Figure 3

Accumulated RNAs in SAMHD1-deficient cells function as immune stimuli. (A,B) PMA-differentiated wild-type THP-1 cells were stimulated with poly dA:dT, poly I:C, an equal amount (5 μg/ml) of isolated total DNA and RNA from wild-type and SAMHD1-deficient cells, or left unstimulated (A). Total RNA isolated from wild-type and SAMHD1-deficient cells were further size-fractionated and an equal amount of RNA from each fraction was used to stimulate PMA-differentiated wild-type THP-1 cells (B), followed by qRT-PCR analysis of IFN-α, IFN-β, IFITM1 and IL6 mRNA levels. (C,D) In vitro RNase activity assay for SAMHD1 immunopurified from undifferentiated THP-1 cells using A20 single-stranded RNA substrates. An isotype-matched control anti-IgG and anti-SAMHD1 antibodies were used for immunopurification. THP1 cells were infected with serial dilution of Vpx-loaded or control SIV VLPs (D). (E) qRT-PCR analysis of IFN-α in wild-type and SAMHD1-deficient cells reconstituted with indicated SAMHD1 wild-type and mutant constructs. (F) Autoradiography of SAMHD1-RNA complex and western blotting of SAMHD1 protein immunoprecipitated from SAMHD1 CLIP. (G) Pie chart showing the distribution of statistically significant peaks (q < 0.001) among the indicated RNA classes. Data were normalized to β-actin expression. In (A), (B) and (E), these data represent the mean ± SEM of triplicate independent experiments (**p ≤ 0.01, ***p ≤ 0.001, ns: not significant, two-tailed Student’s t-test).