PHF5A and U2 snRNP component SF3B1 interact with AAV capsid.

<p><b>(A)</b> HeLa or PHF5A-HA-expressing cell lysates were used to pull-down the HA-tagged PHF5A by anti-HA agarose beads. After 15 washes, the HA-tagged PHF5A was detected by anti-HA antibody. <b>(B)</b> Control or PHF5A-HA-expressing HeLa cells were transduced by AAV2 and AAV9 CMV-Luc vectors (MOI 4 x 10⁵) and total AAV genome copies in the HA pulldown were determined by quantitative real-time PCR. <b>(C)</b> AAV2 CMV-Luc vector (3 x 10¹⁰ genome copies) was unheated or preheated for 30 min at 65°C. PHF5A-HA-over-expressing HeLa cell lysates were then incubated with vectors for 1 hour at 4°C, followed by pulldown of PHF5A-HA. AAV vector genome copies in the precipitates were determined by quantitative real-time PCR. <b>(D)</b> HeLa cells were infected with the AAV2 CMV-Luc vector (4 x 10¹⁰ genome copies/well) for 5 min, 4 or 12 hours. Confocal microscopy analysis was performed to detect the subcellular localizations of AAV vector particles (green) and PHF5A (red). Nuclei were counterstained by DAPI (blue). <b>(E)</b> HeLa cells were infected with AAV2 CMV-Luc vectors (4 x 10¹⁰ genome copies/well) or equivalent amounts of empty AAV2 vectors for 4 hours, and cells were analyzed for co-localization of AAV2 capsid and endogenous PHF5A signals. Prominent co-localized signals were indicated by white arrows. <b>(F)</b> HeLa cells were treated with the PHF5A siRNA for 24 hours, followed by transduction with the AAV2 vector as in E for 4 hours. AAV2 vector particles were detected by anti-AAV2 capsid A20 antibody, and the patterns of cytoplasmic and nuclear accumulations of AAV2 vector particles were compared between control and PHF5A-ablated cells. Representative Z-stack images of the middle sections (slices 3 and 4) from control and PHF5A knockdown cells are shown. <b>(G)</b> Schematic representation for the iodixanol cushion method to enrich cellular factors interacting with particulated AAV capsids. <b>(H)</b> HeLa cell lysates were incubated with AAV2 CMV-Luc vectors (5 x 10¹⁰ genome copies) for 1 hour at 4°C. After centrifugation over 25% iodixanol, three layers (the upper phase, lower phase, and pellet) were separately harvested for Western blotting. AAV capsid proteins VP1, 2 and 3, phospho-SF3B1, and endogenous PHF5A were detected by A20, anti-SF3B1, and anti-PHF5A antibodies, respectively. <b>(I)</b> Same as H for AAV capsid proteins, except that empty AAV2 VP3 only capsids were used for SF3B1 co-precipitation. <b>(J)</b> Control or AAV VP1-over-expressing 293T cell lysates were used to pull-down the AAV VP1 protein by A20 antibody. After 15 washes, the pellets were probed for SF3B1 enrichment by anti-SF3B1 antibody.</p

Meayamycin B increases AAV vector transduction of clinically relevant cell types.

<p><b>(A)</b> Primary mouse islets were infected with AAV8 CMV-GFP in the presence or absence of 2 nM meayamycin B, and GFP expression was monitored for three days. <b>(B)</b> Primary human islets were treated with AAV2 or AAV9 CMV-Luc vectors for 7 hours and then treated with 0, 2, 5 or 10 nM meayamycin B. Luciferase expression was analyzed 48 hours p.i. <b>(C)</b> Neonatal rat cardiomyocytes were infected with AAV2 CMV-Luc or scAAV9 CMV-GFP vectors and treated with meayamycin B, 3 hours p.i. Luciferase activity was measured 3 days p.i., while GFP expression was monitored at 5 days p.i. <b>(D)</b> Porcine hepatocytes were infected with AAV2 or AAV9 CMV-Luc vectors for 8 hours, virus was then removed and cells were treated with 0, 2, or 20 nM meayamycin B. Cells were harvested 48 hours p.i. for the luciferase assay. In A-D, an MOI of 10⁴ was used.</p

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

<p><b>(A)</b> 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>(B)</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. <b>(C)</b> 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. <b>(D)</b> 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. <b>(E)</b> 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. <b>(F)</b> 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>(B-D, F)</b> Data are shown as averages of three independent experiments with error bars representing standard error of the mean. *p<0.05.</p

The U2snRNP complex plays the key role in restricting AAV vector transduction.

<p><b>(A)</b> HeLa cells were transfected with control siRNA, or siRNAs targeting PHF5A, histone 4, U2AF1, SF3B1, SF3B2 and SF3B3 for 24 hours, followed by the AAV9 CMV-Luc vector transduction (MOI 10⁴). Relative luciferase expression was determined 48 hours p.i. <b>(B)</b> Same as A, but luciferase-expressing adenoviral vector was used to transduce siRNA-treated cells (MOI of 3 x 10²). <b>(C)</b> Same as A, except that the siRNA-treated cells were transfected with the vector genome plasmid, pAAV CMV-Luc (0.2 μg/well)for 48 hours. Note that this plasmid was used to generate the infectious AAV9 CMV-Luc vector used in (A). <b>(D)</b> HeLa cells were treated with increasing concentrations of U2 snRNP inhibitor, meayamycin B, followed by transduction with the AAV9 CMV-Luc vector (MOI 10⁴). Relative luciferase expression was determined 48 hours p.i. <b>(E)</b> Same as D, except that a prespliceosome/A complex inhibitor, Isoginkgetin, was used. <b>(F)</b> HeLa cells were treated with indicated spliceosome inhibitors for 8 and 24 hours and levels of unspliced and spliced cellular MAPT (microtubule associated protein tau) transcripts were determined by RT-PCR. <b>(G)</b> HeLa cells were treated with 20 nM meayamycin B at various time points before or after AAV9 CMV-Luc vector infection (MOI 10⁴). Relative luciferase expression was determined 48 hours p.i. <b>(H)</b> HeLa cells were infected by AAV9 CMV-GFP (MOI 10³) or scAAV9 CMV-GFP vectors (MOI 6 x 10³), followed by treatment with 20 nM meayamycin B at 8 hours p.i. Flow cytometry analysis was performed to see GFP-positive cell populations at 48 hours p.i. <b>(I)</b> Co-treatment of HeLa cells with SF3B1 siRNA and Meayamycin B. HeLa cells were treated with the siRNAs for 48 hr, followed by infection with AAV9 CMV-Luc (MOI 10⁴). At 9 hours p.i. Meayamycin B (5nM) was added, and cells were harvested for the luciferase assay 48 hours p.i. <b>(J)</b> Co-treatment of HeLa cells with MG-132 and Meayamycin B. 30 min prior to AAV infection cells were treated with MG-132. 9 hours after infection with AAV9 CMV-Luc (MOI 10⁴), cells were treated with meayamycin B, and harvested for luciferase assay 20 hours later. Due to notable toxicity of MG-132, we needed to harvest cells at this early time point. <b>(K)</b> Influence of dual treatment with human adenovirus 5 infection and SF3B1 disruption on AAV vector infection. HeLa cells were treated with control or SF3B1 siRNAs for 24 hours, followed by infection with AAV2 CMV-Luc (MOI 10⁴) or co-infection with AAV2 CMV-Luc and human adenovirus 5 (MOI 3 x 10⁴) for 48 hours. <b>(L)</b> Influence of adenovirus 5 infection on subcellular localization of SF3B1 in HeLa cells. HeLa cells were infected with human adenovirus 5 (MOI 10⁴) for 24 hours, and SF3B1 in control and infected HeLa cells was visualized by anti-SF3B1 antibody (red). Nuclei were counter-stained by DAPI (blue). (A-E, G, I, J and K) Samples were run in triplicate and results are the average of two independent experiments. *p<0.05.</p

PHF5A blocks AAV vector transduction after second strand synthesis.

<p><b>(A)</b> 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>(B)</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. <b>(C)</b> 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. <b>(D)</b> 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. <b>(E)</b> 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. <b>(F)</b> 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. <b>(G)</b> 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. <b>(H)</b> 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. <b>(I)</b> 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.</p

Three hypoxia-controlled genes associated with Gleason score and prognosis.

<p>Among the hypoxia-regulated genes significantly overexpressed in CaP, cyclin B1 (CCNB1), DLGAP5 and hyaluronan-mediated motility receptor (HMMR) were associated with Gleason score and disease outcome.</p

Transcript levels for CCNB1, DLG7, and HMMR measured in CaP and noncancerous prostate tissue.

<p>Panels on the left compare transcript levels in CaP bulk tissue (full symbols) with the levels measured in benign prostate tissue (open symbols) from men free of CaP (BP) and in benign prostate tissue (BPC) adjacent to CaP of combined Gleason score 6 (gs6). Panels on the right display transcript levels measured in non-neoplastic prostate epithelial cells isolated by laser capture microdissection (LCM) in benign tissues (open symbols): BP, benign prostatic hyperplasia (BPH) and BPC adjacent to CaP of the indicated Gleason score (gs). Full symbols in panels on the right denote transcript levels measured in LCM-isolated CaP cells: high-grade prostatic intraepithelial neoplasia (HGPIN), the cells isolated from areas of combined Gleason scores 6 through 8 and cells isolated from lymph node metastases (met). CCNB1, cyclin B1; DLG7, discs large homolog 7; HMMR, hyaluronan-mediated motility receptor.</p

Receiver operating characteristic (ROC) analysis for DLG7.

<p>The area under the curve was 0.74 (95% CI, 068–0.80). Inset: Scatter plot of the normalized expression values for cases (red) and controls (green).</p