ppat.1009305.s006.tif (1.39 MB)

shRNA-induced HERVK HML-2 env knockdown changes expression of multiple HERV subgroups and results in reduced expression of interferon-stimulated genes.

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posted on 2021-02-08, 19:17 authored by Natallia Mikhalkevich, Ina P. O’Carroll, Rok Tkavc, Kateryna Lund, Gauthaman Sukumar, Clifton L. Dalgard, Kory R. Johnson, Wenxue Li, Tongguang Wang, Avindra Nath, Sergey Iordanskiy

(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.