Enterovirus Infection Restricts Long Interspersed Element 1 Retrotransposition

FIGURE 1. Suppression of long interspersed element 1 (LINE-1) retrotransposition by both EV-D68 and EV-A71. (A) Retrotransposition assay process. EGFP can only be detected when LINE-1 successfully transposes into the genome and the intron is removed during RNA splicing. (B–D) L1RP EGFP plasmids were transfected into HEK293T cells. Cells were then infected with EV-D68 (Fermon) at increasing MOI of 0, 2 × 10–5, 5 × 10–5, 1 × 10–4, 2 × 10–4, 5 × 10–4, and 1 × 10–3 or EV-D68(US/MO/14-18947)/EV-D68(US/KY/14-18953) at increasing MOI of 0, 2 × 10–5, 5 × 10–5, 1 × 10–4, and 2 × 10–4 12 h post-transfection. Retrotransposition-defective LINE-1 (JM111) was used as a negative control. 4 days after transfection, flow cytometry assay was performed to determine EGFP-positive cells. The bar graph of EGFP-positive cells represents retrotransposition efficiency. The asterisks indicate statistically significant differences between groups by Unpaired Student’s t-test (p < 0.01). After flowcytometry assay, the cells were harvested, lysed and immunoblotted by anti-EV-D68 VP1 antibody and anti-α-Tubulin antibody. (E,F) HEK293T cells were transfected with L1RP EGFP plasmids and then infected with…

FIGURE 2. Non-structural proteins of enterovirus impact on LINE-1 retrotransposition. (A,B) Empty vector VR1012 or EV-D68 2A, 2B, 2C, 3A, 3C, 3D, EV-ORF2p-expressing plasmids were co-transfected into HEK293T cells with L1RP EGFP plasmids. JM111 was used as a negative control for the cytometry assay. The bar represents retrotransposition efficiency, with the VR1012-transfected sample set to 100%. The asterisks indicate statistically significant differences between groups by Unpaired Student’s t-test (p < 0.01). All experiments in this figure were performed in triplicate, and each error bar indicates the SD of three replicates in one experiment.

FIGURE 3. EV-D68 2A modulates LINE-1 retrotransposition in an ORF1p and ORF2p dependent manner. (A) L1RP EGFP plasmids and empty vector VR1012 or EV-D68 2A expressing construct were co-transfected into HEK293T cells. JM111 was used as a negative control. Flow cytometry was performed 4th day post-transfection. The bar graph depicts the M ± SD of each experiment, performed in triplicate The asterisks indicate statistically significant differences between groups by Unpaired Student’s t-test (p < 0.01). (B) Empty vector VR1012 or EV-D68 2A expressing construct was co-transfected with the pYX014/pYX017 construct into HEK293T cells. Dual-Luciferase assays were performed according to the manufacturer’s instructions 96 h post-transfection. The bar graph of Firefly and Renilla luciferase ratio represents the retrotransposition frequency, with the VR1012-transfected sample set to 1.0. The asterisks indicate statistically significant differences between groups by Unpaired Student’s t-test (P < 0.01). (C) VR1012 or VR1012-2A plasmids were co-transfected with pc-L1-1FH. Anti-HA antibodies immunoblotted the cell lysate. (D) HEK293T cells were transfected with pEGFP-N1-ORF1-EGFP and VR1012 or VR1012-2A…

FIGURE 4. Inhibition of LINE-1 retrotransposition by EV-D68 3A. (A) L1RP EGFP plasmids and VR1012/VR1012-3A plasmids were co-transfected into HEK 293T cells. JM111 was used as a negative control. Flow cytometry assay was performed 4 days post-transfection. The bar graph depicts the M ± SD of each experiment, performed in triplicate. The asterisks indicate statistically significant differences between groups by Unpaired Student’s t-test (p < 0.01). (B) pYX014/pYX017 constructs and VR1012/VR1012-3A- plasmids were co-transfected into HEK 293T cells. Dual-Luciferase assay was performed 4 days after transfection. The VR1012-transfected group was set to 1.0. The asterisks indicate statistically significant differences between groups by Unpaired Student’s t-test (p < 0.01). (C) VR1012 or VR1012-3A plasmids were co-transfected with pc-L1-1FH. Immunoblot was performed on cell lysate after 48 h. (D) HEK293T cells were transfected with pEGFP-N1-ORF1-EGFP and VR1012/VR1012-3A plasmids. Immunostaining and fluorescence imaging were performed as described in the “Materials and Methods” after 48 h: scale bar, 20 μm. Bar graph represents the percentage of GFP-positive cells from flowcytometry. The…

FIGURE 5. Suppression of LINE-1 retrotransposition by EV-D68 3C. (A) L1RP EGFP plasmids and VR1012/VR1012-3C plasmids were transfected into HEK 293T cells. JM111 was used as a negative control. Flow cytometry was performed 96 h post-transfection. The bar graph depicts the M ± SD of each experiment, performed in triplicate The asterisks indicate statistically significant differences between groups by Unpaired Student’s t-test (p < 0.01). (B) HEK 293T cells were transfected with pYX014 or pYX017 constructs together with VR1012 or VR1012-3C-. Then dual-luciferase assay was performed 4 days post-transfection. The VR1012 transfected sample was set to 1.0. The asterisks indicate statistically significant differences between groups by Unpaired Student’s t-test (p < 0.01). (C) VR1012 or VR1012-3C plasmids were co-transfected with pc-L1-1FH. Western blot was performed after 48 h. (D) pEGFP-N1-ORF1-EGFP and VR1012/VR1012-3C plasmids were co-transfected. Immunostaining and fluorescence imaging were performed as described in the “Materials and Methods” 48 h post-transfection: scale bar, 20 μm. Bar graph represents the percentage of GFP-positive cells from flowcytometry. The asterisks indicate…

FIGURE 6. EV-ORF2p inhibited LINE-1 retrotransposition activity. (A) L1RP EGFP and VR1012/VR1012-EV-ORF2 plasmids were co-transfected into HEK 293T cells. Flow cytometry was performed after 4 days. The bar graph depicts the M ± SD of each experiment, performed in triplicate. The asterisks indicate statistically significant differences between groups by unpaired Student’s t-test (p < 0.01). (B) pYX014 or pYX017 constructs were transfected with VR1012 or VR1012-EV-ORF2p. The dual-luciferase assay was performed after 4 days. The VR1012 group was set to 1.0. The asterisks indicate statistically significant differences between groups by unpaired Student’s t-test (p < 0.01). (C) VR1012 or VR1012-EV-ORF2 plasmids (250, 300, and 350 ng) were co-transfected with pc-L1-1FH (250 ng). Cells were harvested after 48 h, and then immunoblotting was performed. (D) pEGFP-N1-ORF1-EGFP and VR1012/VR1012-EV-ORF2 plasmids were co-transfected. Immunostaining and fluorescence imaging were performed as described in the “Materials and Methods” 48 h post-transfection: scale bar, 20 μm. Bar graph represents the percentage of GFP-positive cells from flowcytometry. Data are represented as the mean ± SD, and the…

FIGURE 7. Diagram indicating that EV non-structural proteins 2A, 3A, 3C, and EV-ORF2p inhibit LINE-1 retrotransposition. LINE-1 is transcribed into LINE-1 RNA, and then the LINE-encoded proteins are translated. LINE-1 proteins interact with LINE-1 RNA to facilitate the assembly of LINE-1 ribonucleoproteins (RNPs). The LINE-1 ORF2p (endonuclease activity) nicks the bottom strand of the target site DNA. The LINE-1 ORF2p (reverse transcriptase activity) reverse transcribed the LINE-1 RNA using the 3’ hydroxyl group generated by LINE-1 ORF2p endonuclease activity as a primer. This process is a target-primed reverse transcription (TPRT). The DNA/RNA heteroduplex was converted to a DNA/DNA duplex and integrated into the target site. However, the underlying mechanisms remain unclear.