Adeno-associated viruses (AAV) have evolved to exploit the dynamic reorganization of host cell machinery during co-infection by adenoviruses and other helper viruses. In the absence of helper viruses, host factors such as the proteasome and DNA damage response machinery have been shown to effectively inhibit AAV transduction by restricting processes ranging from nuclear entry to second-strand DNA synthesis. To identify host factors that might affect other key steps in AAV infection, we screened an siRNA library that revealed several candidate genes including the PHD finger-like domain protein 5A (PHF5A), a U2 snRNP-associated protein. Disruption of PHF5A expression selectively enhanced transgene expression from AAV by increasing transcript levels and appears to influence a step after second-strand synthesis in a serotype and cell type-independent manner. Genetic disruption of U2 snRNP and associated proteins, such as SF3B1 and U2AF1, also increased expression from AAV vector, suggesting the critical role of U2 snRNP spliceosome complex in this host-mediated restriction. Notably, adenoviral co-infection and U2 snRNP inhibition appeared to target a common pathway in increasing expression from AAV vectors. Moreover, pharmacological inhibition of U2 snRNP by meayamycin B, a potent SF3B1 inhibitor, substantially enhanced AAV vector transduction of clinically relevant cell types. Further analysis suggested that U2 snRNP proteins suppress AAV vector transgene expression through direct recognition of intact AAV capsids. In summary, we identify U2 snRNP and associated splicing factors, which are known to be affected during adenoviral infection, as novel host restriction factors that effectively limit AAV transgene expression. Concurrently, we postulate that pharmacological/genetic manipulation of components of the spliceosomal machinery might enable more effective gene transfer modalities with recombinant AAV vectors. Mammalian cells have developed diverse innate/intrinsic immune strategies to counteract viral infections. Post-entry infection steps of a single-strand DNA virus, adeno-associated virus (AAV), are subject to such restrictions. Here, we screened an siRNA library to identify a novel cellular factor involved in AAV restriction. We found PHF5A, a component of the U2 snRNP mRNA splicing factor, blocks expression from recombinant AAV vectors. Disruption of PHF5A expression specifically enhanced AAV vector performance. Moreover, genetic and pharmacological inhibition of other U2 snRNP proteins, but not spliceosome proteins involved in other splicing steps, strongly increased transgene expression from AAV vectors. Further study demonstrated that U2 snRNP proteins recognize incoming AAV capsids to mediate this cellular restriction at the step after second-strand synthesis. In summary, we identify the U2 snRNP spliceosome complex as novel host factors that effectively restrict recombinant AAV vectors. Considering frequent reorganization of host splicing machinery in DNA virus infections, it is conceivable that U2 snRNP plays a role as a broad spectrum antiviral factor and helper viruses have evolved to counteract this restriction through sequestration of snRNP proteins. Viral pathogens are known to reorganize different components of the host cell machinery during the course of infection. For instance, adenoviruses have been shown to induce nuclear reorganization of host splicing factors and mislocalization of the DNA damage response machinery . SimilarAAV has emerged as a promising vehicle to achieve long-term gene expression with low toxicity. Recombinant vectors based on naturally occurring AAV serotype capsids and libraries of engineered capsid mutants have demonstrated unique receptor usages and tissue tropisms, providing versatility for tissue-targeted gene expression ,20,21,22We screened an siRNA library, which covers 600 known and putative human genes in the ubiquitin and proteasome pathways, for AAV vector transduction. We identified 12 candidate genes . DisruptTo rule out possible off-target effects of siRNA, we generated a lentiviral vector expressing an siRNA-resistant, HA-tagged PHF5A mutant, PHF5A-HA-Escape . When enThe AAV CMV-Luc vector construct used in the library screening contained a human beta globin intron. To rule out the possibility of PHF5A modulating the CMV promoter activity or the intronic unit, we first replaced the CMV promoter and intron sequence in the AAV vector genome with an intron-less retroviral SFFV promoter. Disruption of PHF5A increased transduction by multiple AAV serotypes , indicatNext, we examined the influence of PHF5A ablation on multiple stages of AAV vector transduction. No notable effects were observed on AAV cellular or nuclear entry Figs  and S2. PHF5A has been reported to interact with various proteins, including the U2 snRNP proteins, SF3B1, SF3B2, SF3B3 ,35,36, UTo further confirm the role of U2 snRNP proteins in the restriction of AAV vectors, we assessed the influence of pharmacological inhibition of U2 snRNP on expression from AAV vectors. One drug we employed was meayamycin B, a potent SF3B1 inhibitor, synthesized according to the literature . When HeNext, we assessed the interaction between PHF5A and AAV vector components. Upon pull-down of HA-tagged PHF5A from AAV vector-infected cells Figs  and S5, Next, immunohistochemistry was performed in order to identify the subcellular localization of PHF5A and AAV capsid. The A20 anti-AAV2 capsid antibody was used to detect AAV capsids . The majWhen the subcellular localization of the major U2 snRNP component, SF3B1, was assessed, nuclear speckles in the nucleoli and a diffuse cytoplasmic signal were observed . Althougin vivo.Finally, we tested the ability of meayamycin B to boost AAV transduction in various cell types, relevant to gene therapy applications. When primary pancreatic islets were transduced with AAV8 CMV-GFP and treated with 2 nM meayamycin B 3 hours p.i., there were increased numbers of GFP expressing cells in drug treated mouse islets . When prHere, we have demonstrated that PHF5A and U2 snRNP proteins, such as SF3B1, restrict AAV vector transduction through recognition of incoming AAV capsids. Of particular relevance to gene therapy applications, genetic and pharmacological inhibition of PHF5A or U2 snRNP-associated proteins strongly increased the transduction efficiency of AAV vectors. Thus, transient suppression of U2 snRNP or designing AAV vectors to avoid this restriction can provide a novel strategy to achieve efficient AAV vector transduction with reduced vector doses, which in turn could lead to improved safety profiles for AAV-mediated gene therapy applications.Several strategies have demonstrated the potential to enhance AAV vector transduction efficiency, including treatments with genotoxic agents ,44,45, aDissection of the mechanism by which PHF5A and U2 snRNP components block AAV vector transduction might allow rational design of next generation AAV vectors that can potentially circumvent this host restriction machinery. Based on the following observations, we conclude that U2 snRNP restricts AAVs at an early stage of infection. First, optimal enhancement of AAV vector transduction required U2 snRNP inhibition at an early time point post AAV vector infection (3–24 hours p.i.), while washout of a U2 snRNP inhibitor for the following 2 days did not impair the effects. Thus, short-term U2 snRNP suppression appears to change the fate of AAV vectors up to 2 days post-incubation. Secondly, U2 snRNP inhibition showed no enhancing effect when purified AAV vector genomic DNA or AAV vector genome plasmids were introduced by transfection. Third, although PHF5A was predominantly found in the nuclei in uninfected HeLa cells, we found frequent co-localization of AAV vector particles and PHF5A signals, both in the nucleus and the cytoplasm of cells exposed to AAV vectors. We speculate that newly synthesized PHF5A likely interacts with incoming AAV capsids in the cytoplasm. Finally, pull-down and capsid co-precipitation assays using AAV vectors and empty AAV particles indicate that PHF5A and SF3B1 interact with the capsid, likely mediated by domains within the VP3 region. Of note, the use of heated AAV particles, which leads to exposure of the hidden VP1 N-terminal and viral genome release ,40,49, iAlthough the exact mechanism is currently under investigation, one potential path being explored hinges on the involvement of U2 snRNP proteins in chromatin regulation. For instance, Isono et al.  have repBased on the aforementioned reasons, our current working model is the U2 snRNP recognition of incoming AAV capsid, leading to subsequent block of AAV transcription. However, some observations suggest potential U2 snRNP-mediated AAV restriction at a late stage of transduction. For instance, at very late time points in infection in cell cultures, there was still a substantial enhancement in AAV vector (100-fold at 24 hr and 20-fold at 33 hr p.i.). At this late time point, most of the genomes are considered to be in the nucleus, and it is less likely that U2 snRNP can target incoming AAV capsid. One plausible explanation is that U2 snRNP can also target AAV genome-associated capsid in the nucleus for blocking AAV vector expression. Another point is on our Northern blot analysis of vector transcripts upon PHF5A knockdown. We found a notable increase in cytoplasmic AAV transcripts, but lesser degree in nuclear transcripts. Thus it remains possible that U2 snRNP can also target the nuclear export/cytoplasmic accumulation of AAV transcripts. Another caveat of our experimental system was the use of rapidly dividing cells, where some, or even the majority, of vector genomes can be lost at later time points.in vivo applications of meayamycin B for improved AAV gene delivery. Nevertheless, since a low dose meayamycin B substantially increased AAV vector transduction without strongly affecting host RNA splicing in vitro, designing a novel U2 snRNP inhibitor with reduced cytostatic effects and in vivo stability may allow co-administration of the inhibitor with AAV vectors for improved AAV gene therapy with reduced vector doses.In addition to mechanistic analysis, we have compared FR901464 analogs and herboxidiene, and have identified that meayamycin B is the most potent SF3B1 inhibitor . ImportaIn conclusion, we demonstrate that the U2 snRNP spliceosome inhibits AAV vector transduction and genetic/chemical modulation of this machinery improves transduction efficiency. This finding may lead to approaches that might help reduce AAV vector doses in clinical applications. Further understanding the underlying mechanism would provide novel insights into host-virus interactions and could inform the rational or combinatorial design of next generation AAV vectors with improved transduction efficiency and safety profile.Primary human islets were obtained through the Integrated Islet Distribution Program (IIDP) and the use of the cells was approved by the Mayo Institutional Review Board (IRB10429). All animal experiments were conducted according to the National Institute of Health guidelines and approved by the Institutional Animal Care and Use Committee (IACUC A33214 and IACUC A9014).AGGGGTGGCCATCGGAAG; PHF5A-Escape-R, 5'-CTTCCGATGGCCACCCCTGCCTGCTTGCGGC; PHF5A-ZF1m-F, 5'-ACCAGGGGCGCGCTGTGATCGCTGGAGGACCTGGGG; PHF5A-ZF1m-R, 5'-CCCCAGGTCCTCCAGCGATCACAGCGCGCCCCTGGT; PHF5A-ZF2m-F, 5'-GGTCTCTGATGCCTATTATGCTAAGGAGGCCACCATCCAGG; PHF5A-ZF2m-R, 5'-CCTGGATGGTGGCCTCCTTAGCATAATAGGCATCAGAGACC; PHF5A-ZF3m-F, 5'-GGTGCGCATAGCTGATGAGGCTAACTATGGATCTTACCAG; PHF5A-ZF3m-R, 5'-CTGGTAAGATCCATAGTTAGCCTCATCAGCTATGCGCACC.pAAV-CMV-Luc vector genome construct, which drives firefly luciferase expression by a CMV internal promoter followed by the human beta globin intron, was described previously . pAAV-SFHeLa (ATCC), 293T (ATCC), A375 (ATCC) and primary human cardiac fibroblast cells (ScienCell Research Laboratories) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS) (GIBCO) and antibiotics (penicillin 100 U/mL and streptomycin 100 μg/mL) (Corning Cellgro). HeLa cells stably expressing a series of PHF5A mutants were generated by transduction of corresponding lentiviral vector, followed by puromycin selection. Human islets were obtained through Integrated Islet Distribution Program and cultured in RPMI1640 medium supplemented with 10% FBS and antibiotics. Murine islets were harvested through intraductal collagenase perfusion and enzymatic digestion of the pancreas as previously described , and maiHelper-free AAV vectors were produced by transfection of three plasmids as described previously . BrieflyHuman siGENOME Ubiquitin Conjugation Subsets #1 (89 genes), #2 (115 genes) and #3 (396 genes), a SMARTpool siRNA Library in Reverse Transfection Format (RTF) covering 600 gene targets, were purchased from Thermo Fisher Scientific. According to the provided RTF protocol, 5,000 cells/well HeLa cells were seeded, followed by AAV9 CMV-Luc infection at a multiplicity of infection of 100 (gc/cell). 48 hours after infection, luciferase assay was performed using the ONE-Glo Luciferase Assay System (Promega).HeLa cells were seeded in a 96-well plate at 5,000 cells/well for one day. Cells were then transfected with 0.5 μL of 10 μM siRNA using DharmaFECT Transfection Reagents (Thermo Fisher Scientific) according to the manufacturer’s instruction. Following siRNAs were used; control siRNA (siKrt1 5 SI02636732 from Qiagen), siPHF5A#1 and #2 (PHF5A 6 SI04210892 and 7 SI04310621) from Qiagen, siGenome Smart Pool siRNAs for PHF5A-interacting proteins;—siHIST1H4B , siU2AF1 , siSF3B1 , siSF3B2 , siSF3B3 . Twenty four hours post transfection, cells were infected with luciferase- or GFP-expressing vectors for 2 days.Meayamycin B was described previously . IsoginkcDNA synthesis was performed with one μg RNA using RNA to cDNA EcoDry Premix (Clontech). Primers used were as follows: Western blotting was performed as described previously . Followi4) was added for 1, 3 or 6 hours. Cells were harvested in lysis buffer for nuclear fractionation. Nuclear lysates were purified as in “Cell fractionation and analysis of nuclear rAAV genomes” section. 2 μg of DNA sample was run on a 2% agarose gel without ethidium bromide at 50V for 1.5 hours. The gel was prepared for transfer in the following washes with rocking: 0.25N HCl 10 min, rinse ddH20, denaturation buffer  15 min, denaturation buffer 30 min, neutralization buffer  15 min, and neutralization buffer 30 min. The gel was then blotted overnight by capillary transfer with 10x SSC on a positively charge nylon membrane (Roche). DNA was fixed to the membrane by UV-crosslinking and the luciferase probe was hybridized to the DNA overnight at 43.5°C. The membrane was washed and developed according to the protocol (Roche).DIG High Prime DNA Labeling and Detection Starter Kit II (Roche) was used for Southern blotting to detect the luciferase DNA in the AAV vector genome. A luciferase DNA fragment from pSIN-Luc was labeled according to the manufacturer’s instruction. HeLa cells were seeded in a 6-well plate at 200,000 cells per well, followed by transfection with control or PHF5A siRNAs. 24 hours post transfection, AAV9 CMV-Luc (MOI 8 x 105). 36 hours post transduction cells were harvested and nuclear and cytoplasmic RNA was isolated using the PARIS kit (Ambion). 1 μg RNA was run on a formaldehyde gel, washed, and blotted by capillary transfer overnight according to the DIG Northern Starter Kit (Roche). The RNA was fixed to the membrane by UV-crosslinking and the DIG-labeled luciferase DNA probe from Southern blotting was incubated with the pretreated membrane overnight at 50°C. The membrane was washed and developed according to the manufacturer’s instructions.Cells were prepared for Northern blot by seeding at 200,000 cells per well in a 6-well plate, transfected with control and PHF5A siRNAs, followed by transduction with AAV9 CMV-Luc vector  and incubated on ice for 10 min. The lysates were homogenized using a 21-guage needle and syringe, spun at 14,000 rpm for 15 min at 4°C and the supernatant (cytoplasmic fraction) was collected. The pellet (nuclear fraction) was washed with PBS. RNA was eliminated by RNaseA (200U/mL) treatment. A Qiagen QIAamp DNA Mini kit was used to further purify the DNA. Quantitative real-time PCR was performed to determine AAV luciferase genomic copy numbers. To assess encapsidated AAV genomes in the nucleus, the above procedure was followed with the addition of DNase (Invitrogen) treatment at 37°C for 30 min (both control and DNase treated samples) at the beginning of DNA purification and quantitative real-time PCR detection.Cells were resuspended in cytoplasmic lysis buffer  by FuGENE6 (Promega), and luciferase expression was analyzed 48 hours after viral DNA transfection.AAV genomic DNA was isolated using the QIAamp DNA mini kit following the provided Protocols for Viral DNA (Qiagen). The genomic DNA was isolated from 4 x 105) in a 6-well plate for 6 hours at 37°C. Cells were then harvested on ice in RIPA buffer containing protease inhibitor, followed by pull-down with 20 μL anti-HA agarose beads (Pierce). After 15 cycles of washing, pellets were resuspended in 0.5 mL PBS and split into 2 aliquots. One aliquot was used for Western blotting of AAV capsid proteins, and the other was used for the isolation of total DNA by QIAamp DNA Mini kit for RT-qPCR detection of AAV genomic DNA. For pull-down assay using heated AAV particles, 100 μL cell lysate was combined with 3 x 1010 gc of AAV2 CMV-Luc vector particles that were unheated or pre-heated for 30 min at 65°C, followed by precipitation by anti-HA agarose as above.Semi-confluent HeLa cells with or without stable overexpression of the HA-tagged PHF5A were infected with AAV2 or AAV9 CMV-Luc (MOI 4 x 104 cells/well and were infected with AAV2 CMV-Luc (4 x 1010 gc/well) or empty AAV2 (25 μl/well) at 37°C for 5 min, 4 hours, or 12 hours. Cells were fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.3% Triton X-100 for 15 min, and blocked with 5% FBS/PBS for 30 min. Primary antibody was added, and cells were incubated for 1.5 hours at room temperature in a humidified chamber. Secondary antibody followed according to the same procedure. Then cells were washed three times with PBS, treated with DAPI  for 1 min, washed three times with PBS, and mounted with Dako fluorescent mounting media. Confocal microscopy was performed on an LSM 780 confocal microscope (Zeiss). The following primary and secondary antibodies were used for immunocytochemistry of uninfected and AAV infected HeLa cells: anti-AAV particles (A20) mouse monoclonal antibody (American Research Products) at 1:100 followed by FITC-conjugated donkey anti mouse IgG (H+L) (Jackson 715-095-151) 1:500; rabbit anti-PHF5A (Sigma HPA028885-100UL) 1:250 and Alexa Fluor 594-conjugated donkey anti-rabbit IgG (H+L) (Invitrogen A-21207) 1:2000; rabbit anti-phospho-SF3B1  1:500 and Alexa Fluor 594-conjugated donkey anti-rabbit IgG (H+L) 1:500.The Lab-TekII 8-well chamber slides (Thermo Fisher Scientific) were pretreated for 5 min with poly-d-lysine . HeLa cells were plated at 1 x 1010 gc/tube) and purified AAV2 empty, VP3 only particles were left unheated or pre-heated at 65°C for 30 min and placed on ice. HeLa and HeLa-PHF5A-HA cells were harvested by incubating 1 well of a 6-well plate with 500 μL RIPA buffer supplemented with protease inhibitors on ice for 10 min. Cells were harvested by scraping, homogenized using a 21-guage needle and syringe, and spun for 5 min at 13,200 rpm. 400 μL of the cell lysate was added to the virus and samples were rotated for 1 hour at 4°C. The 25% iodixanol solution was prepared using 3.2 mL 1x PBS, 2.8 mL 9:1 Optiprep to 10x PBS, and 0.15% phenol red. 30 μL of the lysate virus mixture was removed and used as an input. The remaining lysate-virus mix was layered on top of 0.5 mL 25% iodixanol. These samples were spun at 4°C for 1 hour at 14,000 rpm. After spinning the clear upper layer, red lower layer, and pellet were harvested for Western blot. The following antibodies were used in this experiment: rabbit anti-PHF5A (Sigma HPA028885) 1:100, rat anti-HA clone 3F10 (Roche 11867423001) 1:500, rabbit anti-VP1, 2, 3  1:250, mouse anti-SF3B1  1:500, rabbit anti-phospho-SF3B1  1:400, rabbit anti-histone H2B  1:1000, and rabbit anti-histone H3  1:1000 in blocking buffer. To note, the membrane that received phospho-SF3B1 primary antibody was blocked in 5% BSA/PBS + 0.05% Tween-20. Antibodies were diluted in 1x TBS + 0.001% Tween-20.AAV2 CMV-Luc vectors (5 x 10HeLa cells at 80% confluency were treated with pre-mRNA splicing inhibitors at the following concentrations: 3-Aminophenylboronic acid (5mM and 1mM), Isoginkgetin (25uM and 12.5uM), and meayamycin B (10nM and 5nM). Eight hours post drug RNA was isolated using TRIzol (Invitrogen), and cDNA synthesis was performed with one μg RNA using RNA to cDNA EcoDry Premix (Clontech). cDNA was amplified by KOD Hot Start DNA Polymerase (EMD Millipore) using primers for MAPT exon 10 5’-AAGATCGGCTCCACTGAGAA-3’ and 5’-ATGAGCCACACTTGGAGGTC-3’.S1 Fig(A) Quantitative real-time RT-PCR was performed to determine the levels of RAB40A and PRICKLE4 transcripts in HeLa cells infected with a control lentiviral vector pLKO.1 or vectors carrying RAB40A and PRICKE4-targeting shRNAs at 48 hours. (B) Upon infection with AAV9 CMV-Luc vector for two days, relative luciferase activity was determined in HeLa cells pre-treated with the shRNA lentivectors. (C) Same as B, but luciferase-expressing adenoviral and lentiviral vectors were used. Data are shown as averages of three independent experiments with error bars representing standard error of the mean. * (p<0.05). For the introduction of shRNAs, cells were transduced with lentiviral control vector (pLKO.1), or vectors carrying shRAB40A or shPRICKLE4 from OpenBiosystems (plasmid names) at estimated MOI of 4. 24 hours post lentiviral transduction, cells were infected with luciferase-expressing vectors. Luciferase expression was measured by the ONE-Glo Luciferase Assay System (Promega) according to the manufacturer’s instruction. Pre-designed primers from Invitrogen were purchased  and used for the qRT-PCR. Over-expression of the PRICKLE4-Escape mutant did not reverse the effects of the PRICKLE4 shRNA in HeLa cells stably expressing an shRNA-resistant PRICKLE4 mutant. Thus, the effects observed with the PRICKLE4 disruption were likely due to off-target effects. We have not tested the effect of over-expression of RAB40A-Escape mutant.(PDF)Click here for additional data file.S2 FigCytoplasmic and nuclear fractions were analyzed 2 hours p.i. to determine subcellular fractionation purity. GAPDH  and Histone H3  antibodies were used as cytoplasmic and nuclear markers to verify the cytoplasmic and nuclear fractions respectively.(PDF)Click here for additional data file.S3 Fig(A) The alignment of PHD finger-like domains of PHF5A and its yeast homolog Rds3 is shown. Five conserved CxxC repeats are underlined. (B) Predicted triquetra knot structure of PHF5A, based on the yeast Rds3 structure , is shown with the three GATA-type zinc fingers. (C) HeLa cells were transduced by lentiviral vectors expressing a series of zinc finger mutants of the PHF5A-HA-Escape, followed by puromycin selection. Expression of HA-tagged PHF5A proteins and their resistance to the PHF5A siRNA were verified by transfecting individual HeLa lines with control or PHF5A siRNAs. (D) Control HeLa cells, HeLa lines stably expressing PHF5A-HA Escape, or PHF5A-HA Escape zinc finger mutants, were pre-treated with control or PHF5A siRNAs for 24 hours, followed by transduction by the AAV9 CMV-Luc vector. Relative luciferase expression was determined 48 h ours p.i. Data are shown as averages of three independent experiments with error bars representing standard error of the mean.(PDF)Click here for additional data file.S4 Fig4) and given meayamycin (20 nM) 9 hours p.i. Luciferase assay was performed 48 hours p.i. (D) HeLa cells were transduced with AAV9 CMV-Luc vectors, in the presence of increasing doses of ABA. Relative luciferase expression was determined 72 hours p.i. (E) HeLa cells were transduction with AAV2 or AAV9 CMV-Luc vectors, followed by 20 nM meayamycin B treatments at various time points . Cells were harvested and relative luciferase expression was determined 72 hours p.i. (F) Co-treatment of HeLa cells with PHF5A siRNAs and Meayamycin B. HeLa cells were treated with the siRNA 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. (G) Influence of dual treatment with human adenovirus 5 infection and meayamicin on AAV vector infection. HeLa cells were infected with AAV2 CMV-Luc (MOI 104) or co-infection with AAV2 CMV-Luc and human adenovirus 5 (MOI 3 x 104), in the presence or absence of meayamycin (20 nM) for 48 hours. (H) Influence of adenovirus 5 infection on subcellular localization of PHF5A in HeLa cells. HeLa cells stably expressing the HA-tagged PHF5A were infected with human adenovirus 5 (MOI 104) for 24 hours, and PHF5A-HA in control and infected HeLa cells was visualized by anti-HA antibody (red). (I) Influence of U2 snRNP inhibition on AAV vector production. AAV2 vectors were made by triple transfection with pHelper, pXX2, and pAAV CMV-Luc, triple transfection with Meayamycin B (5 nM), or double transfection (no pHelper) with Meayamycin B (5 nM). Viruses were added to HeLa cells (2 uL/well) and 48 hours later their infectivity was determined by luciferase assay.(A) HeLa cells were transfected with control or siRNAs targeting the potential PHF5A-interacting proteins. Commercially available specific primers and probe sets were used for the quantitative real-time RT-PCR to determine the transcript levels. From Bio-Rad LMNA, FUS, EEF1A1, EEF2, HIST1H4B, U2AF1, SRSF5, SF3B1, SF3B2, SF3B3, EP400, DDX1, SNRNP200 and PRPF31 primers; from Invitrogen PHF5A primers and probe. The levels of individual transcripts in untreated cells were set as 100%. Error bars represent standard deviation. (B) HeLa cells were transfected with control siRNA, or a series of siRNAs for 24 hours, followed by the AAV9 CMV-Luc vector transduction. Relative luciferase expression was determined 48 h ours p.i. (C) HeLa cells were transduced with AAV2 or AAV9 CMV-Luc (MOI 10(PDF)Click here for additional data file.S5 Fig(A) HeLa or HeLa overexpressing PHF5A-HA lysates were used to pull-down the HA-tagged PHF5A by anti-HA agarose beads. After immunoprecipitation samples were spun for 5 min and the supernatant and pellet were separated and run on a Western blot for HA detection of the PHF5A-HA tagged protein. Relative PHF5A-HA levels were quantified using ImageJ software. (B) Same as A except samples were washed 15 times following IP and washes 5, 10 and 15 were saved an run along side the input and pull-down for Western blotting. The percent PHF5A-HA pulled down relative to the input was calculated by ImageJ software.(PDF)Click here for additional data file.S6 Fig(A) Control and AAV2 vector-treated HeLa cells were monitored for the subcellular localization of endogenous PHF5A. Note increased cytoplasmic PHF5A signals upon AAV2 vector infection. (B) Same as (PDF)Click here for additional data file.S7 Fig(A) Control HeLa cells were infected with the AAV2 vector 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 visualized using Z-stack images. Top (slice 1) to bottom (slice 5) images were shown above. The middle sections (slices 3 and 4) were shown in higher magnification. (B) Same as A, but HeLa cells were pre-treated with the PHF5A siRNA for 24 hours. (C) Control and PHF5A siRNA-treated HeLa cells were infected with the AAV2 CMV-Luc vector for 4 hours, followed by confocal microscopic analysis of AAV2 capsid and PHF5A. Note the PHF5A siRNA-treatment suppressed the PHF5A signals. Again, no notable changes in AAV2 vector particle trafficking were observed.(PDF)Click here for additional data file.S8 Fig(A) HeLa cells were infected with the AAV2 CMV-Luc vector for 5 min, 4 hr, or 12 hr. Confocal microscopy analysis was performed to detect the subcellular localizations of intact AAV vector particles (green) and SF3B1 (red) by specific antibodies. Nuclei were counterstained by DAPI (blue). (B) HeLa cells were infected with the AAV2 CMV-Luc vector for 4 hours, and cells were analyzed for co-localization of AAV2 capsid and endogenous SF3B1 signals. (C) Same as (PDF)Click here for additional data file.


After the 1968 H3N2 pandemic emerged in humans, H3N2 influenza viruses continuously circulated and evolved in nature. An H3N2 variant was circulating in humans in the 1990s and subsequently introduced into the pig population in the 2000s. This virus gradually became the main subtype of swine influenza virus worldwide. However, there were no reports of infections in dogs with this virus.In 2013, 35 nasal swabs from pet dogs were positive for Influenza A virus by RT-PCR. Two viruses were isolated and genetically characterized. In the phylogenetic trees of all gene segments, two H3N2 canine isolates clustered with Moscow/10/99 and most H3N2 swine influenza viruses. These results indicated that two H3N2 CIVs possessed high homology with human/swine influenza viruses, which at the same time exhibited some amino acid substitutions in NA, polymerase basic protein 1 (PB1), and nucleoprotein (NP), which probably were related to the interspecies transmission.These two viruses share the highest homology with swine H3N2, Moscow/99-like viruses, which indicated that these viruses might originate from swine viruses. Influenza A viruses circulate worldwide and are endemic in multiple species from birds to mammals . Historiet al. [During April to November 2013, 261 nasal swabs and 315 blood samples were collected from different pet hospitals, including Nanning, Yulin, Liuzhou, Hechi, Baise, Qinzhou, Wuzhou, Pinxiang in Guangxi Zhuang Autonomous Region (Guangxi) of China. Some of these pet dogs showed similar clinical signs of coughing, sneezing, nasal discharge, and fever (>39.5°C), and others had no respiratory syndrome (Table http://www.ncbi.nlm.nih.gov/BLAST). Sequences comparisons revealed that six gene segments of two H3N2 CIVs had highest homology with the corresponding genes of swine H3N2 influenza viruses (SIVs) from the 2000s, including HA, PB2 and PA genes (99.8–99.9%) of swine/Henan/1/2010 (H3N2), PB1 and NP genes (99.8%) of swine/Shandong/3/2005, and M genes (99.8%) of swine/Guangdong/L22/2010 (H3N2), but NA and NS genes were most closely related to the corresponding gene from swine H1N2 influenza virus, swine/Zhejiang/01/2008 (H1N2) , which is a reassortant of human-like H3N2 viruses, seasonal human H1N1 viruses and classical H1N1 swine viruses . Reverse transcription was carried out under standard conditions with Uni12 primer (5’-AGCAAAAGCAGG-3’). Eight pairs of primers  were useTo characterize more precisely the genetic origin of these two H3N2 CIVs, the phylogenetic trees of eight gene segments  were constructed using MEGA 5.2. Compared with the related reference nucleotide sequences available in GenBank, it was found that canine influenza viruses were mainly divided into human/swine, avian/canine, pdm09-like/canine, and equine/canine lineages based on HA gene  stalk region. However, S44P, V240I and V398I substitutions were found in the NA protein of both CIVs. Besides, substitution R189K in PB1 and A493T in NP genes were present in CIVs differed from swine or human influenza viruses , as previously described . An HI tEmergence of human-like H3N2 influenza viruses in pet dogs, with or without any clinical signs, raises further concerns about whether the viruses have crept into the dog population, and whether novel reassortant influenza viruses will emerge from infection of pet dogs that pose a potential threat to public health. Continued surveillance for influenza viruses in dogs will become essential.


Loss of human orthologs ubiquitination factor E4 B (UBE4B) and lysine-specific demethylase 1 (LSD1), respectively encoding a ubiquitin ligase and a lysine-specific demethylase, promotes the clearance of misfolded proteins in mammalian cells by activating both proteasomal and autophagic degradation machineries. An unbiased search in this pathway reveals a downstream effector as the transcription factor p53, a shared substrate of UBE4B and LSD1 that functions as a key regulator of protein quality control to protect against proteotoxicity. These studies identify a new protein quality control pathway via regulation of transcription factors and point to the augmentation of protein quality control as a wide-spectrum antiproteotoxicity strategy.Protein quality control is essential for clearing misfolded and aggregated proteins from the cell, and its failure is associated with many neurodegenerative disorders. Here, we identify two genes,  A new protein quality control regulatory pathway is identified in which a ubiquitin ligase and a lysine-specific demethylase act together on the transcription factor p53 to control protein degradation systems. Caenorhabditis elegans as a model to understand how cells handle disease-associated misfolded proteins. In a large-scale genetic screen, we discovered two suppressor genes, ufd-2 and spr-5, which encode a ubiquitin ligase and a lysine-specific demethylase, respectively. When these two proteins are inactivated, we observe a marked reduction in the toxicity of several misfolded proteins. ufd-2 and spr-5 are conserved in humans , as are their effects on misfolded proteins. We show that UBE4B and LSD1 regulate the activity of protein degradation machineries including the proteasome and autophagosomes. Using microarrays and biochemical analyses, we identify p53 as a key downstream transcription factor that mediates the action of UBE4B and LSD1 on protein clearance. This work establishes p53 as a regulator of proteome integrity and uncovers a new protein quality control pathway that could potentially be exploited to increase the degradation of misfolded proteins in diseased cells.To function properly, proteins must assume their correct three-dimensional shapes. There are numerous mechanisms within the cell, collectively referred to as protein quality control (PQC), that verify proper folding. If abnormal folding is detected, PQC can either help the protein to refold or target it for degradation. Failures in protein folding and PQC lead to the accumulation of misfolded proteins, which often self-associate into large aggregations that are thought to be the underlying cause of several neurodegenerative diseases. In this study, we use the roundworm  Living organisms endure environmental stress and metabolic errors that inflict damage on macromolecules, including DNA and protein, which are either repaired or removed by quality control programs in the cell. Toxicity resulting from protein misfolding and aggregation, known as proteotoxicity, underlies many degenerative diseases, including those affecting the nervous system, such as Creutzfeldt-Jakob disease, Alzheimer disease, Parkinson disease, Huntington disease, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS) ,2. To guMutant Cu/Zn superoxide dismutase (SOD1), linked to ~20% of familial ALS, represents a simple molecular model for protein misfolding and aggregation. The wild-type (WT) SOD1 protein has a stable β-barrel structure with a two-state folding process, whereas mutant SOD1 proteins show a heightened propensity to aggregate in vitro and in vivo –10. TherCaenorhabditis elegans model that expresses neuronal ALS-linked SOD1 mutant proteins and develops robust movement defects to perform an unbiased genetic screen for potent suppressors of the behavioral defects. We identified mutations in two genes, ufd-2, encoding a ubiquitin ligase, and spr-5, encoding a lysine-specific demethylase, that synergistically attenuate the neurotoxicity of mutant human SOD1 proteins. The actions of the suppressor genes are conserved in Drosophila, and they protect against proteotoxicity initiated by diverse mutant proteins, including TDP-43, FUS, and the polyglutamine (polyQ) tract. Furthermore, we found human orthologs of these modifiers to be part of a pathway regulating protein quality control in mammalian cells. Further analysis showed that this pathway acts through the transcription factor p53, which mediates cellular stress responses. Together, these results identify a new mechanism involving previously unrecognized players, which the cell utilizes to augment protein quality control.Here we took advantage of a C. elegans model of ALS, in which the neuron-directed expression of the ALS-linked, G85R mutant human SOD1 (SOD1G85R) protein leads to its aggregation into misfolded soluble oligomers and larger insoluble aggregates . Wh. Whufd-25(by134) . However1 strain . Total l strains .. Howevenchanged . Finally1 strain , indicatC. elegans models, yellow fluorescent protein (YFP) fusions of several disease-relevant, aggregation-prone proteins, such as SOD1G85R-YFP . RT. RT69]. Embryonic Sprague Dawley rat spinal cord cultures and neuronal survival assays were previously described  Click here for additional data file.S1 FigA) A schematic for protein misfolding and aggregation in relation to protein solubility and quality control. For an aggregation-prone protein, there is a dynamic equilibrium among correctly folded native proteins, its misfolded forms, oligomeric aggregates, and large aggregates. The relative proportion of these species depends on the intrinsic folding properties of the protein and the cellular environment of protein quality control. In a protein solubility assay, large aggregates can be sedimented into the insoluble fraction via extraction and centrifugation, while smaller oligomeric aggregates and other misfolded proteins are retained in the soluble fraction. The misfolded and aggregated proteins are targets of cellular protein quality control machineries including molecular chaperones, the ubiquitin-proteasome system, and autophagy. (B) Western blotting analyses of C. elegans indicate that the ufd-2 and spr-5 mutations do not substantially alter the total protein levels of untagged SOD1G85R. The western blot lanes are from the same gel and exposure. (C) Total protein levels of YFP-tagged SOD1G85R, TDP-43c25, or PolyQ proteins are reduced in double ufd-2 and spr-5 mutant strains, suggesting that larger fractions of YFP-tagged misfolded proteins are removed in the mutant strains. (D) Total protein levels of untagged TDP-43M337V or FUSR521C are not substantially altered in Drosophila with the knockdown of ufd-2/UBE4B (CG9934) or spr-5/LSD1 (Su(Var)3-3).((TIF)Click here for additional data file.S2 FigA) The flow chart of the mammalian cell-based protein solubility assay as described in the Materials and Methods. (B) A representative western blot of total SOD1G85R protein in HEK293T cells treated with UBE4B and LSD1 shRNAs. (C) Quantification of total SOD1G85R protein and its corresponding supernatant (S) and pellet (P) fractions. The quantification is based on the band intensities and the relative amounts of S and P fractions as parts of the total lysate that were loaded on the gel. The percentage of insoluble SOD1G85R protein in the total SOD1G85R protein remains relatively stable at 25%–30%. (D) A representative western blot of the TDP-43Q331K protein solubility assay. HEK293T cells were transfected with a TDP-43Q331K expression plasmid, together with a control shRNA (CTRL) or mixed UBE4B and LSD1 shRNA plasmids. Following cell lysis and fractionation, S and P fractions were run on 15% SDS-PAGE gels. The western blot lanes are from the same gel and exposure. (E) Quantification of TDP-43Q331K in the S and P fractions, containing smaller and larger aggregates, respectively, shows a significant reduction in protein levels caused by the UBE4B and LSD1 knockdown . (F) Western blot analyses of cycloheximide chase show that TDP-43Q331K protein is degraded faster in double UBE4B and LSD1 knockdown cells than in the nontargeting shRNA control cells. (G) Quantification of chase experiments in (F) (n = 2). Data represent means ± SEM. The numerical data used to make this figure can be found in ((TIF)Click here for additional data file.S3 FigA) The heat map of microarray signals of differentially regulated genes (p < 0.05) upon the knockdown of LSD1 alone, UBE4B alone, or both, in triplicates. Hierarchical clustering of the samples indicates that the single UBE4B knockdown induces similar transcriptional changes as the double knockdown, consistent with the pattern of antiproteotoxic activities shown in B) The heat map of p53 transcriptional targets. The hierarchical clustering of the samples demonstrates the same pattern as shown above for all differentially regulated genes. (C) The p53 network is activated in the UBE4B and LSD1 double-knockdown cells . The transcriptional targets with changes consistent with p53 activation are shown, with up-regulated genes in red and down-regulated genes in green.((TIF)Click here for additional data file.S4 FigA) Elevation of MDM2, the p53-targeting ubiquitin ligase, significantly reduced the p53 response element-mediated activity that was induced by the UBE4B and LSD1 knockdown (n = 5). MDM2 is co-transfected with the p53 reporter upon knockdown of UBE4B and/or LSD1. (B) The dimethylation at the K370 residue of the p53 protein (Me2-K370-p53) is increased in double UBE4B and LSD1 knockdown cells. In HEK293T cells treated with UBE4B and LSD1 shRNAs, there is an increase of Me2-K370-p53 relative to the total p53 protein in the nucleus-enriched fraction as shown in the western blots (left) and quantification chart . (C) A constitutively active forkhead responsive element luciferase reporter (FOXO1-AAA) was used to demonstrate that the knockdown of UBE4B and LSD1 specifically induces the FOXO1 transcriptional activity (n = 3). (D) C. elegans locomotion assays indicate the rescuing effects of loss-of-function mutations spr-5(by134);ufd-2(tm1380) (indicated by-/-) on the neurotoxicity of transgenic SOD1G85R protein. Loss-of-function mutation daf-16(mu86) partially reversed locomotion rescue of spr-5(by134);ufd-2(tm1380) mutations (n > 40). Data represent means ± SEM. The numerical data used to make this figure can be found in ((TIF)Click here for additional data file.S5 FigA) A schematic of a luciferase-based proteasomal activity assay. Isolated cytosol is mixed with a peptide substrate, Suc-LLVY-luciferin. The chymotrypsin-like activity of proteasomes cleaves off the Suc-LLVY peptide, releasing the amino-luciferin, which produces strong chemiluminescence in the presence of luciferase and ATP. The detected chemiluminescence is used to quantify the proteasomal activity. (B) Inhibition of the proteasomal activity reverses the reduction of insoluble SOD1G85R protein in the pellet fraction induced by the UBE4B and LSD1 knockdown. Western blots are shown for insoluble SOD1G85R proteins from MG132-treated (20 μM) or untreated (DMSO) HEK293T cells with knockdown of UBE4B and LSD1 or nontargeting shRNA controls. Cells were treated with MG132 for 48 h and lysed 72 h post-transfection. (C) Quantification of SOD1G85R levels from western blots as shown in (B), n = 2. Data represent means ± SEM. (D) The flow chart of an autophagic activity assay to measure LC3 cleavage based on a luciferase (GLuc) reporter. Cells were transfected with a set of plasmids to knockdown LSD1 and UBE4B  and to express the GLuc reporters and SEAP . SEAP is constitutively secreted and serves as a transfection normalization control. SOD1G85R is expressed concurrently to match the condition with the increased burden of misfolded proteins, as described earlier (E) A schematic of the LC3 cleavage and Gluc release assay. A cleavable fusion protein, Actin(Act)-LC3-GLuc, or its uncleavable negative control, Act-GLuc, is anchored to the actin cytoskeleton inside the cell. When Act-LC3-GLuc is cleaved by the autophagy-associated protease ATG4B, the GLuc fragment is released from its actin anchor and rapidly secreted out of the cell. The activity of GLuc in the cell medium is assayed over a period of several days using the Dual Luminescence Assay kit. The numerical data used to make this figure can be found in ( earlier . (E) A s(TIF)Click here for additional data file.S6 FigA) The p53 small molecule activator, Tenovin-1 , significantly decreased the levels of misfolded SOD1G85R but not WT SOD1 proteins in both supernatant and pellet fractions as compared with vehicle-treated controls (VEH) (n = 4). Representative western blots are shown in G85R solubility assay in HEK293T cells is described in Materials and Methods. (B) Western blots of cycloheximide chase experiments with Tenovin-1-treated or untreated HEK293T cells. (C) Quantification of the chase experiments shows increased SOD1G85R degradation in Tenovin-1-treated cells (n = 2). (D) Another p53 small molecule activator, CP-31398 (4 μg/ml), also significantly decreased the levels of misfolded SOD1G85R proteins but not WT SOD1 proteins in both supernatant and pellet fractions (n = 3). Representative western blots are shown in E) Western blots of cycloheximide chase experiments with CP31398-treated or untreated HEK293T cells. (F) Quantification of the chase experiments shows increased SOD1G85R degradation in CP31398-treated cells (n = 2). Data represent means ± SEM. (G) Knockdown of p53 blocked the improved clearance of misfolded SOD1G85R proteins by Tenovin-1 or CP-31398. HEK393T cells were transfected with SOD1G85R and treated with p53-activating drugs Tenovin-1 or CP31398. Protein aggregation assays were performed to evaluate the levels of SOD1G85R proteins in the supernatant (S) and pellet (P) fractions. The reduction of SOD1G85R aggregation in cells treated with Tenovin-1 or CP-31398 is dependent on p53, as the knockdown of p53 abolishes the ability of the drugs to remove aggregates. (H) p53-activating drugs activate autophagy as indicated by LC3 protein levels. HEK293T cells treated with Tenovin-1 or CP-31398 for 24 h were lysed in 1% SDS buffer, and LC3-I and LC-II levels were analyzed by western blots. For both Tenovin-1 and CP-31398, the LC-II levels are augmented with increasing drug concentrations. Both panels were from the same gel and western blot with identical imaging settings. The numerical data used to make this figure can be found in ((TIF)Click here for additional data file.S7 FigA) Stable knockdown of p53 partially blocks the improved clearance of SOD1G85R proteins conferred by the knockdown of UBE4B and LSD1. A stable cell line with inducible knockdown of p53 via an integrated shRNA (in a pR4R3-TET-PURO vector) is used to conditionally remove p53 upon the induction of doxycycline (DOX). The protein solubility assay was used to analyze the SOD1G85R protein levels in supernatant (S) and pellet (P) fractions. The knockdown of UBE4B and LSD1 substantially enhances the clearance of misfolded SOD1G85R proteins, but this effect is partially reversed by the DOX-induced knockdown of p53 (+DOX). The western blots for the S and P fractions are from the same gels. (B) Transient knockdown of p53 blocks the improved clearance of SOD1G85R proteins conferred by the knockdown of UBE4B and LSD1. The protein solubility assay was performed as in (A), except with the knockdown of p53 achieved through shRNA transient transfection and with LSD1 and UBE4B shRNAs in a pR4R3-NEO vector targeting their respective 3′ UTRs (see C) Loss of p53 blocks increased SOD1G85R protein clearance conferred by the knockdown of UBE4B and LSD1 in HCT116 cells. HCT116 cells with either the p53 knockout or WT genotype were treated with the UBE4B and LSD1 shRNAs or nontargeting controls. Left: western blot analyses of the insoluble SOD1G85R proteins show that loss of p53 increases the levels of the mutant SOD1 proteins, and GAPDH is a loading control. Right: quantification of the western blots indicates that p53 is required for the effect of UBE4B and LSD1 on SOD1G85R protein clearance (n = 3). Data represent means ± SEM. The numerical data used to make this figure can be found in (UTRs see . (C) Los(TIF)Click here for additional data file.S1 MovieG85R-expressing C. elegans with severe locomotor defects is shown side by side with an isolated suppressor mutant (M1) that exhibits significantly improved movement. The animals were age matched and videotaped as day 1 adults.An SOD1(MP4)Click here for additional data file.S1 TableG85R, as described in the Results and the Materials and Methods. This table contains six sheets (A–F) that summarize the upstream regulator analysis. (A) Activated upstream regulators with z-score ≥ 2.0 for each of the three knockdown conditions, LSD1 alone, UBE4B alone, and (UBE4B+LSD1), as compared with the control. A z-score over 2.0 indicates that the particular upstream regulator is significantly activated because its target genes are differentially regulated accordingly in the experimental dataset. Note that the expression levels of the upstream regulators are often not changed. (B) Upstream regulators in the double UBE4B+LSD1 knockdown cells, with stringent p-values of overlap (p < 0.005) but any z-score. Activation/inhibition of upstream regulators with z-score between -2.0 and +2.0 is considered statistically nonsignificant and therefore not predicted. Upstream regulators with z scores ≥2.0 and ≤–2.0 are considered in an “activated” or “inhibited” state, respectively. (C) Expression analysis of 130 p53 target genes that are differentially regulated in the UBE4B and LSD1 double knockdown compared with the control. 49 “activated” genes are those whose actual changes are in agreement with the predicted direction in the event of p53 activation. 35 “inhibited” genes are those whose actual changes are opposite to the predicted direction in the event of p53 activation. Another 44 “affected” genes are those that are differentially regulated in the experiment but without IPA-predicted directions.  Lists of all upstream regulators in each of the three knockdown conditions, LSD1 alone, UBE4B alone, and both, as compared with the control.The human transcriptome microarray analysis was carried out in triplicates of four conditions, control shRNA (CTRL), LSD1 knockdown alone, UBE4B knockdown alone, and UBE4B+LSD1 double knockdown, all in the presence of misfolded SOD1(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 Text(DOC)Click here for additional data file.


Streptomyces. Two parallel strategies were exploited to forge the carbazole core: 6π-electrocyclization/aromatization and indole C2–H bond activation/Heck annulation. The construction of their trans-decalin motifs relied on two diastereochemically complementary radical cyclization reactions mediated by Ti(III) and Mn(III), respectively. The C23 hydroxyl of oridamycin B was introduced by an sp3 C–H bond oxidation at a late stage. On the basis of the chemistry developed, the dimeric congener dixiamycin C has been synthesized for the first time. Evaluation of the antiviral activity of these compounds revealed that xiamycin A is a potent agent against herpes simplex virus–1 (HSV-1) in vitro.Indolosesquiterpenoids are a growing class of natural products that exhibit a wide range of biological activities. Here, we report the total syntheses of xiamycin A and oridamycins A and B, indolosesquiterpenoids isolated from  Using a single strategy to make a number of related intermediates is a useful strategy in the total synthesis. Here, the authors report the synthesis of a number of natural products, employing two diastereomerically complementary metal-catalyzed cyclizations as the key step to access a number of frameworks. Several1–4) and indosespene (6). The syntheses of 2–4 were accomplished for the first time. A 6π-electrocyclization strategy and an oxidative Heck annulation strategy were developed for assembling the carbazole scaffold. The antiviral properties of the synthetic natural products were evaluated in vitro, and 1 exhibits potent inhibitory activity against herpes simplex virus–1 (HSV-1).In this paper, we report the total syntheses of xiamycin A, oridamycins A and B, dixiamycin C  and indosespene (6), our retrosynthetic analysis of 1 (2425262728307) and indosespene ester (8) are considered as the key intermediates for the two strategies. These functionalized decalin systems are expected to arise from a cascade cyclization of dienylepoxide precursors, such as 9 and 10. This type of radical cyclization may be initiated by reduction of the epoxide by a Ti(III) species323334353637383940414243444546142 and 3) from an alternative precursor. Successful execution of the route outlined above would also be anticipated to provide access to indosespene (6) and dixiamycin C (4) through minor modifications.The synthetic challenge of the xiamycin and oridamycin families may be separated into two distinct problems, namely the stereocontrolled assembly of the sis of 1  began wi1). Starting from optically active epoxide 11 (96% ee)N-oxyl (AZADO) oxidation5112 in 76% overall yield. On the basis of seminal work on the Ti(III)-catalysed bioinspired epoxypolyene cyclization by Cuerva and Oltra38394012. Under the optimized conditions , the desired trans-decalin 13 was obtained as a single diastereomer. TBS protection followed by acetyl deprotection gave 14 (44% yield for the three steps), which was subjected to a sequence of DMP oxidation, Grignard addition (with reagent 15)iPr2NEt) to furnish triene 16 in 75% overall yield. On microwave irradiation , 16 underwent a thermal 6π-electrocyclization and a subsequent aromatization to generate carbazole 17 (80% yield). Similarly, when using Grignard reagent 18 with a bromine substituent, we readily obtained compound 19 via the corresponding triene 20. This bromocarbazole served as an appropriate substrate for the crucial C–N bond formation in our dixiamycin C synthesis (vide infra). Desulfonylation of 17 followed by TMSE deprotection of the resulting compound 21 afforded xiamycin A (1) in a good overall yield, the structure of which was confirmed by X-ray crystallographic analysis  conditions. However, the compound 13 with an equatorial carboxylic substituent was detected as the major product, presumably due to the fast rotation of the C15–C16 bond to adopt the sterically favoured conformation (5556575825 (ref. 3/Cu(OAc)2 in DMSO effected the radical cyclization with an acceptable yield of decalin 26 . This compound was subjected to a sequence similar to that described above to afford triene 27 with good overall efficiency, via the intermediacy of aldehyde 28. Under thermal conditions (120 °C) and in presence of air, 27 was converted to carbazole 29 (83% yield) in one pot. Sequential ketone reduction (NaBH4) and desulfonylation (Mg/MeOH) provided compound 30 in 83% overall yield. Final TMSE deprotection led to the completion of the first synthesis of (±)-oridamycin A (2).We then focused on the synthesis of oridamycin A bearing an axial carboxylic substituent on C16 . The cisormation  for the 29 2 (20 mol%), PhI(OAc)2]6031 with good overall efficiency. It should be noted that stoichiometric Pd-mediated Csp3–H bond oxidation reactions have been exploited in terpenoid synthesis416232 with acceptable overall efficiency. Removal of TMSE with TASF provided (±)-oridamycin B (3) in 91% yield. The spectroscopic data of the synthetic samples of oridamycins A and B matched those reported for the natural products.We accomplished the synthesis of oridamycin B for the first time by adjusting the C23 oxidation state of 29 , taking 1) fulfilled the strategy of indole C2–H activation/Heck-type annulation (12 with N-Boc-3-(tributylstannyl)indole 3334 , which underwent a Ti(III)-catalysed radical cyclization under the same conditions as above to give decalin 35 as a single diastereomer in 60% yield2 and p-benzoquinone at 50 °C  and xiamycin A (1), respectively. The structure of 6 was verified by X-ray crystallographic analysis  mediated cyclization. N-Sulfonylation of 3-geranylindole 37 (ref. 2Cl) and subsequent allylic oxidation  gave alcohol 38 in 78% overall yield, which underwent bromination  followed by alkylation with the dianion generated from β-ketoester 39  to furnish 36 (80% yield for the two steps). Under the similar conditions used for the cyclization of compound 25  afforded carbazole 41 (65% yield), which was subjected to a two-step sequence of ketone reduction and TMSE deprotection to give (±)-oridamycin A (2). Sanford Csp3–H bond oxidation was exploited again for the synthesis of oridamycin B from 41; deactivation of the carbazole motif with an electron-withdrawing N-protecting group was required. Thus, 41 was subjected to NH2OMe condensation and Boc protection to afford the oxidation precursor. The same conditions  as used before delivered acetate 42 smoothly (67% yield for the three steps). Global deprotection of the oxime, acetyl and Boc groups under acidic conditions followed by reduction of the exposed carbonyl group furnished the late intermediate 32 in 54% overall yield, which was converted to (±)-oridamycin B (3) through the removal of TMSE group.The above strategy was further applied to alternative syntheses of oridamycins A and B . In thispound 25 , decalinanalysis . Desulfo19 and 21, we investigated a variety of conditions for the transition metal promoted C–N bond formation. Buchwald’s protocol43, K3PO4, 110 °C] proved to be optimal to provide dimer 44. Deprotection of the benzenesulfonyl and TMSE groups furnished dixiamycin C (4) in a good overall yield, via the intermediacy of 45. The spectra and physical properties of the synthetic dixiamycin C were consistent with those reported for the natural products.Having synthesized the monomeric natural products, we directed our attention to the synthesis of dixiamycin C . Startin1), oridamycins A (2), dixiamycin C (4), indosespene (6) and sespenine1450 assay1, 4, 6 and sespenine can suppress HSV-1 propagation in a dose-dependent manner. Notably, xiamycin A (1) is the most potent compound that reduces >95% of virus propagation at a concentration of 0.50 μM. Indosespene (6) significantly inhibits HSV-1 propagation at the concentration of 5.0 μM and displays essentially no antiviral activity at the concentration of 0.50 μM. Dixiamycin C (4) and sespenine show moderate inhibitory activity against HSV-1 at 5.0 μM but no activity at 0.50 μM. For the anti-HCV test (using a replicon assay), only dixiamycin C (6) exhibited inhibitory activity at a high concentration , N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexamethylphosphoramide (HMPA), triethylamine (Et3N), N,N-diisopropylethylamine (iPr2NEt) and pyridine were distilled from calcium hydride and stored under an argon atmosphere. Methanol (MeOH) was distilled from magnesium and stored under an argon atmosphere. Acetone was dried over drierite and distilled before use. Reagents were purchased at the highest commercial quality and used without further purification, unless otherwise stated. Solvents for chromatography were used as supplied by Titan Chemical. Reactions were monitored by thin layer chromatography (TLC) carried out on S-2 0.25 mm E. Merck silica gel plates (60F-254) using UV light as a visualizing agent and aqueous ammonium cerium nitrate/ammonium molybdate or basic aqueous potassium permanganate as a developing agent. E. Merck silica gel  was used for flash column chromatography. Preparative thin layer chromatography separations were carried out on 0.25 or 0.50 mm E. Merck silica gel plates (60F-254). NMR spectra were recorded on Bruker AV-400, DRX-600 or Agilent 500/54/ASP instrument and calibrated by using residual undeuterated chloroform (δH=7.26 p.p.m.) and CDCl3 (δC=77.16 p.p.m.), or undeuterated methanol (δH=3.31 p.p.m.) and methanol-d4 (δC=49.00 p.p.m.), as internal references. IR spectra were recorded on a Thermo Scientific Nicolet 380 FT-IR spectrometer. Melting points (m.p.) are uncorrected and were recorded on a SGW X-4 apparatus. High-resolution mass spectra (HRMS) were recorded on a Bruker APEXIII 7.0 Tesla ESI-FT mass spectrometer.All reactions were carried out under an argon atmosphere with dry solvents under anhydrous conditions, unless otherwise noted. Tetrahydrofuran (THF), toluene and 1,4-dioxane were distilled immediately before use from sodium-benzophenone ketyl. Methylene chloride .Accession codes: The X-ray crystallographic coordinates for structures  reported in this Article have been deposited at the Cambridge Crystallographic Data Centre (CCDC), under deposition number CCDC 948203, 949054 and 948503. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.Supplementary Figures 1-82, Supplementary Tables 1-10, Supplementary Methods and Supplementary References


NEIL1 recognizes and cleaves mainly oxidized pyrimidines from DNA. The past decade has witnessed the identification of an increasing number of post-translational modifications (PTMs) in BER enzymes including phosphorylation, acetylation, and sumoylation, which modulate enzyme function. In this work, we performed the first comprehensive analysis of phosphorylation sites in human NEIL1 expressed in human cells. Mass spectrometry (MS) analysis revealed phosphorylation at three serine residues: S207, S306, and a third novel site, S61. We expressed, purified, and characterized phosphomimetic (glutamate) and phosphoablating  mutants of the three phosphorylation sites in NEIL1 revealed by the MS analysis. All mutant enzymes were active and bound tightly to DNA, indicating that phosphorylation does not affect DNA binding and enzyme activity at these three serine sites. We also characterized phosphomimetic mutants of two other sites of phosphorylation, Y263 and S269, reported previously, and observed that mutation of Y263 to E yielded a completely inactive enzyme. Furthermore, based on sequence motifs and kinase prediction algorithms, we identified the c-Jun N-terminal kinase 1 (JNK1) as the kinase involved in the phosphorylation of NEIL1. JNK1, a member of the mitogen activated protein kinase (MAPK) family, was detected in NEIL1 immunoprecipitates, interacted with NEIL1  BER is initiated by a lesion-specific DNA glycosylase, which catalyzes the cleavage of the N-glycosidic bond, generating an apurinic/apyrimidinic (AP) site –3. In huPost-translational modifications (PTMs) such as phosphorylation, methylation, alkylation, ubiquitylation, and sumoylation play critical roles in many aspects of DNA metabolism, cell-cycle regulation, and other cellular processes , 16. PTMin vitro activity assays .–40.38–40Escherichia coli cells (Novagen), followed by IPTG induction (0.6 mM) for 4 hrs at 25°C. The cell pellet was resuspended in a buffer containing 50 mM sodium phosphate, pH 8.0, 150 mM NaCl, 10 mM imidazole pH 8.0, 10% (v/v) glycerol, 5 mM β-Me, and 1 mM PMSF and sonicated. The clarified cell lysate was added to pre-equilibrated TALON cobalt resin (Clontech). The proteins were eluted using 250 mM imidazole in the above buffer and diluted two fold into a buffer containing 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10% (v/v) glycerol, and 1 mM DTT. A HiTrap SP-FF column  with a linear NaCl gradient (300 mM– 1 M) was used to elute the enzymes and the resulting fractions were exchanged into a buffer containing 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 10% (v/v) glycerol, and 1 mM DTT. A Superdex 200 gel-filtration column  was used as a final purification step after which the enzyme was concentrated and flash frozen in LN2 for long term storage at -80°C. A QuikChange XL II site-directed mutagenesis kit (Stratagene) was used to introduce the S207E, S207A, S306E, S306A, S61E, S61A, S207E/S306E, S207A/S306A, S207E/S306E/S61E, and S207A/S306A/S61A mutations in the NEIL1 plasmid. All mutant enzymes were expressed and purified in a manner similar to the WT enzyme  with these exceptions: Double and triple mutants : 25°C, 0.4 mM IPTG, 3 hours; Y263E/Y263F: 16°C, 0.4 mM IPTG, overnight.The full-length human NEIL1 construct was cloned and purified as described previously . The pro2. A pCC384 vector containing NEIL1 with a C-terminal streptavidin binding protein (SBP) tag  was transiently transfected into the HEK293T cells using Turbofect (Thermo Fisher) as detailed in the manufacturer’s protocol. The cells were supplemented with fresh media 16 hrs post transfection and were washed and scraped into cold PBS 48 hrs later. Nuclear extracts were prepared as described previously  dATP, for 30 min at 37°C. The phosphorylation reaction was terminated by addition of 25 mM EDTA and heat inactivation. The end-labeled DNA was separated from the [γ-32P] dATP by ethanol precipitation and diluted in 9 pmoles of the appropriate non-labeled damage-containing oligodeoxynucleotide and 10 pmoles of the complementary oligodeoxynucleotide to a final concentration of 250 nM in 10 mM Tris—HCl (pH 8.0) and 50 mM NaCl. In order to create an AP site, a double-stranded uracil-containing oligodeoxynucleotide was treated with 2 units of uracil DNA glycosylase  for 30 min at 37°C.The 35-mer oligodeoxynucleotides used for the glycosylase/lyase activity assays were purchased from Midland Certified Reagent Co.  and purified by urea PAGE. The sequence of the damage-containing strand was eviously  in the fGlycosylase/lyase assays were performed with 20 nM lesion-containing substrate and increasing concentrations of enzyme in 20 mM HEPES pH 7.5, 150 mM NaCl, 2 mM EDTA with 200 μg/ml BSA at 25°C for 30 mins. The reactions were stopped with the addition of an equal volume of formamide loading buffer  to assay for both glycosylase and lyase activities. The reaction products were separated from the uncleaved substrates with a 12% (w/v) denaturing polyacrylamide gel and quantified with an isotope imaging system .32P] dATP, for 30 min at 37°C. The same amount of complimentary DNA was added to the mixture and allowed to anneal to the labeled damaged strand thereby yielding 100% labeled duplex. Samples (without loading dye) containing 10 pM DNA and varying amounts of enzyme (0–600 nM) were loaded onto a pre-run 10% native polyacrylamide gel. 2x loading dye was loaded in one lane as a marker. The gels were run for 2 hrs at 10 mA and then transferred to Whatmann 3 mm paper, covered with plastic wrap and placed on a gel drier at 80°C for 1 hr. The dried gels were exposed to a K-screen overnight and imaged with a Molecular Imaging System (BioRad).Gel shift analysis was performed using 35-mer double stranded oligos with tetrahydrofuran in the lesion position using the same sequence context as above. The furan-containing strand (1 pmol/μl) was labeled with T4 PNK in presence of [γ-®-FL PVDF membrane (Millipore). The membrane was washed (2X for 10 min each) with 1X PBS and 1 mM DTT. The membrane was then incubated in PBS with 6M guanidine-HCl and 1 mM DTT for 30 min at room-temperature with gentle shaking. The proteins on the membrane were then gradually refolded using serial dilutions of guanidine-HCl (in PBS with 1 mM DTT) to a final concentration of 0.09M guanidine-HCL . The first 5 steps were done at room temperature for 30 minutes. The penultimate and final steps were performed overnight at 4°C. The membranes were then blocked for 1 hr using PBS blocking reagent (LI-COR Biosciences) and further incubated with a pre-prepared HEK293T cell lysate  overnight. The membranes were then washed (3X) with cold PBS containing 0.05% Tween-20 and then processed using a standard Western blot protocol (LI-COR Biosciences). An anti-mouse JNK1 purified monoclonal antibody  and a rabbit polyclonal NEIL1 antibody  were diluted 1:1000  in blocking reagent. The blots were then incubated with anti-mouse/rabbit secondary antibody  and scanned using an Odyssey CLx imaging system (LI-COR biosciences).Samples for far-Western analysis were boiled in SDS-sample buffer and loaded on a 4–12% precast gradient gel (Novex) and run for 1 hr at a constant 180 V. The gels were transferred to an Immobilon32P ATP (Perkin Elmer) and 25 ng/μl purified active mouse JNK1 . Reactions  were incubated at 32°C for 30 minutes. The reaction was stopped by the addition of 5 μl 6X SDS-PAGE sample buffer and boiled at 95°C for 5 minutes. Samples were resolved by SDS-PAGE, the gel was dried, and phosphorylation via γ-32P incorporation was visualized using autoradiography. MS analysis was used to verify sites of phosphorylation. Samples for MS were incubated without γ-32P ATP and stained using Coomassie Blue. Bands were excised and digested with trypsin followed by MS/MS analysis performed as described above.NEIL1-WT and phosphomimetic or ablating mutants (10 μM) were incubated in 1X kinase buffer , supplemented with 2 mM ATP , 0.2 μl γ-In order to identify PTMs in the NEIL1 DNA glycosylase, a construct of NEIL1 tagged at the C-terminus with streptavidin binding peptide (SBP) was transfected into HEK293T cells. NEIL1-SBP was isolated using streptavidin magnetic beads, run on an SDS-PAGE gel, and stained with Coomassie blue . The sinBased on the MS data obtained, we focused on the three serine sites, S207, S306, and S61. We generated phosphomimetic (glutamate) and phosphoablating  variants at each of the three serine positions in NEIL1 . Table .In addition to NEIL1 harboring single point mutations, we created double S207E/S306E (DE) and triple S207E/S306E/S61E (TE) mutations, as well as the corresponding double and triple Ala mutations  were performed using an uncleavable abasic site analog, tetrahydrofuran, substrate paired opposite C with increasing amounts of either NEIL1-WT or a phosphomimetic/ phosphoablating mutant. Free DNA and shifted bands representing complex (C) formation were quantified. A representative EMSA reaction for the WT enzyme is shown  accompan~2–20 nM , S3B Fig~2–20 nM . In suchSince mutants of S207, 306, and 61, possessed both glycosylase and lyase activities under the conditions tested above , we usedwww.phosphonet.ca) [The mitogen-activated protein kinase (MAPK) family of kinases phosphorylate Ser or Thr residues in response to extracellular stimuli and environmental stresses . These konet.ca) . For allonet.ca) , 54.in vitro. NEIL1 and the phosphomimetic and ablating mutants were transferred and immobilized on a membrane after SDS-PAGE analysis, denatured with guanidine HCl, gradually refolded, and incubated with HEK293 WCE. Equal loading (50 pmol) of all mutants was attempted as indicated by the Coomassie stained gel  on HEK293T cells overexpressing a NEIL1-FLAG construct. Western blot analysis revealed that both isoforms of JNK1, p46 and p54, are present in HEK293T whole cell extracts (WCE), and are detected in the NEIL1-IP . Using fin vitro. Purified NEIL1 was treated with active JNK1 kinase in the presence of γ-32P ATP. SDS-PAGE analysis of the samples indicates that NEIL1-WT was phosphorylated in vitro  cleaves both SP:C and AP:C substrates in a manner similar to unphosphorylated NEIL1 . Based on kinase predictions from the PhosphoNET server, the kinases most likely to be involved in the phosphorylation of Y263 and S269 are the ITK/ BTK kinases and the mTOR/ FRAP kinases, respectively. ITK and BTK are cytoplasmic tyrosine kinases belonging to the Tec family of kinases [in vivo. In summary, (1) we identified three sites of phosphorylation in the human NEIL1 glycosylase, S207, S306, and S61 using a systematic mass spectrometry approach, (2) we demonstrated that phosphorylation has no significant effect on the enzyme’s DNA binding and enzymatic function using single, double, and triple phosphomimetic mutants, and (3) we identified a kinase that is involved in the phosphorylation of NEIL1 at the three sites identified here. Since phosphorylation at the these sites does not alter the enzyme’s activity in vitro, we surmise that this modification could be involved in mediating NEIL1’s interaction with other proteins, in determining subcellular localization of the enzyme, or in modulating function in a particular disease. A database search using the catalogue of somatic mutations in cancer (COSMIC) revealed that S207 when mutated to isoleucine is a confirmed somatic mutation in breast carcinoma [Our current analysis did not reveal phosphopeptides for residues Y263 and S269, reported previously . Factors kinases , 65 wher kinases . In the arcinoma , 68. TheS1 Fig(TIFF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S1 File(DOC)Click here for additional data file.


Sp) in its T-loop by the CDK-activating kinase (CAK). While modification of T161 promotes association with the cyclin partner, phosphorylation of T14 inhibits the CDK1-cyclin complex. This inhibition is further enforced by the modification of Y15 by Wee1 in the presence of DNA lesions. In S.pombe, the dominant inhibition of Cdc2 is provided by the phosphorylation of Y15 and only a small amount of Cdc2 is modified at T14 when cells are in S phase. Unlike human cells, both inhibitory modifications are executed by Wee1. Using the novel IEFPT technology, which combines isoelectric focusing (IEF) with Phos-tag SDS electrophoresis (PT), we report here that S.pombe Cdc2 kinase exists in seven forms. While five forms are phosphorylated, two species are not. Four phospho-forms associate with cyclin B (Cdc13) of which only two are modified at Y15 by Wee1. Interestingly, only one Y15-modified species carries also the T14 modification. The fifth phospho-form has a low affinity for cyclin B and is neither Y15 nor T14 modified. The two unphosphorylated forms may contribute directly to the DNA damage response as only they associate with the DNA damage checkpoint kinase Chk1. Interestingly, cyclin B is also present in the unphosphorylated pool. We also show that the G146D mutation in Cdc2.1w, which renders Cdc2 insensitive to Wee1 inhibition, is aberrantly modified in a Wee1-dependent manner. In conclusion, our work adds support to the idea that two distinct Cdc2 pools regulate cell cycle progression and the response to DNA damage.The activity of Cdc2 (CDK1) kinase, which coordinates cell cycle progression and DNA break repair, is blocked upon its phosphorylation at tyrosine 15 (Y15) by Wee1 kinase in the presence of DNA damage. How Cdc2 can support DNA repair whilst being inactivated by the DNA damage checkpoint remains to be explained. Human CDK1 is phosphorylated by Myt1 kinase at threonine 14 (T14) close to its ATP binding site before being modified at threonine 161 (T167 S.pombe Cdc2 (CDK1) can override the cell cycle control by Wee1 kinase . To. Tocdc2.extracts . We alsoextracts .cdc2.T14A cells. This identified Cdc2-Hf as a strong candidate for carrying the T14 and the Y15 modifications as the intensity of the F species significantly declined in the cdc2.T14A mutant. The second Y15 modified form, Cdc2-He, was not affected in the mutant strain , the anti-Y15P antibody identified only the two most negative species E and F . This imt strain . Unfortu mitosis  and thercdc13-HA strain to find out which Cdc2 forms associate with it. This assay indicates T167 phosphorylation as this modification is required for the stable formation of the Cdc2-cyclin B complex [CDC2-cyclin B complex [Since we could not test the T167 phosphorylation directly, we decided to immunoprecipitate cyclin B (Cdc13) from an affinity tagged  complex . As show complex , Cdc13 aS.pombe [chk1-HA strain [chk1-HA pull down material contained only the two unphosphorylated forms Cdc2-Ua and Cdc2-Ub, but none of the phosphorylated species. Activation of Chk1 by the DNA damage checkpoint in the presence of CPT was evident from the two phosphorylated forms of the kinase which migrate closer to the alkaline end of the strip as reported previously [We reported recently two Cdc2 protein complexes in S.pombe . A largeA strain . The celeviously . This unS.pombe CDK1 (Cdc2) kinase is restricted to the three amino acids T14, Y15 and T161Hs (T167Sp) [S.pombe cells [The phosphorylation of human and (T167Sp) , 38. Earbe cells . How thecdc2.T14A mutant (Sp (T161Hs) is not essential for cyclin B binding [S.pombe Cdc2-cyclin B complex for its roles in S phase.Our first key finding is the observation that only two Cdc2 forms are modified at tyrosine 15 (Y15). This identifies Cdc2-He and Cdc2-Hf as the target of Wee1 and Mik1 kinase in cell cycle regulation Figs  and 4C. A mutant . It is tA mutant  strongly binding , it sign binding . Further binding , and rea binding . It seem binding , the samcdc2.T14A mutant  appears on the Phos-Tag gel   . This fo(Cdc2-M) . Its abumutation . Taken tw mutant  and thatw mutant . One pos and the response to DNA damage (Cdc2-Ua + Cdc2-Ub) . Further


Kip2 (CDKN1C), a negative regulator of proliferation and a tumor suppressor, is controlled by members of the NAP1 family, we tested the hypothesis that NAP1L1 may have a mechanistic role in regulating pancreatic NEN proliferation through regulation of p57Kip2.The chromatin remodeler NAP1L1, which is upregulated in small intestinal neuroendocrine neoplasms (NENs), has been implicated in cell cycle progression. As p57p < 0.05) in the pancreatic neuroendocrine neoplasm cell line BON in vitro (p < 0.0001) and resulted in significantly smaller (p < 0.05) and lighter (p < 0.05) tumors in the orthotopic pancreatic NEN mouse model. Methylation of the p57Kip2 promoter was decreased by NAP1L1 silencing (p < 0.05), and expression of p57Kip2 (transcript and protein) was upregulated. For methylation of the p57Kip2 promoter, NAP1L1 bound directly to the promoter . In 43 pancreatic NEN samples (38 primaries and 5 metastasis), NAP1L1 was over-expressed in metastasis (p < 0.001), expression which was inversely correlated with p57Kip2 (p < 0.01) on mRNA and protein levels. Menin was not differentially expressed.NAP1L1 silencing (siRNA and shRNA/lipofectamine approach) decreased proliferation through inhibition of mechanistic  target of rapamycin pathway proteins and their phosphorylation (p57Kip2 promoter methylation.NAP1L1 is over-expressed in pancreatic neuroendocrine neoplasm metastases and epigenetically promotes cell proliferation through regulation of  Pancreatic neuroendocrine neoplasms (pNENs) are an aggressive form of cancer that are derived from a heterogeneous population of endocrine cells . While aNAP1L1). NAP1L1 belongs to a family which is thought to be involved in nucleosome assembly and exchange of histone H2A-H2B dimmers ). The forward primers encompass SP1 and CTIP2 binding sites involved in HDAC-mediated transcriptional regulation [To investigate the status of gulation . Unmethyp57Kip2 promoter, we performed ChIP using the Pierce Agarose ChIP Kit (Thermo Scientific). Briefly, we fixed the crosslinks between DNA and protein by formaldehyde, lysed the cells, and digested chromatin using micrococcal nuclease. Of these DNA fragments, 10% was used as input control. After immunoprecipitation with a ChIP-grade-NAP1L1-antibody , DNA fragments were purified and qPCR performed using primers for detecting the p57Kip2 promoter (see above). We used normal rabbit IgG as negative and anti-RNA polymerase II antibody and GAPDH promoter primers as positive controls. The p57Kip2 promoter expression in NAP1L1-bound DNA fragments was calculated relative to the control.To examine whether NAP1L1 directly bound to the post hoc test where appropriate. Binary comparisons were made using a 2-tailed Mann–Whitney U test. Correlations were undertaken using Spearman’s correlation. A p value of <0.05 was designated as significant. Statistical significance is indicated by an asterisk and described in the figure legends. Bars without an asterisk did not reach significance.All statistical analyses were performed using Microsoft Excel and Prism 5 . Comparisons between more than two groups were performed using the Kruskal-Wallis test, followed by the Dunn’s BrdU: bromdesoxyuridin; CgA: chromogranin A; ChIP: chromatin immunoprecipitation; HDAC: histone deacetylase; INA: internexin alpha; MEN-1: multiple endocrine neoplasia 1; mTOR: mechanistic  target of rapamycin; NAP1L1: nucleosome assembly protein 1-like 1; NF-1: neurofibromatosis 1; pNENs: pancreatic neuroendocrine neoplasms; RPL28: ribosomal protein L28; RPS24: ribosomal protein S24; S6K1: S6 kinase 1; TSC: tuberous sclerosis complex; VHL: Von Hippel-Lindau disease; 4E-BP1: eukaryotic initiation factor 4E-binding protein.The authors declare that they have no competing interests.MK, IM, and SS generated the main idea of the work and developed the study design, both conceptually and methodologically. AT and DA carried out methylation studies, isolation of mRNA and protein as well as Western blot and PCR. BL, MK, and SS contributed to analysis and interpretation of the data. HSW and MB contributed materials. SS and MK wrote the manuscript. BL, HSW, MB, and IM were involved in interpretation of data and critical revision for important intellectual content. All authors read and approved the final manuscript.


H9N2 subtype influenza viruses have been detected in different species of wild birds and domestic poultry in many countries for several decades. Because these viruses are of low pathogenicity in poultry, their eradication is not a priority for animal disease control in many countries, which has allowed them to continue to evolve and spread. Here, we characterized the genetic variation, receptor-binding specificity, replication capability, and transmission in mammals of a series of H9N2 influenza viruses that were detected in live poultry markets in southern China between 2009 and 2013. Thirty-five viruses represented 17 genotypes on the basis of genomic diversity, and one specific “internal-gene-combination” predominated among the H9N2 viruses. This gene combination was also present in the H7N9 and H10N8 viruses that have infected humans in China. All of the 35 viruses preferentially bound to the human-like receptor, although two also retained the ability to bind to the avian-like receptor. Six of nine viruses tested were transmissible in ferrets by respiratory droplet; two were highly transmissible. Some H9N2 viruses readily acquired the 627K or 701N mutation in their PB2 gene upon infection of ferrets, further enhancing their virulence and transmission in mammals. Our study indicates that the widespread dissemination of H9N2 viruses poses a threat to human health not only because of the potential of these viruses to cause an influenza pandemic, but also because they can function as “vehicles” to deliver different subtypes of influenza viruses from avian species to humans. Avian influenza viruses continue to present challenges to human health. Recently the H7N9 and H10N8 viruses that are of low pathogenicity for poultry have caused human infections and deaths in China. H9N2 influenza virus have been isolated worldwide from wild and domestic avian species for several decades, and their low pathogenic nature to poultry made them a low priority for animal disease control, which has allowed them to continue to evolve and spread. Here, we investigated a series of H9N2 influenza viruses that were detected in live poultry markets in southern China. We found that these viruses are able to preferentially bind to the human-type receptor, and some of them can cause disease and transmit between ferrets by respiratory droplet. All the transmissible H9N2 viruses have a similar internal gene constellation, which was also present in the H7N9 and H10N8 viruses. Our study indicates that the widespread dissemination of H9N2 viruses poses a threat to human health not only because of the potential of these viruses to cause an influenza pandemic, but also because they can function as “vehicles” to deliver different subtypes of influenza viruses from avian species to humans. Avian influenza viruses of several subtypes continue to present challenges to human health. The H5N1 influenza viruses have caused 380 fatal cases among the 641 documented human infections in 16 countries During the last several decades, H9N2 influenza viruses have been isolated worldwide from wild and domestic avian species Although the H9N2 viruses have been detected in chickens and ducks in many provinces in China since 1993 To investigate the genetic relationship of the viruses from different times and places, we sequenced the genomes of 35 viruses that were collected from 2009 to 2013 from 12 provinces in southern China . The amiSeveral amino acid changes related to the increased replication or virulence of avian influenza viruses in mammals The six internal genes of the H9N2 viruses showed distinct diversity, with PB2, PB1, PA, nucleoprotein (NP), M, NS genes of the 35 viruses sharing 84.8%–99.4%, 86.4%–99.6%, 87.9%–99.5%, 92.5%–99.6%, 94.1%–99.9%, and 91.3%–99.6% identity, respectively, at the nucleotide level. The PB2 genes formed six groups in their polygenic trees , and theOn the basis of this genomic diversity, the viruses examined in this study were divided into 17 genotypes . Of noteReceptor-binding preference has important implications for influenza virus replication and transmission The amino acid change Q226L is reported to contribute to the human-type receptor binding of H9N2 virus By using plasmid-based reverse genetics, we constructed a reassortant virus containing the HA and NA genes of the "early" H9N2 isolate A/chicken/Guangxi/9/99 (CK/GX/9/99) and the six internal genes of the A/Puerto Rico/8/1934 (H1N1) (PR8) virus and designated it as rCK/GX/9/99. Then, we introduced the avian influenza virus-like amino acids I and H into the HA gene at positions 155 and 183, respectively, to create the mutants we designated as rCK/GX/9/99-HAT155I and rCK/GX/9/99-HAN183H, respectively. Receptor binding analysis indicated that, similar to the wild-type CK/GX/9/99 virus , the rCK10TCID50, 22 viruses were also detected in the nasal turbinates of mice, with titers ranging from 0.7 to 5.3log10TCID50  of the Chinese Academy of Agricultural Sciences (CAAS) .All experiments with live H9N2 viruses were conducted within the enhanced animal biosafety level 2+ (ABSL2+) facility in the HVRI of the CAAS. The animal isolators in the facility are hyper-filtered. The researchers who work with mice and ferrets wear N95 masks and disposable overalls; they shower on exiting the facility.2.The H9N2 viruses used in this study were isolated from poultry in different regions of China between 2009 and 2013. Virus stocks were grown in specific pathogen-free (SPF) chicken eggs. Madin-Darby canine kidney (MDCK) cells used for virus titration were cultured in DMEM  medium with 4% fetal bovine serum (FBS). 293T cells were cultured in DMEM medium with 10% FBS. All cells were incubated at 37°C with 5% COViral gene amplification and sequencing was carried out as described previously O-phenylenediamine  in PBS containing 0.01% H2O2 for 10 min at room temperature. The reaction was stopped with 0.05 ml of 0.5 M H2SO4. The optical density at 490 nm was determined in a microplate reader (BIO-RAD). Dose-response curves of virus binding to the glycopolymers were analyzed by using a single site binding algorithm and curve fitting by GraphPad Prism to determine the association constant values (Ka). Each value presented is the mean ± SD of three experiments, which were each performed in triplicate.Receptor specificity was analyzed by use of a solid-phase direct binding assay as described previously with modified using two different glycopolymers: α-2, 3-siaylglycopolymer [Neu5Acα2-3Galβ1-4GlcNAcβ1-pAP (para-aminophenyl)-alpha-polyglutamic acid (α-PGA)] and the α-2, 6-sialylglycopolymer [Neu5Acα2-6Galβ1-4GlcNAcβ1-pAP (para-aminophenyl)-alpha-polyglutamic acid (α-PGA)] http://www.agilent.com) was used to create specific mutations in the HA gene by using the following primers: forward: 5′AGCATGAGATGGTTGATTCAA AAGGACAAC, reverse: 5′AGCGTTGTCCTTTTGAATCAACCATCTCAT (for HAT155I mutation); and forward: 5′ CATCACCCACCCACCTTCATGTGGGGCATA, reverse: 5′TCGGTGGGTGGGTGA GTATGCCCCACATGT (for HAN183H mutation). The HA (with or without the mutations) and NA genes of the CK/GX/9/99 virus and the six internal genes of the PR8 virus were used to generate the viruses as previously described The HA and NA segments of CK/GX/9/99 were inserted into the bidirectional transcription vector pBD as described previously 2 and inoculated intranasally (i.n.) with 106.0 EID50 of test viruses in a volume of 50 µl. Three mice were euthanized on day 3 p.i., and the nasal turbinates, lungs, kidneys, spleens, and brains were collected for virus titration in MDCK cells. The remaining five mice in each group were monitored daily for 14 days for weight loss and survival.Groups of six-week-old female BALB/c mice  were anesthetized with CO6.0 EID50 of test viruses in a 500 µl volume (250 µl per nostril). The ferrets were euthanized on day 4 p.i. and the nasal turbinates, tonsils, trachea, lung, spleen, kidneys, liver, and brain were collected for virus titration in MDCK cells. The lung tissue was also collected for histologic study as described previously Four-month-old female ferrets  that were serologically negative for influenza viruses were used in these studies. The animals were anesthetized via intramuscular injection of ketamine (20 mg/kg) and xylazine (1 mg/kg). To examine virus replication, groups of two ferrets were anesthetized and inoculated i.n. with 106.0 EID50 of test virus and housed in specially designed cages inside an isolator as described previously For the respiratory droplet transmission studies, groups of three ferrets were inoculated i.n. with 10AGTAGAAACAAGGTCGTTTTTAAA). PCR fragments of each virus were pooled in equal concentrations, and libraries were created for each virus by using the Ion Xpress Plus Fragment Library Kit (Life Technologies). Sequencing runs were performed by using the Ion Torrent personal genome machine . Sequence reads were sorted by the Ion Xpress Barcode Adaptors 1–18 (Life Technologies). Reads were aligned to the PB2 reference sequence of each virus by using CLC Genomics Workbench 5.0.1. The threshold for mutation detection was manually set at 1%.Viral RNA was extracted and converted to cDNA by use primer 5′AGC RAA AGC AGG. Specific amplification of a 1000-nucleotide PB2 fragment covering codons 627 to 701 was applied by use a pair of specific primers Click here for additional data file.Figure S2Phylogenetic analyses of the seven genes of H9N2 viruses. Phylogenetic analyses of the seven genes of the avian H9N2 influenza viruses isolated between 2009 and 2013. Trees were generated by using the neighbor-joining method (N-J Method) with the MEGA 5.0 program, and the tree topology was evaluated by means of 1000 bootstrap analyses. The trees of NA (A), PB2 (B), NP (E), M (F), and NS (G) were rooted to A/Equine/Prague/1/56(H7N7), the PB1 (D) and PA (E) trees were rooted to A/Equine/London/1416/73(H7N7). The regions of the nucleotide sequences used for the phylogenetic analysis were: NA, 87 to 1263; PB2, 185 to 2290; PB1, 31 to 2233; PA, 25 to 2129; NP, 35 to 1457; M, 56 to 960; and NS, 75 to 800. Sequences of viruses with names in black were downloaded from available databases; viruses with names in blue were sequenced in this study. Abbreviations are as follows: Br, Brambling; CAn, Canine; CK, Chicken; DK, Duck; Env, Environment; GS, Goose; Pf, Peregrine falcon; Pg, Pigeon; QA, Quail; SW, Swine; Ty, Turkey; AH, Anhui; BJ, Beijing; CA, California; CS, Chang Sha; CQ, Chongqing; DH, Donghu; FJ, Fujian; GD, Guangdong; GX, Guangxi; GZ, Guangzhou; HB, Hebei; HZ, Hangzhou; HN, Henan; HK, Hong Kong; HuB, Hubei; HuN, Hunan; JL, Jilin; JS, Jiangsu; SD, Shandong; SH, Shanghai; TZ, Taizhou; WI, Wisconsin; XJ, Xinjiang; XZ, Xuzhou; YZ, Yangzhou; YN, Yunnan; ZJ, Zhejiang.(PDF)Click here for additional data file.Figure S3Characterization of the receptor-binding properties of H9N2 viruses. The binding of the viruses to two different biotinylated glycans  was tested. The data shown are the means of three repeats; the error bars indicate the standard deviations.(PDF)Click here for additional data file.Figure S4Histological lesions caused by H9N2 viruses in the lungs of ferrets. Ferrets were euthanized on day 4 p.i. with 106EID50 of test virus, and the lungs were collected for pathological study. The lungs of DK/ZJ/C2046/12 virus-inoculated animal showed only mild histopathological changes  (A), whereas the lungs of CK/HuN/C4136/10 (B), CK/CQ/C1258/11 (C), CK/HuB/C4196/09 (D), CK/ZJ/SC324/13 (E), and CK/SH/SC197/13 (F) virus-inoculated ferrets showed severe pathological lesions (H&E staining). Viral antigen was detected in the epithelial cells of bronchus and alveoli by means of immunohistochemical (IHC) staining . Images A–F were taken at ×100 magnification; images G and H were taken at ×400 and ×200 magnification, respectively.(PDF)Click here for additional data file.Table S1Mutations detected in the H9N2 viruses that contribute to the increased binding to human-type receptors, transmission, replication, and virulence in mammals, as well as to resistance to amantadine and rimantadine.(PDF)Click here for additional data file.Table S2Virus binding affinity to sialylglycopolymers.(PDF)Click here for additional data file.Table S3Virus selection scenarios for the ferret study.(PDF)Click here for additional data file.Table S4The body temperature increase and body weight loss of each of the ferrets inoculated with or exposed to the different H9N2 influenza viruses.(PDF)Click here for additional data file.Table S5Assessment of the presence of the 627K and 701N mutations in the PB2 segment of stock viruses and viruses recovered from the nasal washes of H9N2 influenza virus-infected or -exposed ferrets by use of deep sequencing.(PDF)Click here for additional data file.