An siRNA Screen Identifies the U2 snRNP Spliceosome as a Host Restriction Factor for Recombinant Adeno-associated Viruses
Fig 2
PHF5A blocks AAV vector transduction after second strand synthesis.
(A) HeLa cells pre-treated with control or PHF5A siRNAs for 24hr were transduced with AAV9, 2, 6 or 8 vectors (MOI 104) expressing luciferase under the control of the SFFV retroviral promoter with no splicing unit. Relative increase in luciferase expression was determined 48 hours p.i. Averages of three independent experiments were shown. Error bars represent standard error of the mean. (B) Melanoma A375 cells and primary human fibroblasts were pre-treated with siRNAs for 24 hours, followed by transduction with the AAV2 CMV-Luc vector (MOI 104) for 48 hours. (C) HeLa cells were pre-treated with siRNAs for 24 hours and infected by the AAV9 CMV-Luc vector (MOI 8 x 104). Total cytoplasmic and nuclear DNA were isolated and AAV luciferase vector genome copies were determined by quantitative real-time PCR at 2, 6 and 24 hours p.i. All samples were prepared in duplicate, and results represent the average of three separate experiments. (D) Same as C, but total DNA at 6 hours p.i. was used to determine total and DNase-resistant AAV genome copies and assess the percent DNase-resistant AAV genomes. Samples were in duplicate and results show the average of two independent experiments. (E) siRNA-treated HeLa cells were infected with AAV9 CMV-Luc vector (MOI 8 x 104) for 1, 3 or 6 hours. Total nuclear DNA samples were used to detect the vector-derived single-stranded and double stranded monomers by Southern blotting. (F) HeLa cells were transfected with siRNAs for 24 hours, followed by infection with a GFP-expressing self-complementary (sc) AAV9 vector (MOI 2 x 104) for 48 hours. Flow cytometry analysis was performed to quantify GFP-positive cell populations. The graph represents percentage of GFP-positive cells from the R2-gated population. (G) HeLa cells were pre-treated or untreated with siRNAs for 24 hours, followed by transduction with the AAV9 CMV-Luc vector (MOI 4 x 105) for 36 hours. Nuclear and cytoplasmic RNA samples were subject to the Northern blotting analysis for detection of the luciferase transcripts. (H) HeLa cells were treated with no siRNA, control or PHF5A siRNAs for 24 hours and then transduced by AAV9 CMV-Luc (MOI 2 x 105). Thirty-six hours p.i., cells were harvested and levels of luciferase transcripts were determined by RT-qPCR. (I) HeLa cells were pre-treated with control or PHF5A siRNAs for 24 hours, followed by transfection with purified AAV CMV-Luc genomic DNA (0.1 μg/well). Luciferase activities were determined at 48 hours p.i. *p<0.05.