shRNA-induced HERVK HML-2 env knockdown changes expression of multiple HERV subgroups and results in reduced expression of interferon-stimulated genes.
(A) Relative count of HERVK HML-2 env RNA in THP1 cells infected with lentivial vector pLKO.1 puro expressing indicated shRNA, selected with 0.5 μg/ml puromycin. RNA was isolated and relative env RNA abundance was measured by RT-qPCR. The fold change RNA count (ΔΔCt) was calculated in relation to β actin reference gene. Error bars indicate ±SD of three independent biological replicates. (B) Transcription, measured by RT-qPCR, of randomly selected differentially expressed HERVK proviruses, identified by transcriptomic analysis, in THP1 cells expressing control (grey) or shRNA-Env (red), 48h post-irradiation. In panels A and B, error bars: ±SD of three independent biological replicates; * p<0.05, ** p<0.01, two-tailed paired t test. (C) Ratio of HML-2 RNA bound to anti-dsRNA antibodies to RNA input. RT-qPCR of RNA IP complexes with rJ2 and 9D5 antibodies, 48h post-irradiation. Error bars: ±SD of five independent biological replicates; * p<0.05, paired Wilcoxon test. (D) Heatmap depicting expression of interferon-stimulated genes (ISG) shown on Fig 5E in THP1 cells exposed to 5Gy γIR, 48h post-exposure: cells expressing shRNA-Env vs control shRNA, measured by PCR array of total cellular RNA samples. Rows: ISG codes; columns: samples. Color codes are shown on the left panel.