Fig 1. Screening of the siRNA library for proteasomal pathway genes identifies PHF5A as a factor blocking the transduction by AAV9 vector.

(A) Screening of the siRNA library was carried out by reverse transfection of HeLa cells with siRNAs, followed by infection with luciferase-expressing AAV9 vectors (AAV9 CMV-Luc) at a multiplicity of infection (MOI) of 10⁴, and assessment of luciferase expression. Screening of the library identified 12 candidate genes that increased transduction by AAV9 vectors over 10-fold. Further studies were carried out in HeLa cells transfected/transduced with specific siRNAs or shRNA lentivectors for each of the 12 genes to verify the screening candidates. (B) Quantitative real-time RT-PCR was performed to determine the levels of PHF5A transcripts in cells treated with control or PHF5A siRNAs at 48 hours. (C) HeLa cells were transfected with control or PHF5A siRNAs for 24 hours, followed by infection with AAV9 CMV-Luc vectors (MOI 10⁴) for an additional 48 hours. The luciferase assay was performed in order to determine relative luciferase activities in treated cells. (D) Same as C, except that a luciferase-expressing adenoviral vector at an MOI of 3 x 10² or an HIV-1-based lentiviral vector (MOI 0.3) were used to infect siRNA-treated HeLa cells. (E) Lentiviral vector pSIN-PHF5A-Escape with the PHF5A-HA Escape transgene was generated through introduction of three silent mutations in the PHF5A siRNA#1-targeted sequence. Western blotting was performed to verify the expression of the PHF5A-HA-Escape and its resistance to the PHF5A siRNA#1 treatment. Anti-PHF5A antibody was used to detect endogenous and over-expressed PHF5A-HA, while anti-HA antibody detected the HA-tagged PHF5A. (F) HeLa cell lines stably expressing the PHF5A-HA-Escape mutant were generated through lentiviral transduction of the escape mutant, followed by puromycin selection. Upon treatment with the PHF5A siRNA and AAV9 CMV-Luc vector (MOI 10⁴), luciferase expression was determined in control HeLa and PHF5A-HA-Escape-expressing HeLa cells. (B-D, F) Data are shown as averages of three independent experiments with error bars representing standard error of the mean. *p<0.05.