Fig. 2

Regulatory activity of SARS-CoV-2 induced LTR69_Dup69. A LTR69 repeats (orange) were inserted into enhancer reporter vectors expressing Gaussia luciferase (black) under the control of a minimal promoter (grey). HEK293T cells were co-transfected with the indicated reporter vectors expressing Gaussia luciferase and a control vector expressing firefly luciferase for normalization. A previously described LTR12C repeat with known enhancer activity served as positive control. Two days post transfection, reporter luciferase activity was determined and normalized to the activity of the control luciferase. Mean values of three to four independent experiments, each performed in triplicates are shown. Error bars indicate SEM (* p < 0.05, ** p < 0.01, **** p < 0.0001). B LTR69_Dup69 was also inserted downstream of the Gaussia reporter gene. Reporter gene expression was determined as described in (A). Mean values of three to seven independent experiments, each performed in triplicates are shown. Error bars indicate SEM (* p < 0.05). C Integrative Genomics Viewer (IGV) snapshot (hg38) illustrating the localization of LTR69_Dup69 (orange) within an intron of the PTPRN2 gene (green) and adjacent to the ENS00000289418 gene (red). D, E Expression of (D) ENS00000289418 and (E) PTPRN2 in SARS-CoV-2-infected vs. uninfected Calu-3 and A549-ACE2 cells. Cells were infected with an MOI of 2 and harvested 24 h post infection. Mean values of two to three independent experiments ± SEM are shown. F Nucleotide sequence of LTR69_Dup69. The presence of putative binding sites for STAT1, NF-κB subunits and IRF3 is highlighted in bold and underlined. G HEK293T cells were co-transfected with the LTR69_Dup69 reporter vector expressing Gaussia luciferase, a control vector expressing firefly luciferase for normalization and increasing amounts of the indicated stimuli. Two days post transfection, reporter luciferase activity was determined and normalized to the activity of the control luciferase. Mean values of three independent experiments, each performed in triplicates are shown. Error bars indicate SEM (* p < 0.05, ** p < 0.01, ns not significant). H ChIP-seq data of TNF-α-stimulated A549 cells [20] illustrating the enrichment of RELA binding within and adjacent to LTR69_Dup69