransposons.

Fig. 1.

Subcellular localization of human APOBEC3 proteins. (A) HeLa cells were transfected with plasmids expressing the indicated wild-type or mutant APOBEC3 protein bearing a C-terminal HA epitope tag. At 48 h after transfection, cells were fixed and incubated with a mouse monoclonal anti-HA antibody, followed by tetramethylrhodamine B isothiocyanate-conjugated goat anti-mouse antiserum, and visualized by fluorescence. (B) Nucleocytoplasmic shuttling was visualized by using heterokaryons (45). Briefly, COS7 cells were transfected with a plasmid expressing A3B-HA. Two days later, the COS7 cells were mixed with HeLa-GSN cells, which express GFP fused to a NLS (31). The mixed culture was then treated with cycloheximide and fused by using polyethylene glycol. A3B-HA was detected as described above. Nuclei were visualized by staining with DAPI; GFP was detected by intrinsic fluorescence.

Fig. 2.

Inhibition of LINE-1 retrotransposition and HIV-1ΔVif infectivity by APOBEC3 proteins. (A) The effect of APOBEC3 proteins on HIV-1ΔVif infectivity was quantified as described in ref. . Data are presented relative to a culture lacking any APOBEC3 protein (positive), which was designated as 100% activity. Data are the average of at least three experiments, with standard deviation indicated. The relative expression of each APOBEC3 protein was comparable (Fig. 6). Because the effect of APOBEC3 proteins on the infectivity of Vif-deficient HIV-1 was measured by using 293T cells, although their effect on LINE-1 retrotransposition was measured in HeLa cells, these results are only qualitatively comparable. (B) Representative experiment showing the relative number of G418-resistant colonies obtained after selection of HeLa cells transfected with the LINE-1 retrotransposition indicator construct pJM101/L1.3 or the negative (NEG) control construct pJM105/L1.3 (lacking a functional ORF2 gene) in the presence or absence (POS) of the indicated APOBEC3 proteins.

Fig. 3.

Effect of mutations on the CDA activity and retrotransposition inhibitory activity of A3A and A3B. (A) This assay measures the ability of the indicated wild-type or mutant A3B and A3A proteins to enhance mutagenesis in bacteria (40). Plasmids encoding the indicated proteins were introduced into E. coli, and their expression was activated by using isopropyl β-d-thiogalactoside. The level of mutation induced by each protein was then assessed by plating the bacteria on plates containing rifampicin and counting the number of resistant colonies. (B) Effect of the indicated wild-type or mutant A3A and A3B proteins on retrotransposition of human LINE-1 or the murine LTR retrotransposon IAP. This analysis of IAP retrotransposition frequency was performed as described in ref. . The average of three independent experiments is shown.

Fig. 4.

A3A and A3B inhibit Alu retrotransposition. HeLa cells were cotransfected with an Alu construct tagged with a retrotransposition indicator cassette, an APOBEC3 expression plasmid, or a control plasmid (pK/β-arr) and expression plasmids encoding wild-type LINE-1 or a LINE-1 lacking ORF1. Three days after transfection, cells were subjected to G418 selection, and neo-resistant colonies were stained and counted 12 days later. Data are presented relative to the culture cotransfected with pK/β-arrestin (Control), which was set at 100%. Data are the average of two experiments performed in duplicate, with standard deviation indicated.

Fig. 5.

Analysis of A3A and A3B mRNA expression in hES cells. RT-PCR was used to assess the expression of LINE-1, Sox2, A3A, and A3B mRNA in three undifferentiated hES cell lines (H9p47, BG01p53, and hSF-6p48). A positive control for the RT-PCR is shown beside the gel panel for A3A. The procedures used for hES cell culture and RT-PCR analyses are described in Supporting Methods, which is published as supporting information on the PNAS web site.