Inhibition of LINE-1 and Alu retrotransposition by exosomes encapsidating APOBEC3G and APOBEC3F

Abstract

Human cytidine deaminases, including APOBEC3G (A3G) and A3F, are part of a cellular defense system against retroviruses and retroelements including non-LTR retrotransposons LINE-1 (L1) and Alu. Expression of cellular A3 proteins is sufficient for inhibition of L1 and Alu retrotransposition, but the effect of A3 proteins transferred in exosomes on retroelement mobilization is unknown. Here, we demonstrate for the first time that exosomes secreted by CD4(+)H9 T cells and mature monocyte-derived dendritic cells encapsidate A3G and A3F and inhibit L1 and Alu retrotransposition. A3G is the major contributor to the inhibitory activity of exosomes, however, the contribution of A3F in H9 exosomes cannot be excluded. Additionally, we show that exosomes encapsidate mRNAs coding for A3 proteins. A3G mRNA, and less so A3F, was enriched in exosomes secreted by H9 cells. Exosomal A3G mRNA was functional in vitro. Whether exosomes inhibit retrotransposons in vivo requires further investigation.

Fig. 1. Effect of A3 proteins and exosomes on L1 retrotransposition

293T cells were transfected with a pL1_RP tagged with an EGFP retrotransposition indicator cassette and HA- or V5-tagged A3 expression plasmids. Cells transfected with pL1_RP-EGFP only were exposed to exosomes (20 µg/ml) secreted by H9, SupT1, M-DC, or isolated from peripheral blood plasma (Blood exo). (A) After 5 days, cells were observed by fluorescence microscopy and (B) trypsinized, fixed with 3% paraformaldehyde and subjected to flow cytometry analysis. The retrotransposition level in cells transfected with pL1_RP-EGFP (in the absence of A3 proteins or exosome treatment) was set as 100%. EF05J = negative control with inactive pL1_RP-EGFP. Error bars, mean values ± SDs of triplicate samples. Similar results were obtained in 4 independent experiments. (C) Expression of A3G proteins in transfected cells (left panel) or in exosomes (right panel) was analyzed by SDS-PAGE followed by immunoblotting with anti-HA or anti-V5. A3G in exosomes was detected with anti-A3G antibodies.

Fig. 2. Expression of A3G and A3F in exosomes

Exosomes were purified from cell culture supernatants or from blood plasma of 2 different healthy blood donors. Cell and exosome lysates (20 and 10 µg/lane, respectively) were separated by SDS-PAGE and analyzed by Western blotting for the presence of A3G, A3F, and exosome markers Rab7 and Alix. In addition to the 70–75 kDa form of Alix detected predominantly in cells, higher molecular forms of Alix (95–105 kDa) were detected, particularly in exosomes. A slower migrating form of A3G was specifically detected in H9 exosomes. Note that A3F was detected in exosomes purified from blood plasma of donor 2 but not donor 1.

Fig. 3. Expression of A3 mRNAs in CD4⁺ T cells and exosomes

Total RNA was extracted from H9 and SupT1 cells and from exosomes secreted by these cells and subjected to real-time RT-PCR. Levels of A3 mRNAs in cells (A) and in exosomes (B) were normalized against GAPDH mRNA levels and were expressed relative to A3C mRNA levels that were set as 1.0 in both H9 cells and H9 exosomes. Error bars, mean values ± SD of triplicate samples. (C) A3G mRNA present in H9 exosomes is functional. PolyA⁺ mRNA (1 µg) isolated from H9 exosomes (exo) was subjected to an in vitro translation using a human in vitro protein expression assay. Samples without RNA (Ø, negative control) or containing GFP mRNA (positive control) were included. Translation reaction products were separated by SDS-PAGE and analyzed by Western blotting for the presence of A3G and GFP. Molecular weight markers (in kDa) are shown. NS, non-specific protein.

Fig. 4. Effect of exosomes on L1-mediated Alu retrotransposition

HeLa cells were cotransfected with an Alu construct tagged with the neo^Tet retrotransposition indicator cassette and an L1 construct (Alu + L1), with or without an A3G expression vector. Cells transfected with Alu + L1 were exposed with different exosomes (20 µg/ml). Three days post-transfection, cells were subjected to selection in G418 for 14 days and resistant colonies were fixed, stained with crystal violet (A) and counted (B). As negative controls, cells were mock transfected (cells only) or transfected with L1 alone, Alu neo^Tet alone (Alu) or together with irrelevant GFP gene (Alu + GFP). Retrotransposition efficiency was expressed relative to the positive control (Alu + L1), which was set as 100%. Values represent the means ± SD of triplicate samples. Similar results were obtained in 4 independent experiments.