Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells.The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the  The current model of HCV egress is that virions assemble at lipid droplets, envelope at the ER and then likely exit the hepatocyte via the secretory pathway in association with apolipoproteins. To gain a more detailed insight into infectious HCV release, we combined an RNAi analysis of host factors that are required for infectious HCV secretion with live cell imaging of HCV core trafficking. Using this approach, we identified numerous components of the secretory pathway that are both required for infectious HCV release and co-traffic with HCV core. The dynamics of HCV core trafficking, both in terms of frequency of transport, particle velocity, and the corresponding run lengths were quantified. We observe that dynamic core movements in the periphery require NS2, a viral protein required for virion assembly. Core co-traffics with multiple components of the secretory pathway, including the Golgi, recycling endosome, microtubules, VAMP1 secretory vesicles, and ApoE. This study identifies new molecular determinants of HCV secretion and describes the dynamics of their movements with HCV core in real time. Flaviviridae family that contains a single-stranded positive RNA genome (∼9600bp). This is translated into a polyprotein, which is processed by viral and host proteases into 3 structural proteins  and 7 non-structural (NS) proteins . The discovery of HCV strains that are infectious in cell culture has enhanced the appreciation of the functions of these proteins in all stages of the viral life cycle Hepatitis C virus (HCV) is a member of the Following replication, genomic RNAs in complex with viral and possibly cellular proteins are thought to transit to lipid droplets (LD), where core protein localizes and virion assembly occurs The events following HCV assembly are less clear. The egress of flaviviruses through the secretory pathway is well documented and may be similar to the mechanism by which HCV is secreted This study is a continuation of two previous RNAi studies in which membrane trafficking pathways were interrogated for their roles in HCV entry and replication This study is a part of a larger analysis, which interrogated a previously described unique siRNA library that targeted known membrane trafficking pathways p<0.001)  and viral titers were quantified via limiting dilution in at least two independent experiments, measured in duplicate, and normalized to at least 4 replicates of cells silenced with an irrelevant siRNA (siIRR) . The siRp<0.001) .In this study, genes that were required for infectious HCV production, but not HCVpp entry or replicon replication, were further tested for a role in infectious HCV secretion. We repeated the RNAi analysis and quantified intra- and extra-cellular HCV infectivity following HCV infection of siRNA-treated cells. The siRNA screen identified multiple components of the secretory pathway, from ER to Golgi trafficking (SAR1A), to Golgi structure and function (CYTH3), cargo sorting and vesicle budding from TGN , regulators of TGN to plasma membrane trafficking , and the v-snare, VAMP1. This suggests that HCV virions egress through the secretory pathway from the TGN to recycling endosomes to the plasma membrane. A role for ER to Golgi trafficking genes, such as SAR1A, ARF3, and CYTH3, in the egress of infectious HCV is supported by previous data showing that brefeldin A treatment of infected cells accumulates intracellular infectious HCV To study the dynamics of HCV core trafficking in Huh-7.5 cells, a TC tag was fused in frame following amino acid three of the structural protein core in a derivative of J6/JFH-1, a chimeric genotype 2a isolate. The TC sequence of CCPGCC can coordinate biarsenical dyes, FlAsH and ReAsH. The TC-core virus replicates comparably to wild type , with a We first tested the specificity of labeling by examining whether FlAsH TC-core fluorescence overlapped with core detected by standard immunofluorescence. TC-core RNA was electroporated into Huh-7.5 cells and three days later the cells were treated with FlAsH biarsenical dye, then processed for immunofluorescence using a monoclonal antibody against HCV core . Both anbona fide HCV virions. RNA localization was examined in wild type infected cells using “Click-iT” technology (Invitrogen) and cells were stained using an anti-core antibody  protein 2, NS2, is required for HCV assembly by acting as a scaffold bridging structural and non-structural proteins mpetent, , while ompetent, .HCV capsid assembly is dependent on the association of core with the lipid droplet We next examined whether the dynamic TC-core movements observed previously  requiredThe dynamics of TC-core trafficking were examined by distance from origin analysis. The cumulative distance traveled (in any direction) by a TC-core puncta during an imaging session was measured and plotted for each virus from a single particle. The distance from origin plots show core puncta in TC-core and bicistronic expressing cells had extensive total movements ranging from 3-17 microns per minute from the point of origin during an imaging session . Core pu2 = .9924, which is consistent with microtubule associated transport. The average velocity of moving TC-core puncta was .1336 µm/second ± .010139  movements with few processive runs and reversals where particles switched direction. The average run length displacement of wild type TC-core moving puncta was 2.457 µm ± .1215  by microtubule directed transport cis- face of the Golgi)  or GM130 (e Golgi) . We obseThe role of exit from the Golgi in TC-core trafficking was assessed by quantifying the effects on TC-core movements in the presence of inhibitors of two genes identified in the RNAi screen: brefeldin A (BFA), which inhibits ARF3, and PIK93, which inhibits PI4KB. BFA treatment inhibits secretion of viral particles de novo forming HCV particles during secretion instead of being acquired extracellularly. We determined the percentage of TC-core puncta moving with ApoE-GFP to be 81.25%±8.98, n = 26/32 of moving TC-core puncta. We also investigated colocalization of core with ApoB. Although some colocalization of core and ApoB was observed in the proximity of lipid droplets, little colocalization between core and ApoB occurred in the periphery  was identified as a cofactor in the release of extracellular infectious HCV in our RNAi screen. VAMP1 can complex with syntaxins and SNAP25 to form a SNARE complex involved in docking and/or fusion of vesicles at the plasma membrane Finally, we tested whether silencing the Golgi lipid kinase PI4KB would decrease the association of core with VAMP1. siRNA treated cells were transfected with GFP-VAMP1, infected with wild type HCV for two days and processed for core and GFP-VAMP1 imaging. We observed extensive colocalization of core and GFP-VAMP1 (65.1±2.5% (n = 366)) in siIRR treated cells . HoweverFlaviviridae , which does not occur in hepatocytes, and vaccinia virus displays slower saltatory movements (1-3 µm/sec)  and apical  plasma membrane domains, which are separated by tight junctions. The association of apical membranes from neighboring cells forms bile caniculi. With complex polarity, also comes complex trafficking of proteins to apical and basolateral membranes. Two pathways are used to direct proteins to the apical membrane in hepatocytes, a direct route or through transcytosis after reaching the basolateral membrane. HCV is known to spread via two distinct routes, extra-cellular release and cell-cell spread We plan to revisit the trafficking studies in a more relevant polarized hepatocyte cell culture system. Hepatocytes Huh-7.5 cells were grown in Dulbecco's modified high glucose media  supplemented with 10% fetal bovine serum , nonessential amino acids , and 1% penicillin-streptomycin (Invitrogen). Viral RNA was electroporated into Huh-7.5 cells as described previously CCPGCCMEP) tetracysteine (TC) tag GAATTCTAATACGACTCACTATAGACCAAC and 5′ GTGCACGGTCTACGAGACCTCCCGGGGAAC (EcoRI and ApaLI sites underlined). The 3′ region was amplified using 5′ GTGCACCATGAGCACATTTCTCAATTGTTGTCCTGGCTGTTGTATGGAACCTAATCCTAAACCTCAAAGAAAAACCAAC and 5′ CGTACG CCAAGATCATGGTAGCCGTGGAAC . The 5′ and 3′ regions were digested with ApaLI and ligated together to form the full-length core region with the tag inserted. The TC-core fragment was subsequently cloned into JFHxJ6NS23 using the EcoRI and BsiWI sites and the TC tag insertion was confirmed by sequencing. The bicistronic construct was constructed by insertion of a HCV genotype 2a IRES – NS2 fragment into a subgenomic replicon, pSGR-JFH1-neo, containing the encephalomyocarditis virus (EMCV) IRES through the HCV replicase genes NS3-NS5B GAATTCTAATACGACTCACTATAGACCTGCCCCTAATAG and 5′ GTTTAAACTTAAAGGAGCTTCCACCCCTTGGAGGTGTAGCCATCAGCTGGACC (EcoRI and PmeI sites are underlined). The subsequent fragment was subcloned in the replicon resulting in the bicistronic construct. The NS2 deletion construct was made by two-part subclone. NS2 was deleted by insertion of the HCV genotype 2a IRES-p7 genes into a subgenomic replicon as stated above. The IRES-p7 region was PCR amplified by using the same EcoRI primer as above and 5′ GTTTAAACTTAAGCATAAGCCTGTTGGGGCAATGCTAGGAGCAGTAGGCTAACC (PmeI site underlined) which goes through p7. The resulting PCR fragment was cloned in the subgenomic replicon stated above. This resulted in the NS2 deletion virus in a bicistronic backbone. The TC-tagged core was introduced by digestion of the TC-core virus with EcoRI and BsiWI to liberate a DNA fragment containing TC-core through part of glycoprotein E1. Untagged core in the NS2 deletion backbone was replaced with the TC-core fragment using the corresponding EcoRI and BsiWI restriction sites. This resulted in a TC-core NS2 deletion (ΔNS2) construct. The resulting clone was sequenced to verify in frame insertion of the tag and presence of the deletion.pJFHxJ6-CNS2C3 (WT) is a HCV genotype 2a infectious clone where core through the first transmembrane domain of NS2 of a JFH-1 backbone was replaced with J6 genotype sequence GAATTCATGAAGGTTCTGTGGGCTGCAC (EcoRI site is underlined) and 5′ GGATCCCGTGATTGTCGCTGGGCACGT . The PCR product was cloned into pEGFP-N2 at the EcoRI and BamHI sites to create ApoE-GFP, and then confirmed by sequencing. Rab11A was PCR amplified from a human ORF clone, accession number CV027643 (Open Biosystems), using the primers 5′ AAGCTTCGATGGGCACCCGCGACGACGAGTACGACACC (HindIII site is underlined) and 5′ GGTACCTTAGATGTTCTGACAGCACTGCACCTTACC (KpnI site is underlined). The PCR product was cloned into pmGFP-C1, resulting in GFP-Rab11A and confirmed by sequencing. VAMP1 ORF was PCR amplified from a human ORF clone, accession number CV029376 (Open Biosystems), using the primers 5′ AGATCT ATGTCTGCTCCAGCTCAGCCACCGC (BglII site is underlined) and 5′ AAGCTT TCAGCGATACTTACTTACAATAACGC (HindIII site is underlined). The resulting PCR product was cloned into pEGFP-C1 using the BglII and HindIII sites to result in GFP-VAMP1 and confirmed by sequencing.Apolipoprotein E was PCR amplified from a human ORF clone, accession number EL733202 (Open Biosystems), using the primers 5′ in vitro as described ACTTCATTAGCGGCATCCAATAC; reverse 5′-CGGCACTGAATGCCATCAT; probe 5′-6FAM-CAGGATTGTCAACACTGCCAGGGAACC - (Iowa Black), amplifies from the NS4 gene. PCR conditions were 50°C for 30 minutes, 95°C for 6 minutes, and (95°C for 15 seconds followed by 60°C for 1 minute) x 50 cycles.WT, TC-core, bicistronic, and ΔNS2 RNAs were transcribed 6 Huh-7.5 cells in 0.05 ml of PBS pH 7.4 were electroporated with 125 picomoles of siRNA for 5 pulses of 770 volts for 99 microseconds with one- second intervals on a BTX 830 electroporator with 96-well attachment. Cells were infected 72 hours after electroporation with a multiplicity of 0.5 infectious HCV particles per cell for 6 hours, rinsed with media, then maintained for 2 days at 37°C. 48 hours after infection, the supernatants were collected and analyzed for infectious virus via limiting titer dilution as described previously The siRNAs used are listed in For visualization of core localization during silencing, cells were silenced with siRNAs followed by infection and immunofluorescence. Briefly, Huh-7.5 cells were electroporated (as above) with 2 pre-validated RAB11a siRNAs , 2 pre-validated PIK4B siRNAs , or irrelevant siRNA and silencing was established for 48 hours. Electroporated cells were infected with HCV then maintained for 2 days, followed by seeding onto coverslips, fixation, and immunofluorescence staining.HCV TC-core infected cells stained with biarsenical dye were fixed in 4% paraformaldehyde for 10 min, permeabilized in 0.2% Triton X-100 in PBS for 20 minutes, and blocked with 0.1% Tween-20 and 10% goat serum in PBS for 30 min. Cell were washed with PBS plus 0.1% Tween-20 (PBST) after blocking and between antibody incubations. Primary and secondary antibodies were diluted in PBST. For detection of core, a mouse monoclonal antibody  was used at a dilution of 1∶50, mouse monoclonal antibody to NS5A (9E10) was used at a dilution of 1∶1000, dsRNA antibody  was used at 1∶1000, and a human monoclonal antibody to E2 (Steve Foung) was used at 1∶250. GFP fusion proteins were assessed for correct localization by incubation with antibodies. Anti-Apolipoprotein E (Abcam) was used at a dilution of 1∶250, anti-Vamp1 (Abcam) was used at a dilution of 1∶250, anti-Rab11a (Invitrogen) was used at a dilution of 1∶250, anti-GM130 (Abcam) was used at 1∶250 to label the Golgi network, and anti-ApoB was used at a dilution of 1∶250. Alexa-488 or Alexa-594 conjugated IgG  secondary antibodies were diluted 1∶1000. Golgi-GFP (Invitrogen) was used to label the TGN by the recommended directions. Bodipy 493/503 was used to label neutral lipids (lipid droplets) in both fixed and live cell images at a concentration of 20 µg/ml.FlAsH or ReAsH labeling was performed per the instructions provided in the TC-tag detection kit (Invitrogen). At 72 hours post electroporation, cells were washed once with Opti-MEM (Invitrogen) and labeled with biarsenical dye (1.25 µM) in Opti-MEM. Cells were incubated at 37°C for 30 minutes, then washed two times with 1X BAL  wash buffer  supplemented with 500 µM EDT in Opti-MEM for 5 minutes. The wash buffer was removed and the cells were washed one time using Opti-MEM followed by incubation with prewarmed media.Tracking of TC-core in the presence of drug inhibitors of the secretory pathway was performed as follows: TC-core electroporated cells were incubated with DMSO, GolgiPlug containing BFA , or PIK93  for 2 hours prior to staining with ReAsh. DMSO or drug was present during time of staining and in the imaging media.Infected cells were incubated with Click-iT RNA Alexa Fluor 488 Imaging Kit (Invitrogen) to visualize RNA. Briefly, infected cells were seeded on round coverslips and incubated with Click-iT kit as per manufacturer's instructions in the presence of actinomycin D (1 µg/ml). Cells were processed for immunofluoresence after Click-iT labeling.All images were acquired with an Olympus DSU spinning disk confocal microscope fitted with a 100×1.45 N.A oil-immersion objective. FlAsH labeled core was detected using the EGFP filter set (ex: 480/25 and em: 525/40), whereas ReAsH labeled core was visualized with the DsRed filter set (ex: 565/25 and em: 620/60) coupled to a Hamamatsu back-thinned EM-CCD high speed/sensitivity camera. Slidebook software was used for image acquisition and processing .Immunofluorescence imaging of TC-core with viral or host markers was accomplished by acquiring sequential static images with DsRed, EGFP, and 350 filter set sequential exposures.Live cell imaging of TC tagged HCV was performed by growing infected cells on polylysine treated 35 mm glass bottom Fluorodishes (World Precision Instruments). Cells were stained with biarsenical dye as above except imaging media (DMEM-F12- Invitrogen) supplemented with 10% fetal bovine serum, 0.1 mM nonessential amino acids, 1% penicillin-streptomycin, and 25 mM HEPES was added following the final wash. Imaging chambers were sealed with parafilm before imaging. Cells were maintained on a heated stage set at 37°C. Timelapse imaging of TC-core particles and GFP labeled host proteins was taken by sequential imaging every 2 seconds with 200 ms exposures for each channel, DsRed and GFP respectively. In some cases cells were incubated with transferrin-488  or phrododextran  prior to imaging as per manufacturer's instructions.Individual core puncta transport velocities and run lengths were calculated by kymograph analysis. Briefly, the multiple kymograph plugin for ImageJ was used to generate kymographs. Runs were defined as uninterrupted diagonal lines and the distance traveled (length) and the average velocity (slope) were measured. The frequency of TC-core puncta movement was measured by quantifying the number of TC-core puncta displaying a run length greater than 1 µm as a percentage of total cellular TC-core puncta signal using the analyze particles tool in ImageJ per imaging session. Distance from origin plots were generated by using the Manual Tracking plugin for Image J, measuring the distance traveled (in any direction) between frames for a respective TC-core puncta. Values were added and plotted against time. Manual Tracking plugin was also used to generate the distance versus time plots. Distance between timepoints was plotted versus time.p-values for replication and TCID50/mL titer curves. The velocity and run length plots were also done using Prism software where the data was modeled to a Gaussian distribution or a decaying exponential.Prism software was used to plot and determine Figure S1Cell viability following electroporation with the indicated siRNAs. Viability of siRNA-treated cells was measured at 5 days post electroporation in two distinct experiments (A) and (B) by a luminescence-based assay (Promega) that measures intracellular ATP levels. SEM is shown.(TIF)Click here for additional data file.Figure S2Intra- and extra-cellular infectious virus following siRNA treatment. Relative levels of intra-and extra-cellular infectious virus levels from (TIF)Click here for additional data file.Figure S3Biarsenical dyes do not affect virus release. Huh-7.5 cells were infected with wildtype virus for 48 hours then incubated with ReAsh dye for 30 minutes. ReAsh dye was removed and cells were washed with 1x BAL buffer supplemented with 500 µM EDT. Cells were incubated in fresh media and supernatants were collect at 2, 8, 24, 48 hours post ReAsh incubation and titered.(TIF)Click here for additional data file.Figure S4Immunofluorescence of GFP fusions of ApoE, Vamp1, and Rab11a. Huh7.5 cells were transfected with ApoE-GFP, GFP-Vamp1, or GFP-Rab11a. Cells were fixed and processed for immunofluorescence using antibodies directed against ApoE (top), Rab11a (middle), GM130 (middle bottom) or Vamp1 (bottom) and corresponding secondary antibodies (red). Scale bar is 10 µm.(TIF)Click here for additional data file.Figure S5TC-core cotransports with ApoE-GFP. Huh-7.5 cells were electroporated with TC-core RNA and transfected with ApoE-GFP at 48 hpe. Cells were stained with ReAsh (red) at 72 hpe then imaged. Boxed region contains TC-core puncta shown in montage in (TIF)Click here for additional data file.Figure S6ApoB does not colocalize with TC-core puncta. Huh7.5 cells were infected with TC-core virus and stained with ReAsh dye (red) at 72 hours post infection followed by processing for immunofluorescence using an ApoB antibody (green). Scale bar is 10 µm.(TIF)Click here for additional data file.Figure S7TC-core cotransports with GFP-Rab11a. Huh-7.5 cells were electroporated with TC-core RNA and transfected with GFP-Rab11a at 48 hpe. Cells were stained with ReAsh (red) at 72 hpe then imaged. Boxed region contains TC-core puncta shown in montage in (TIF)Click here for additional data file.Figure S8TC-core cotransports with Alexa Fluor 488 transferrin. Huh-7.5 cells were electroporated with TC-core RNA and stained with ReAsh (red) at 72 hpe then incubated with Alexa Fluor 488 transferrin (green). Cells were immediately imaged. Boxed region contains TC-core puncta shown in montage in (TIF)Click here for additional data file.Figure S9TC-core cotransports with GFP-VAMP1. Huh-7.5 cells were electroporated with TC-core RNA and transfected with GFP-VAMP1 at 48 hpe. Cells were stained with ReAsh (red) at 72 hpe then imaged. Boxed region contains TC-core puncta shown in montage in (TIF)Click here for additional data file.Figure S10TC-core cotransports with dextran. Huh-7.5 cells were electroporated with TC-core RNA and stained with FlAsh (green) at 72 hpe then incubated with dextran (red). Cells were immediately imaged. Boxed region contains TC-core puncta shown in montage in (TIF)Click here for additional data file.Figure S11TC-core produced in infected cells co-transports with host secretory pathway components. Huh-7.5 cells were infected with TC-core virus followed by transfection with either ApoE-GFP (A), GFP-Rab11a (B), or GFP-Vamp1 (C) plasmids at 24 hours post infection (hpi). Cells were incubated with ReAsh at 72hpi followed by live cell confocal microscopy. Shown are time-lapse montages of alternating DsRed (200ms) and EGFP (200ms) exposures taken every 2 seconds for several minutes. Kymographs are shown to the right of each montage.(TIF)Click here for additional data file.Figure S12ApoE-TC-core colocalization at core accumulations. Huh-7.5 cells were electroporated with TC-core RNA and transfected with ApoE-GFP at 48 hours post electroporation. Shown is ApoE (green) and TC-core (red) colocalization at a crescent shaped core accumulation (inset), presumably at a lipid droplet. Scale bar 10 µm.(TIF)Click here for additional data file.Figure S13Vamp-1 and ApoE vesicle dynamics. Huh-7.5 cells were transfected with ApoE-GFP or GFP-VAMP1 constructs. 200ms GFP exposures were taken by confocal microscopy every 2 seconds. Single GFP puncta were tracked using the manual tracking plugin for Image J. Plotted are average (A) velocity (µm/sec) and (B) run length (µm). Overall, 16 VAMP1-GFP vesicles  and 15 ApoE-GFP vesicles  were tracked. Error bars indicate standard error of the mean.(TIF)Click here for additional data file.Table S1Genes and siRNAs tested in the RNA interference screen.(DOC)Click here for additional data file.Table S2Real time RT-PCR assays for quantifying host gene RNA levels.(DOC)Click here for additional data file.Video S1Dynamics of TC-core puncta. Snapshot of this Video is shown in (AVI)Click here for additional data file.Video S2TC-core dynamics. Montage of single TC-core puncta transport shown in (AVI)Click here for additional data file.Video S3TC-core dynamics in bicistronic background. Montage of single TC-core puncta transport shown in (AVI)Click here for additional data file.Video S4TC-core dynamics in ΔNS2 background. Montage of single TC-core puncta transport shown in (AVI)Click here for additional data file.Video S5TC-core dynamics and microtubules stained with Tubulin Tracker green. Shown as montage in (AVI)Click here for additional data file.Video S6TC-core dynamics in the presence of nocodazole. Shown as a montage in (AVI)Click here for additional data file.Video S7TC-core traffics with ApoE-GFP. Shown as a montage in (AVI)Click here for additional data file.Video S8TC-core traffics with GFP-Rab11a. Shown as a montage in (AVI)Click here for additional data file.Video S9TC-core traffics with Alexa Fluor 488 transferrin. Shown as a montage in (AVI)Click here for additional data file.Video S10TC-core traffics with GFP-VAMP1. Shown as a montage in (AVI)Click here for additional data file.Video S11TC-core traffics with dextran. Shown as a montage in (AVI)Click here for additional data file.Video S12TC-core traffics with Ribogreen labeled RNA. Shown as montage in (MOV)Click here for additional data file.Video S13TC-core in infected cells traffics with ApoE-GFP. Shown as a montage in S11.(AVI)Click here for additional data file.Video S14TC-core in infected cells traffics with GFP-Rab11a. Shown as a montage in S11.(MOV)Click here for additional data file.Video S15TC-core in infected cells traffics with GFP-Vamp1. Shown as a montage in S11.(MOV)Click here for additional data file.


Peroxisome proliferator-activated receptor beta/delta (PPARD) is a crucial and multifaceted determinant of diverse biological functions including lipid metabolism, embryonic development, inflammatory response, wound healing and cancer. Recently, we proposed a novel function of porcine PPARD (sPPARD) in external ear development. A missense mutation (G32E) in an evolutionary conservative domain of sPPARD remarkably increases external ear size in pigs. Here, we investigated the underlying molecular mechanism of the causal mutation at the cellular level. Using a luciferase reporter system, we showed that the G32E substitution reduced transcription activity of sPPARD in a ligand-dependent manner. By comparison of the subcellular localization of wild-type and mutated sPPARD in both PK-15 cells and pinna cartilage-derived primary chondrocytes, we found that the G32E substitution promoted CRM-1 mediated nuclear exportation of sPPARD. With the surface plasmon resonance technology, we further revealed that the G32E substitution had negligible effect on its ligand binding affinity. Finally, we used co-immunoprecipitation and luciferase reporter assays to show that the G32E substitution greatly reduced ubiquitination level by blocking ubiquitination of the crucial A/B domain and consequently decreased transcription activity of sPPARD. Taken together, our findings strongly support that G32E is a functional variant that plays a key role in biological activity of sPPARD, which advances our understanding of the underlying mechanism of sPPARD G32E for ear size in pigs. DGAT1 K232A ABCG2 Y581S IGF2 intron3 g.3072 G>A MSTN 3′UTR g.6723 G>A PLAG1 variants affecting bovine stature Deciphering the genetic architecture of complex traits is a big challenge for geneticists. To date, only a handful of causal variants underlying quantitative traits have been unequivocally identified in domestic animals All the procedures involving animals are in compliance with the care and use guidelines of experimental animals established by the Ministry of Agriculture of China. The ethics committee of Jiangxi Agricultural University specifically approved this study.PPARD allele as described previously with minor modifications sPPARD full-length cDNA clone (Genechem) as template, site-directed mutagenesis generated single-amino-acid substitutions including G32E, K16R, K17R, K18R and K16-18R of PPARD by overlap extension PCR. Primers for amplification of the above products are given in 2SO. Leptomycin B (Beyotime) was prepared in ethanol with a final concentration of 50 nM.One-generation primary chondrocytes were isolated from external ears of 1-week old piglet with the wild-type 4 cells per well in mediums as described below. Twenty-four hours later, cell numbers were measured with a counting chamber to determine plating efficiency. The remaining cells were treated in triplicate with 0.1% DMSO, 0.1 µM and 1 µM GW0742 (a specific ligand of PPARD). Cell numbers were determined at daily intervals, and the remaining cells were retreated with fresh mediums each day for up to 7 days.The one-generation primary chondrocytes were seeded onto 6-well tissue culture dishes (Nunc) at 3×105 cells/well) were seeded in 24-well plates. When cells were 90% confluent, cells were co-transfected with pcDNA4A-His-sPPARD and –G32E mutant expression vectors, PPREx3-tk-Luc and pCX-nLacZ constructs using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, cells were treated with GW0742 overnight and then measured for firefly luciferase on a LUMAT LB 9507 luminometer (Berthold Technologies) and for β-galactosidase control activity on an iMark microplate absorbance reader (BioRad) according to standard protocols. The relative luciferase activity was determined by the ratio of firefly luciferase to β-galactosidase activity. Each reporter plasmid was transfected in triplicate, and the reporter assay was performed three times independently.PK-15 cells  as described previously aK) and a dissociation rate constant (dK). The equilibrium dissociation constant (DK) was calculated by the ratio of rate constant (d/KaK).When HEK293T cells were 90% confluent, pcDNA4A-His-sPPARD and -G32E mutant expression vectors were transfected into cells in 10-cm dishes by Lipofectamine 2000 (Invitrogen). Thirty-two hours after transfection, fusion proteins were purified from cell lysate by affinity column chromatography over His-Bind resin  according to the supplier's instruction. Protein concentration was measured by the standard Bradford method 2HPO4/NaH2PO4 (pH 8.0), 0.05% Tween 20 and 10 mM imidazole [pH 8.0]. A total of 5 mg protein was incubated with His-select nickel affinity gels (Sigma) overnight. Beads were washed with urea buffer containing additional 10 mM imidazole (pH 8.0) twice and then with native wash buffer (0.1 M Na2HPO4/NaH2PO4 (pH 8.0), 0.05% Tween 20 and 20 mM imidazole (pH 8.0)). Protein was dissolved in Laemmli buffer (BioRad), loaded on 10% SDS-PAGE gels and analyzed by Western blotting with antibodies against tubulin (Beyotime), His tag (Beyotime) and HA tag (Beyotime) Expression vectors pcDNA4A-His-sPPARD, -G32E, -K16R, -K17R, -K18R or -K16-18R and HA-ub were co-transfected into HEK293T cells by Lipofectamine 2000 (Invitrogen). At 24 hour post-transfection, cells were treated with 10 µM PS341 for 4 hours or 5 µM GW0742 overnight. Then cells were lysed in a urea buffer consisting of 8 M urea, 0.1 M NaP-value≤0.05 was treated as significance.Statistical analysis was performed using a two-tailed student's test. in vitro, especially after 5-days treatment .PPARD is now widely accepted to suppress cartilage growth of bones. Its activation by endogenous ligands promotes chondrocyte apoptosis in growth plate P<0.05) increased transcription activity of wild-type sPPARD compared to the mutant, indicating that the glutamic acid 32 substitution attenuated ligand-induced transcription activity of sPPARD . These observations indicate that the G32E substitution promotes nuclear export of sPPARD.Subcellular compartmentalization is an important regulatory mechanism of protein activity especially for transcription factors http://www.cbs.dtu.dk) . Taken together, we proposed that the G32E substitution might promote the interaction of NES of sPPARD with CRM1 to stimulate nuclear export of sPPARD.von Knethen et al in vitro using the SPR technology. The results showed that GW0742 interacted with sPPARD in a dose-dependent manner (DK) of 44.9 nM for the wild-type sPPARD and 97.9 nM for the mutant, respectively  in the presence of puromycin and PS341. Because the proteasome only interact with target proteins covalently attached to ubiquitin (ub) chains Like most transcription factors, the concentration of PPARs is rigidly controlled, at least in part, through ubiquitin-proteasome pathway P<0.05) reduced transcription activity compared to the wild-type receptor, indicating the ubiquitination of the A/B domain is involved in gene activation (The ubiquitin-proteasome system (UPS) has various non-proteolytic functions, such as regulating transcription activities To determine whether the G32E substitution attenuate ubiquitination of sPPARD, we cotransfected HEK293T cells with HA-ub and two forms of His-tagged PPARD constructs (pcDNA4A-His-sPPARD and -G32E mutant constructs), which were then cultured in the absence (DMSO) or presence (GW0742) of ligand for 12 hours. The G32E mutant showed reduced ubiquitination upon both treatments compared to the wild-type receptor  in promoters of target genes and share a highly similar mechanism of action First, we confirm that the mutation reduces ligand-dependent transcription activation of sPPARD. Second, our analysis of subcellular localization shows that the G32E substitution activates CRM1-mediated nuclear export of sPPARD. Further, we reveal a novel function of ubiquitinations in the A/B domain of sPPARD as a non-proteolytic signal, which is required for efficient ligand-induced transactivation. The G32E substitution proximal to these ubiquitins sites reduces ubiquitination of sPPARD and consequently inhibits PPARD-dependent transactivation. Our data reveals, for the first time, some ubiquitin sites of PPARs in addition to UPS influencing transcription activity, which was poorly understood before this study.The above-mentioned findings together with our previous report The genomic region harboring sPPARD G32E is of great interest in pig genetics, because significant QTL for diverse traits related to growth, carcass length, skeletal morphology and fat deposition have been consistently evidenced in this region using different crosses between Chinese indigenous pigs and Western commercial breeds Table S1PCR primer sequences.(DOC)Click here for additional data file.Figure S1Ligand activation of sPPARD leads to inhibition of chondrocytes growth. Pinna cartilage-derived primary chondrocytes were seeded onto 6-well tissue culture dishes and cultured over 7 days in the presence of 0.1% DMSO, 0.1 µM and 1 µM GW0742. Cell numbers were quantified daily with a counter chamber. Values represent the mean ±S.E.M. from triplicate and independent samples. An asterisk (*) indicates significant (P<0.05) difference between the GW0742-treated group and the DMSO-treated control.(TIF)Click here for additional data file.Figure S2The G32E substitution in the A/B domain of sPPARD activates cytosolic localization in primary chondrocyte cells. Cells were transfected with pEGFP-C1-wild-type sPPARD (A) and -G32E mutant (B) by electroporation. sPPARD subcellular localization was analyzed by GFP fluorescence at 24 hour post-transfection. Cell nuclei were counterstained with DAPI.(TIF)Click here for additional data file.Figure S3A classical short leucine-rich nuclear export signal was identified between amino acids 343–352 in the C-terminal E/F domain of sPPARD by the NetNES software. NES score was calculated from the Markov Model (HMM) and Artificial Neural Network (NN) scores. If the NES score exceeds the threshold, those amino acid residues are predicted to be a nuclear export signal.(TIF)Click here for additional data file.


The mechanisms by which microRNAs (miRNAs) affect cell fate decisions remain poorly understood. Herein, we report that miR-200a can suppress the differentiation of mouse embryonic stem (ES) cells into endoderm and mesoderm. Interestingly, miR-200a directly targets growth factor receptor-bound protein 2 (Grb2), which is a key adaptor in the Erk signaling pathway. Furthermore, high levels of miR-200a dramatically decrease Grb2 levels and suppress the appearance of mesoderm and endoderm lineages in embryoid body formation, as well as suppressing the activation of Erk. Finally, Grb2 supplementation significantly rescues the miR-200a-induced layer-formation bias and the Erk suppression. Collectively, our results demonstrate that miR-200a plays critical roles in ES cell lineage commitment by directly regulating Grb2 expression and Erk signaling. Embryonic stem (ES) cells are derived from the inner cell mass (ICM) of blastocysts, one of the early stages of embryonic development. These cells retain the features of pluripotency and self-renewal while serving as the progenitors of all cell types –3. The rPrevious studies show that miR-200 family members are emerging as important regulators of cell proliferation, differentiation and metastasis –31. The Growth factor receptor-bound protein 2 (Grb2) is a key adapter protein in intracellular signal transduction pathways, in which it links activated cell surface receptors to downstream targets by binding to specific phosphotyrosine-containing and proline-rich sequence motifs ,42. DeleIn this paper, we report that miR-200a was highly expressed in ES cells and gradually decreased in expression during embryoid body (EB) formation and that miR-200a suppressed endoderm and mesoderm lineage commitment. We further identified Grb2 as a direct regulatory target of miR-200a. In EB, knockdown of Grb2 with a specific shRNA had an identical effect to treatment with miR-200a. Finally, a rescue assay showed that exogenous Grb2 could reverse the miR-200a-induced endoderm and mesoderm suppression. Similarly, the extracellular signal-regulated kinase (Erk) signaling, when activated with assistance from Grb2, also rescued miR-200a-induced effects. Taken together, these results suggest that miR-200a might control cell fate decisions affecting the early endoderm and mesoderm layers in a manner that is partly dependent on Erk signaling, by regulating Grb2 expression levels.4 cells in 2 ml medium without LIF. HEK293T cells were cultured in DMEM (Gibco) that was supplemented with 100 U/ml of penicillin/streptomycin (Invitrogen) and 10% FBS (Gibco).The mouse ES cell line ES-E14TG2a, purchased from the American Type Culture Collection (ATCC CRL-1821), was maintained on gelatin-coated plates in high glucose-Dulbecco’s Modified Eagle Medium  that was supplemented with 15% (vol/vol) ES qualified-fetal bovine serum , 2 mM L-glutamine (Hyclone), 1x nonessential amino acids (Hyclone), 50 mM beta-mercaptoethanol (Gibco) and 500-1000 U/ml of leukocyte inhibitory factor . ES cells were trypsinized and split every 2 days, and the culture medium was changed daily. For the formation of EB, ES cells were plated in 3.5 cm dishes at a density of 5 × 104 cells per well. For miRNA overexpression or knockdown experiments, miRNA mimics or inhibitors and the scrambled negative control were gently mixed with Opti-MEM (Gibco) and X-tremeGENE 9 Transfection Reagents (Roche) according to the manufacturer’s instructions. At 12-72 hours post-transfection, the cells were harvested for Western blotting or quantitative real-time PCR analysis.miR-200a mimics and inhibitors (including the Negative control) were purchased from Ribo. ES cells were seeded into 6-well plates at a density of 5 × 10The miR-200a sequence was obtained from the mirbase website and cloned into the AgeI and EcoRI sites of the pLKO.1 vector. To construct a miR-200a sponge vector, the following oligoribonucleotides for the miR-200a sponge were designed and synthesized: miR-200a sponges forward oligo, CCGGTACATCGTTACTCTCAGTGTTACCGACATCGTTACTCTCAGTGTTAGCGACATCGTTACTCTCAGTGTTAG; reverse oligo, AATTCTAACACTGAGAGTAACGATGTCGCTAACACTGAGAGTAACGATGTCGGTAACACTGAGAGTAACGATGTA. After an annealing step at 95 °C for 4 min, the sponge oligoribonucleotides were inserted into the pFUW plasmid between the BamHI and AgeI sites. For luciferase reporter assays, a pGL3-Grb2-3’UTR-WT vector was constructed. A 3’ UTR segment of wild-type Grb2 mRNA, which contained the putative target sites of miR-200a, was amplified and cloned into the SacI and XbaI sites downstream of the luciferase reporter gene in pGL3. A pGL3-Grb2-3’UTR-Mutant vector, which carried a mutation in the complementary site for the seed region of miR-200a, was generated from the pGL3-Grb2-3’UTR-WT vector by mutation PCR. The Grb2 expression vector was created by cloning the Grb2 coding sequence into the BamHI and AgeI sites of pFUW.Cells were lysed in RIPA buffer that contained a protease inhibitor cocktail. Estimation of lysate protein concentrations was performed with a BCA Protein Assay Kit (Pierce). Approximately 100 µg of lysate was resolved on a 12% SDS/PAGE gel and transferred to NC membranes (Bio-Rad). The membranes were blocked with blocking solution (3% BSA in TBST) and incubated with primary antibodies. The following primary antibodies were used: mouse anti-Oct4 , rabbit anti-Sox2 , rabbit anti-Nanog , rabbit anti-Grb2 , rabbit anti-Erk , rabbit anti-p-Erk  and rabbit anti-GAPDH . The blots were subsequently incubated with either HRP-conjugated anti-rabbit IgG or HRP-conjugated anti-mouse IgG . Labeled proteins were detected with the eECL Western Blot Kit (Cwbio).Total RNA was extracted from ES cells and EBs with TRIzol reagent (Takara) according to the manufacturer’s instructions. cDNA synthesis was performed from 250 ng of total RNA in a single step (37 °C for 15 min) with the Takara reverse transcription kit. Quantitative real-time PCR was performed with iTaq reagent (Bio-Rad) and a STRATAGENE Mx3005p Real-Time PCR Cycler. The following PCR cycle conditions were used: initial denaturation for 30 sec at 95 °C, followed by 40 cycles of 5 sec at 95 °C and 30 sec at 60 °C. Mature miRNA primers, including stem-loop RT and special miRNA primers, were purchased from Ribo. Real-time PCR primers are shown in 4 cells per well in 24-well plates. Cells were transfected via Lipofectamine 2000 Transfection Reagent (Invitrogen) and Opti-MEM (Gibco) with 50 nM miRNA mimics, 400 ng of the luciferase vector (pGL3 constructs) and 20 ng of the Renilla vector. At 24 hours post-transfection, the cells were harvested, and the luciferase activity was measured with a dual-luciferase reporter assay (Promega) according to the manufacturer’s instructions.At 12 hours prior to the transfections, HEK293T cells were plated at a density of 5 × 103). Three days later, ES cells were fixed in 4% paraformaldehyde for 3 min at room temperature and then rinsed in PBS solution for 5 min. ES cells were stained with a staining solution of with 0.4% N,N-dimethylformamide (Sigma) and 0.06% Red Violet LB salt (Sigma). The result was obtained after 30 min of incubation at room temperature.ES cells were cultured at a clonal density , anti-alpha-smooth muscle actin , anti-Nestin (Millipore) and anti-Tuj1 (Millipore), which were diluted 1:1000 in PBS with 10% FBS (Gibco). Subsequently, cells were incubated for one hour at room temperature with the secondary Alexa Fluor 546-conjugated antibody (Invitrogen). The cells were counterstained with Hoechst 33342 to visualize the cell nuclei. Images were captured with a Nikon Eclipse Ti–S fluorescence microscope.Cells were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, permeabilized with 0.1% Triton X-100 (Amresco) for 8 min, and blocked by incubation with 10% FBS (Gibco) in PBS for one hour to prevent nonspecific binding. The cells were incubated overnight at 4 To determine whether miR-200 family is responsive to ES cell differentiation, we analyzed miR-200 mumbers  expression during ES cell spontaneous differentiation by quantitative real-time PCR. We found that miR-200a expression was markedly decreased during EB formation  receptor signaling pathway and thus is involved in multiple aspects of cellular function. FGF, which controls the responsiveness of differentiation regulators, is of particular importance to mesoderm and endoderm formation . Gene kn to bFGF . Our dat-/- ES cells, T expression is downregulated and pluripotency markers Oct4 and Nanog are maintained and they fail to differentiate into lateral mesoderm cells under mesoderm differentiation condition [Previous studies have demonstrated that Grb2 transmitted FGF signaling to Erk and thus controlled the basal balance between the ES cell state and inductive differentiation . Autoactondition . In neurondition ,63. Our Taking these findings together, we postulate that Erk signaling and miR-200a maintain a balance in specific cell fate decisions such that Erk signaling regulates differentiation into the mesoderm and endoderm lineages and miR-200a suppresses differentiation into these lineages. The link between these factors is Grb2, which delivers activation signals to Erk. Our findings suggest that miR-200a mediates Grb2 expression, thereby blocking Erk activation, which leads to the arrest of endoderm and mesoderm lineage differentiation and promotes ectoderm lineage commitment.Figure S1(A) Quantifications of Grb2 in miR-200a and Sponge 200a treated ES cells in (TIF)Click here for additional data file.Table S1Real-Time PCR primers used in this study.(DOC)Click here for additional data file.


Cigarette smoke (CS) has been reported to induce autophagy in airway epithelial cells. The subsequent autophagic cell death has been proposed to play an important pathogenic role in chronic obstructive pulmonary disease (COPD); however, the underlying molecular mechanism is not entirely clear. Using CS extract (CSE) as a surrogate for CS, we found that it markedly increased the expressions of both LC3B-I and LC3B-II as well as autophagosomes in airway epithelial cells. This is in contrast to the common autophagy inducer  that increases LC3B-II but reduces LC3B-I. Further studies indicate that CSE regulated LC3B at transcriptional and post-translational levels. In addition, CSE, but not starvation, activated Nrf2-mediated adaptive response. Increase of cellular Nrf2 by either Nrf2 overexpression or the knockdown of Keap1 (an Nrf2 inhibitor) significantly repressed CSE-induced LC3B-I and II as well as autophagosomes. Supplement of NAC (a GSH precursor) or GSH recapitulated the effect of Nrf2, suggesting the increase of cellular GSH level is responsible for Nrf2 effect on LC3B and autophagosome. Interestingly, neither Nrf2 activation nor GSH supplement could restore the repressed activities of mTOR or its downstream effctor-S6K. Thus, the Nrf2-dependent autophagy-suppression was not due to the re-activation of mTOR-the master repressor of autophagy. To search for the downstream effector of Nrf2 on LC3B and autophagosome, we tested Nrf2-dependent genes  that are also increased by CSE treatment. We found that P62, but not NQO1, could mimic the effect of Nrf2 activation by repressing LC3B expression. Thus, Nrf2->P62 appears to play an important role in the regulation of CSE-induced LC3B and autophagosome. Autophagy is a regulated catabolic process, by which cellular components are sequestered in the vesicular system and later delivered to lysosomes for degradation and recycling of biogenic components in vitro western blot or in vivo punctate formation) as the hallmark and surrogate for autophagic activity.Most of the ATG proteins are highly conserved in mammals. Class III phosphoinositide 3-kinase (PI3K) and ATG6 initiate the formation of autophagosomes. Furthermore, two ubiquitination-like conjugation systems are required for autophagosome formation. One of these systems mediates the conjugation of ATG12 to ATG5 Despite the overwhelming studies on autophagy in diseases, very few have been carried on non-malignant airway diseases. Recently, several studies have demonstrated that cigarette smoke (CS) or cigarette smoke extract (CSE) induces autophagy in lung cells 3-Methyladenine (3-MA), E64D, Pepstatin A were purchased from Sigma-Aldrich . Antibodies against LC3B, p62 and HO-1 were from MBL International . Nrf2, Keap1 and Actin antibodies were purchased from Santa Cruz Biotechnology . Antibodies against p-mTOR (Ser 2448) and p-S6K (Thr 389) were purchased from Cell signaling technology . The plasmids of Nrf2 and GFP-LC3B were kind gifts from Dr. Jingbo Pi  and Dr. Tamotsu Yoshimori , respectively. P62 plasmid and two ARE-reporters  (Promega). At 24 hrs post-transfection, the transfected cells were treated with CSE for 24 hrs or immersed in the starve media for 6 hrs. Then, the cells were lysed with passive lysis buffer (Promega) and both firefly and Renilla luciferase activities were measured with the dual-luciferase reporter assay system purchased from Promega. Firefly luciferase activity was normalized to Renilla luciferase activity. The experiment was carried out in triplicate and expressed as the mean ± the standard deviation (SD).BEAS-2B cells were transfected with GFP-LC3B. At 24 hrs post-transfection, the cells were treated with or without CSE for 24 hrs. Fluorescence Images were acquired by using of a confocal microscope . Six random areas (20X) were selected for the quantification on each slide, and triplicate samples were included for each experimental group. Cells containing “punctate dot” were counted and expressed as percentage of the total cells.Total cellular proteins were collected and western blot analysis were performed based on the methods described previously t method using housekeeping gene GAPDH. The purity of amplified product was determined from a single peak of a dissociation curve. Efficiency curves were performed for each gene of interest relative to the housekeeping gene, based on the manufacturer's instructions. Results were calculated as fold induction over control, as described previously Real-time PCR was performed as described previously GTAAGAAGCCAGATGTTAA) AAGAGTATGAGCTGGAAAAACGGGCGTGGCTGTCCTCAAT) TGAACGGTGCTGTCATGTAGGACATCACAGGTAAACTG, #2: GAACCTCAACTGACATATAGCATTGAAGTTGATATCGA) TM 2000  based on manufacturer's instruction. Successful knockdown of the target was confirmed by realtime RT-PCR and western blot.Two different siRNAs were used for the knockdown of each gene. siRNA targeting Nrf2  according to the manufacturer's instruction. Briefly, cells were rinsed twice with warm phenol red-free medium and then loaded for 30 min with CM-H2DCFDA (3 μM) in medium without phenol red. The cells were rinsed three times for removal of the extracellular dye, and the medium was replaced with phenol red-free medium. Then the cells were loaded into a live cell imaging incubator  at 37°C in a 5% CO2 atmosphere. Live cell images were continuously recorded before and after CSE treatment by confocal microscopy .Reactive oxygen species (ROS) production was determined using chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate  was used for analyses of the data.Experimental groups were compared using a two-sided Student's Starvation-induced autophagy has been extensively studied. Recently, CSE has also been shown to induce autophagy in airway cells th lane, th lane, th lane, th Lane, th Lane, Since LC3B is the critical player of autophagy and its dynamics in CSE treated cells were clearly different from the classical autophagy inducer-starvation, we examined the potential molecular mechanism underlying CSE-induced LC3B-I and II. As shown in th lane, th lane, th lane, th lane, th lane, th lane, Since CSE elevated both LC3B and Nrf2, we decided to examine the role of Nrf2 in the regulation of LC3B. We artificially increased Nrf2 level by overexpressing Nrf2. In these cells, LC3B-I and LC3B-II were significantly decreased  mTOR . The repNrf2 is a master regulator of antioxidant response partly through increasing the cellular reduced glutathione (GSH) level via activating several key GSH synthesizing enzymes nd lane, th lane, th lane, th lane, th lane, th lane, th-8th lanes, To further understand the molecular mechanism underlying Nrf2-mediated LC3B repression, we searched for downstream effectors of Nrf2. HO-1, an Nrf2 downstream gene, has been shown to modulate CSE-induced LC3B CSE-induced autophagy appears to be different from the autophagy induced by conventional stimuli such as starvation. The conventional stimuli mostly increase the conversion from LC3B-I to LC3B-II, while the overall amount of LC3B appears to be constant. In contrast, CSE significantly increase the total amount of LC3B. The induction of LC3B-I appears to be much earlier than the increase of autophagy (as shown by the increase of LC3B-II). However, it is unclear whether CSE accelerates the autophagy biogenesis, which leads to more LC3B-I to LC3B-II conversion; or large amount of LC3B-I induced by CSE drives more LC3B-II conversion through a concentration-dependent passive mechanism. To investigate these possibilities, we blocked autophagy biogenesis using class III PI3K inhibitor 3-MA. There was no change in LC3B-I and II levels in untreated cells, but 3-MA significantly decreased LC3B-II in CSE-treated cells without affecting LC3B-I. In addition, when lysosomal protease activity was blocked, LC3B-II level was increased. These suggest that CSE did increase the autophagy biogenesis. It is surprising that, even without CSE treatment, LC3B was still recycled through lysozyme but is 3-MA insensitive. This process is perhaps mediated through other non-canonical autophagic pathways. In our pulse/chase study, we further demonstrate that CSE-induced LC3B is reversible. In the CSE-treated cells, autophagosome/lysosome-mediated post-translational degradation pathway plays an important role in maintaining LC3B level. The excessive LC3B-I was cleared within 6 hrs by autophagy. The increase of LC3B-I protein  by CS (or CSE) exposure appears not to be restricted in the lung epithelial cells In addition to its traditional pro-survival role in the cellular response to stress conditions. excessive autophagy can also lead to cell death (type II) Another major distinction between CSE- and starvation-induced autophagy is the involvement of ROS and Nrf2 in the former. Nrf2 has been well established as the master regulator of cellular antioxidant pathway mainly via the activation of a battery of antioxidant gene expression Recently, Fujita, K et, al. indicates that Nrf2-P62 is required for TLR4 mediated aggresome-like induced structure (ALIS) formation In summary, we have, for the first time, demonstrated that Nrf2 system negatively regulates CSE-induced LC3B expression and autophagosomes partly through the elevation of P62. The elucidation of this important interaction between Nrf2 systems and autophagy will advance our understanding of the pathogenic effect of CS-associated chronic airway diseases and reveal novel drug targets for the development of effective treatments.


In silico V3 sequence based coreceptor usage prediction algorithms are a relatively rapid and cost effective method for determining HIV-1 coreceptor specificity. In this study, we elucidated the V3 sequence determinants of C-HIV coreceptor usage, and used this knowledge to develop and validate a novel, user friendly, and highly sensitive C-HIV specific coreceptor usage prediction algorithm.The majority of HIV-1 subjects worldwide are infected with HIV-1 subtype C (C-HIV). Although C-HIV predominates in developing regions of the world such as Southern Africa and Central Asia, C-HIV is also spreading rapidly in countries with more developed economies and health care systems, whose populations are more likely to have access to wider treatment options, including the CCR5 antagonist maraviroc (MVC). The ability to reliably determine C-HIV coreceptor usage is therefore becoming increasingly more important. V3-C), which we show has superior sensitivity for determining CXCR4 usage by C-HIV strains compared to all other available algorithms and prediction rules, including Geno2pheno[coreceptor] and WebPSSMSINSI-C, which has been designed specifically for C-HIV.We characterized every phenotypically-verified C-HIV gp120 V3 sequence available in the Los Alamos HIV Database. Sequence analyses revealed that compared to R5 C-HIV V3 sequences, CXCR4-using C-HIV V3 sequences have significantly greater amino acid variability, increased net charge, increased amino acid length, increased frequency of insertions and substitutions within the GPGQ crown motif, and reduced frequency of glycosylation sites. Based on these findings, we developed a novel C-HIV specific coreceptor usage prediction algorithm (CoRSeqV3-C is now openly available for public use at http://www.burnet.edu.au/coreceptor. Our results show that CoRSeqV3-C is the most sensitive V3 sequence based algorithm presently available for predicting CXCR4 usage of C-HIV strains, without compromising specificity. CoRSeqV3-C may be potentially useful for assisting clinicians to decide the best treatment options for patients with C-HIV infection, and will be helpful for basic studies of C-HIV pathogenesis.CoRSeq Human immunodeficiency virus type 1 (HIV-1) gains entry into immune cells by binding to CD4 and one of two coreceptors, CCR5 or CXCR4 ,7. The ein silico predictive algorithms capable of predicting viral coreceptor usage have been developed, principally for B-HIV variants, offering a rapid, simplistic and inexpensive alternative to cell based entry assays.The CCR5 antagonist maraviroc (MVC) is presently used as an antiretroviral (ARV) for treatment of HIV-1 infected subjects who have no detectable CXCR4-using virus in their plasma -19. ThusStudies have shown that the major determinants of HIV-1 CCR5 and/or CXCR4 usage lie within the third variable loop (V3) of the viral surface envelope glycoprotein, gp120 -25. CurrIn this study we analyzed all the phenotypically characterized patient-derived C-HIV V3 sequences currently available in the Los Alamos HIV Database in order to identify potential V3 sequence characteristics capable of differentiating CXCR4-using from R5 HIV-C Envs. We selected one representative CXCR4-using and/or R5 V3 sequence per subject in order to avoid biasing the results by the resampling of highly related sequences. In total, we assembled 69 unique CXCR4-using and 473 unique R5 C-HIV V3 sequences.CXCR4-using C-HIV V3 sequences were found to be significantly longer (range 32–37 amino acids) than the R5 C-HIV V3 sequences  , V1 length (p=0.0415 by a Mann Whitney U Test), the number of PNGS within V1 (p=0.0363 by a Mann Whitney U Test), and length of the V4 region (p<0.0001 by a Mann Whitney U Test). These findings suggest that regions outside of the V3 loop may also be involved in determining coreceptor usage of C-HIV strains, and therefore could be used to predict coreceptor specificity. However, the limiting number of phenotypically characterised CXCR4-using C-HIV sequences containing regions other than V3 (n<40) may limit the significance of these findings, and also the practicality of including these characteristics in new C-HIV coreceptor usage prediction algorithms.V3-C). When a query V3 sequence is submitted to CoRSeqV3-C, which is now hosted at http://htpp://www.burnet.edu.au/coreceptor and presently available for use as a research tool, the sequence is aligned against the HXB2 V3 sequence, and a series of questions are addressed , which consisted of every phenotypically characterized C-HIV V3 sequence currently available, allowing multiple sequences per patient (143 CXCR4-using and 1213 R5 C-HIV Envs). The other coreceptor usage prediction algorithms assessed were the Geno2pheno[coreceptor] algorithm , WebPSSMSINSI-C and 11/25/V3 charge. Here, the investigators identified multiple amino acids within gp41 that could be used to differentiate R5 from CXCR4-using C-HIV Envs, suggesting their inclusion in a coreceptor specificity prediction algorithm could improve predictive accuracy. However, a study by Thielen et al.  and WebPSSMSINSI-C algorithms for predicting coreceptor specificity. While we found the 11/25/V3 charge rule to be more accurate than the 11/25 rule and the 11/24/25 rule, it was less accurate than Geno2pheno[coreceptor], WebPSSMSINSI-C, CoRSeqV3-C and the HIV-B specific WebPSSMx4r5-B algorithms. A recent study, by Lin et al. [[coreceptor], WebPSSMSINSI-C, or CoRSeqV3-C algorithms.A study by Raymond et al.  found thn et al. , utilizin et al. . In our V3-C by determining the combination of V3 characteristics and amino acids specific to R5 or CXCR4-using C-HIV Envs that most accurately predict the coreceptor usage of all the currently available phenotypically characterized C-HIV V3 sequences. Future performance evaluation with additional phenotypically characterized and independent V3 sequences is required to more completely evaluate the sensitivity and specificity of CoRSeqV3-C, which will be conducted as more C-HIV V3 sequences are deposited into the Los Alamos HIV database. We showed that CoRSeqV3-C was maximally sensitive and specific for predicting CXCR4 usage when data was restricted to just one representative sequence per patient, which is a desirable quality. Importantly, as more C-HIV V3 sequences become available, CoRSeqV3-C can be reviewed for the potential inclusion of other V3 characteristics and/or amino acids in order to further maximize its sensitivity and specificity. Thus, unlike previously developed algorithms, CoRSeqV3-C is adaptable. CXCR4-using C-HIV Envs were first reported in patients on ART, and studies have suggested that ART provides a selective environment that may drive the emergence of CXCR4-using variants  using a false-positive rate of 10% (http://coreceptor.bioinf.mpi-inf.mpg.de/), as well as the C-HIV and B-HIV specific WebPSSM algorithms (http://indra.mullins.microbiol.washington.edu/webpssm/). The 11/25 rule predicts CXCR4-usage based on the presence of Arg, Lys or His at position 11 and/or 25 within the V3 region of gp120 [The of gp120 . The 11/of gp120 ,25,39,40of gp120  predictsof gp120  predictshttp://www.hiv.lanl.gov/content/sequence/ENTROPY/entropy.html). Potential N-linked glycosylation sites were predicted using the Los Alamos Database N-Glycosite tool (http://www.hiv.lanl.gov/content/sequence/GLYCOSITE/glycosite.html).Sequence alignments were developed using CLC Main Workbench version 6.5. Sequence variability at each amino acid position was determined using the Los Alamos Database Entropy-One tool .PRG is a member of the ViiV Australia Scientific Advisory Board, and has received honoraria from ViiV and financial support for travel to conferences. The other authors declare that they have no competing interests.KC performed the sequence analysis. KC and LRG designed the algorithm. MRJ supplied HIV-1 sequences and helped interpret the results. JS, MJC and PRG helped interpret the results. KC and PRG wrote the manuscript. All authors helped edit the manuscript and have read and approved the final version.Identification of amino acid alterations capable of improving the sensitivity or specificity of a prototype algorithm that predicts C-HIV CXCR4 usage based on the 11/25 rule, the presence of a V3 crown alteration and/or a two amino acid insertion.Click here for file


Colorectal cancer is one of the most common malignant tumors worldwide. Loss of imprinting (LOI) of the insulin-like growth factor 2 (IGF2) gene is an epigenetic abnormality observed in human colorectal neoplasms. Our aim was to investigate the feasibility of using the IGF2 imprinting system for targeted gene therapy of colorectal cancer.We constructed a novel oncolytic adenovirus, Ad315-E1A, and a replication-deficient recombinant adenovirus, Ad315-EGFP, driven by the IGF2 imprinting system by inserting the H19 promoter, CCCTC binding factor, enhancer, human adenovirus early region 1A (E1A) and enhanced green fluorescent protein (EGFP) reporter gene into a pDC-315 shuttle plasmid. Cell lines with IGF2 LOI (HCT-8 and HT-29), which were infected with Ad315-EGFP, produced EGFP. However, no EGFP was produced in cell lines with maintenance of imprinting (HCT116 and GES-1). We found that Ad315-E1A significantly decreased cell viability and induced apoptosis only in LOI cell lines in vitro. In addition, mice bearing HCT-8-xenografted tumors, which received intratumoral administration of the oncolytic adenovirus, showed significantly reduced tumor growth and enhanced survival.Our recombinant oncolytic virus targeting the IGF2 LOI system inhibits LOI cell growth in vitro and in vivo, and provides a novel approach for targeted gene therapy. Malignant neoplasms are responsible for nearly 7.5 million deaths representing 13% of all mortality and 5% of the global burden of disease in terms of disability-adjusted life years lost. ConventGenomic imprinting is an epigenetic modification of a gene based on its parental origin, which results in monoallelic expression. For mosAdenoviruses rapidly infect a broad range of human cells, including proliferating and quiescent cells, which tend to yield high levels of adenoviral particles. In addiIn our previous study, a recombinant replication-defective adenovirus carrying the IGF2 imprinting system and the DT-A gene was successfully constructed, in which Ad-DT-A effectively kills tumor cells showing IGF2 LOI. ReplicaOne of the critical functions of the human adenovirus early region 1A (E1A) protein is to activate transcription of early viral genes. This reThe oncolytic adenovirus Ad315-E1A was successfully constructed in this study, which was regulated by the IGF2 imprinting system. A control virus, Ad315-EGFP, was also constructed  reporter gene. After infection of cells with Ad315-EGFP (10 plaque-forming units [PFU]/cell) for 24 h, EGFP expression was observed in LOI cells lines HCT-8 and HT-29. However, in the HCT116 cell line, which maintains normal IGF2 imprinting, only weakly positive EGFP expression was observed  that were infected with Ad315-E1A and H101 (10 PFU/cell). Cells were harvested at 24 h after infection to determine E1A mRNA expression by RT-PCR, and E1A protein expression was determined by western blot at 48 h after infection. As shown in FigureWe were interested in examining whether Ad315-E1A treatment would effectively suppress tumor cell proliferation. For this purpose, the cytotoxic effects of the oncolytic adenovirus expressing the E1A gene were investigated in all cell lines. As shown in FigureWe further examined apoptosis in all cell lines with varied IGF2 imprinting using flow cytometry at 72 h after infection. Ad315-EFGP infection served as a negative control to enable us to evaluate the cytopathic effect of adenoviral infection itself. As shown in Figure3, respectively. Although no significant difference existed between PBS- and Ad315-EGFP-treated groups, significant antitumor efficacy was observed in the Ad315-E1A-treated group compared with that in other groups (p < 0.05). Moreover, the mean survival times for mice treated with PBS, Ad315-EGFP or Ad315-E1A were 37, 40 and 88 days, respectively. Ad315-EGFP treatment did not increase the survival time, compared with that of PBS treatment (p > 0.05), but Ad315-E1A treatment prolonged the survival time compared with that of treatment with PBS or Ad315-EGFP -stained xenograft sections from Ad315-E1A-treated mice showed pathological signs of tumor necrosis obviously exceeding that of other groups Figure. To bettIn agreement with E1A protein expression, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assays revealed large numbers of apoptotic cells in tumors infected with Ad315-E1A Figure. In miceIn the present study, we successfully constructed a novel replication-selective adenovirus, Ad315-E1A, and a replication-deficient adenovirus, Ad315-EGFP. Our data show a significant effect of Ad315-E1A against tumor growth both in vitro and in vivo. This observation suggests that replication-selective adenoviruses driven by the IGF2 imprinting system may be used as a novel anti-cancer agent with a high therapeutic potential.Using viruses as a genetic carrier offers an attractive alternative or promising integration with traditional therapeutic regimens for cancer treatment. Among vIGF2 encodes the mitogenic anti-apoptotic peptide IGF-II that is overexpressed in several human cancers cells. In colorectal cancer, it has been reported that LOI of IGF2 occurs in 44% of informative colorectal cancers that are linked to microsatellite instability. AdditioBased on these findings, we theorized that we could build a kind of oncolytic adenovirus specific for replication in LOI tumor cells for colorectal cancer therapy. In this proof-of-concept study, we constructed adenoviral vectors, Ad315-E1A and Ad315-EGFP, which carry the three enhancer elements, DMD and promoter based on IGF2 LOI. Initially the utility of our expression system was tested using EGFP reporter assays. The results showed that green fluorescence was detected only in LOI cells. By increasing the multiplicity of infection and extending the time of infection, we found that the results were unchanged. To further demonstrate the selective efficacy of the LOI system, we used the oncolytic adenovirus H101, which has been clinically approved in China to treat malignancies, as a positive control for specific lysis of tumor cells by targeting the inactivated p53 in tumors. We used RT-PCR and western blotting to detect E1A expression at mRNA and protein levels, respectively, in Ad315-E1A-infected cells, and confirmed E1A expression in LOI colorectal cell lines. Furthermore, in H101-infected cells, E1A mRNA and protein was only detected in the p53 mutant cell line HT-29. All of the above results indicate that the replication-selective system that we used is practicable and effective.In this study, the therapeutic potential of Ad315-E1A was tested by an MTT assay and flow cytometry, and the results clearly support the rationale of our hypothesis, in which Ad315-E1A effectively suppressed the growth of tumor cells and induced obvious cytotoxicity in LOI tumor cells. Therefore, we compared the therapeutic effect between IGF2 regulatory sequences and H101 for colorectal cancer treatment. The data showed that there was no detectable cytotoxicity in HCT-116 and GES-1 cells infected with Ad315-E1A or H101 (10 PFU/cell), whereas Ad315-E1A infection achieved significant growth inhibition of the IGF2 LOI and p53-active cell line (HCT-8) relative to that by H101 infection. In addition, both oncolytic viruses exhibited superior efficiency to inhibit growth of the HT-29 (p53 mutant and LOI) cell line. These results suggest that the IGF2 LOI system in our viral vector enabled efficient adenovirus replication in host cells using the E1A endogenous promoter.Using the human colorectal cancer cell line HCT-8 as a model, we showed that this therapeutic strategy significantly increased cancer cell death and apoptosis caused by virus replication in vivo. Despite Ad315-E1A treatment significantly inhibiting tumor growth and prolonging the survival of tumor-bearing mice, the tumors were not eliminated completely. Previous research also found that oncolytic viruses have a limited potential to eradicate tumors when used as a monotherapy-32. ThusIn summary, our results show that an oncolytic adenovirus regulated by the IGF2 LOI system confers a significant anti-tumor effect by induction of apoptosis in vitro and in vivo in human colorectal cancer cells. LOI exists in a wide variety of tumors, and although more investigations are required for future use, our preliminary data support the use of oncolytic viruses in the context of the IGF2 LOI system as a novel approach for cancer therapy.2.HCT-8, HCT116, HT-29 (human colon cancer cell lines) and GES-1  were obtained from the Shanghai Cell Collection, Chinese Academy of Sciences. The HEK293 cell line (human embryonic kidney cells containing the E1A region of the adenovirus) was obtained from Microbix Inc. . HEK293, GES-1, and HCT-8 cell lines were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum . HCT116 and HT-29 cells were maintained in RPMI 1640  supplemented with 10% FBS. Cells were maintained in a humidified incubator at 37°C with 5% COThe original adenoviral shuttle plasmid that we used in this study was pDC315, and AD5 PBHGLOX1, 3CRE . Mouse H19 enhancer exons 1 (258 bp) and 2 (360 bp) were amplified by PCR from mouse genomic DNA and then linked as a single fragment by PCR. The enhancer was cloned into the pDC315 plasmid using restriction endonucleases Xbal and EcoRI. Subsequently, mouse DMD exons 1–2 (429 bp), 3 (207 bp) and 4 (156 bp) were also amplified by PCR from genomic mouse DNA and then linked as a single fragment by PCR. The DMD was cloned downstream of the enhancer using restriction endonucleases EcoRI and NheI. The mouse H19 promoter (302 bp) was amplified by PCR from mouse genomic DNA using the following primers: forward, 5’ GCGCTAGCCCACCGTTCTATGAAGGGCTTC 3’ (containing a NheI site) and reverse, 5’ AAGGATCCTCATCAGCGCCCATCTCTAGCC 3’ (containing a BamHI site), and then cloned downstream of the DMD using restriction endonucleases NheI and BamHI. The human adenovirus E1A sequence (1013 bp) was amplified by PCR from a TOP-K plasmid, which was kindly provided by Dr. Ji-Fan Hu , using the following primers: forward, 5’ CCCGGATCCGGGCCCTATGAGACATATTATCT 3’ (containing a BamHI site) and reverse, CGCGTCGACCGCAATCACAGGTTTACACCTTA 3’ .The EGFP reporter gene from the pEGFP-C1 vector  and the E1A gene were then inserted downstream of the H19 promoter using restriction endonucleases BamHI and SalI to construct pDC315-enhancer-DMD-H19-EGFP and pDC315-enhancer-DMD-H19-E1A. Inserts were confirmed by DNA sequencing. The adenovirus Ad315-E1A was constructed by homologous recombination techniques using pDC315-enhancer-DMD-H19-E1A and the adenovirus packaging plasmid PBHGLOX1, 3CRE in HEK293 cells with Lipofectamine 2000 . A standard replication-deficient adenovirus, Ad315-EGFP, was constructed by cotransfection of the adenovirus shuttle vector containing EGFP with an E1A/B-deleted adenoviral backbone vector.Adenoviruses were plaque purified, propagated in HEK293 cells, and purified again by a CsCl gradient according to standard techniques. Functional particle titers of all adenoviruses were determined by a plaque assay using HEK293 cells. The positive control adenovirus H101 was kindly provided by Dr. Sheng-Fang Ge (Shanghai Jiao Tong University School of Medicine).Cells were seeded in 96-well plates at a density of 10,000 cells/well for a 3--2, 5-diphenyl tetrazolium bromide (MTT) assay, or in 6-well plates at a density of 1000,000 cells/well for RT-PCR and flow cytometric analysis. Then, cells were incubated with various concentrations of Ad315-EGFP, Ad315-E1A, and H101 in serum-free DMEM at 37°C for 90 min. After incubation, serum-free DMEM containing the viruses was replaced with normal growth medium. The infected cells were maintained at 37°C until use in assays.EGFP expression was examined at 24 h after infection with adenoviral vectors (10 PFU/cell) under an Axioskop 2 microscope  with a fluorescent filter set (excitation 450–490 nm). E1A mRNA expression was determined by RT-PCR. Total RNA was extracted using Trizol (Invitrogen Life Technologies) according to the manufacturer’s instructions. First strand cDNA synthesis was performed in a total volume of 25 μl containing 2 μg RNA, 0.5 μg primer and 200 U M-MLV reverse transcriptase. cDNA was subsequently amplified in 50 μl reaction volumes containing 0.4 μmol/l of each E1A primer and 1.25 U Taq DNA polymerase . The amplification conditions were pre-denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 40 s, 60°C for 40 s and 72°C for 60 s, and a final extension of 72°C for 7 min. PCR products were electrophoresed on a 1% agarose gel containing ethidium bromide and then visualized under UV light.Cells were harvested and lysed by three cycles of freeze/thawing at −80°C. Cell lysates were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes . Membranes were probed with a mouse monoclonal antibody against E1A  and then a horseradish peroxidase-conjugated goat anti-mouse IgG. Proteins were visualized using Lumi-Light Western Blotting Substrate .Cytotoxicity was assessed using an MTT assay that was performed as described previously using a Cell Counting Kit-8 . BrieflyQuantitative evaluation of apoptosis was performed using flow cytometry after double staining with an annexin V-fluorescein isothiocyanate apoptosis detection kit to discrimination between early apoptotic (annexin V-positive) and necrotic ( annexin V/propidium iodide double-positive) cells. The cells  were cultured in 6-well plates, and then infected with Ad315-E1A at 10 PFU/cell. Cell apoptosis was analyzed at 72 h after infection with Ad315-EFGP (10 PFU/cell) serving as a negative control.6 HCT-8 cells into the right flank of 4–6-week-old female athymic nude mice, which was approved by the Experimental Animal Center of University of Yangzhou, Yangzhou, China. The average tumor volume was measured by the formula: volume = (length × width2) / 0.5. When tumors reached approximately 100 mm3, xenografted mice were randomly divided into three treatment groups with eight mice in each group. The Ad315-E1A group received intratumoral injections of 1 × 108 Ad315-E1A viral particles three times every other day. Tumors injected with Ad315-EGFP viral particles at the same dosage served as a viral vector control, and those injected with PBS served as a negative control. The tumor size was measured by vernier calipers every 3 days. Mice were then euthanized by cervical dislocation at a predetermined interval of observation. Tumors were dissected out and stored in 40% formalin. For histological analysis, fixed tumors were embedded in OCT compound and then cut into 5–7 mm sections using a Cryocut microtome . Mouse survival was recorded in a separate experiment. Animal experiments were performed in accordance with institutional guidelines for animal care by Nanjing Medical University.Tumor xenografts were established by subcutaneous injection of 5 × 10To immunohistochemically stain the E1A protein, the tumor and neighboring tissues were fixed in 10% formalin and embedded in paraffin for staining. Tumor or tissue sections were incubated at 4°C overnight with a mouse anti-human E1A antibody  at a dilution of 1:50. Sections were rinsed in PBS-T (0.05% Triton X-100 in PBS), followed by incubation with a goat anti-mouse secondary antibody at a 1:500 dilution for 1 h at room temperature. Sections were subsequently incubated with streptavidin-horseradish peroxidase (BD Biosciences) and diaminobenzidine substrate to develop the colorimetric reaction. The number of E1A-positive cells was counted in five random fields at ×400 magnification under a light microscope and averaged. Only cells with distinct staining were counted. The positivity rate was used to grade the expression levels.Apoptosis of tumor cells was detected using a TUNEL assay performed with an In Situ Cell Death Detection Kit  following the manufacturer’s instructions. To stain apoptotic cells, tumor samples were fixed with 10% formaldehyde and paraffin-embedded sections were prepared. The number of TUNEL-positive cells was counted in five random fields at ×400 magnification under a light microscope, and the apoptosis index for each field was calculated as the percentage of TUNEL-positive cells relative to the total number of cells.t-test and one-way analysis of variance. Differences among the results of in vivo survival experiments were assessed by the Kaplan-Meier method. Results were considered statistically significant at p < 0.05.Experimental data were presented as the mean ± standard deviation (SD) and assessed using the Student’s The authors declare that they have no competing interests.SW, YP, and ZN designed the study and wrote the protocols. ZN performed most of the experiments. BH, LG, and LC contributed to administrative, technical or material support. RL, YX, and TG managed the literature searches and analyses. GS undertook the statistical analysis. ZN drafted the manuscript. AH and JH provided critiques of the manuscript. All authors read and approved the final manuscript.


Chromobacterium violaceum. A PSI-BLAST search using the CV_2116 sequence as a query identified only one hit (E = 2e−07) corresponding to a hypothetical protein OR16_04617 from Cupriavidus basilensis OR16, which failed to provide insight into the function of CV_2116. The CV_2116 gene was cloned into the p15TvLic expression plasmid, transformed into E. coli, and 13C- and 15N-labeled NMR samples of CV_2116 were overexpressed in E. coli and purified for structure determination using NMR spectroscopy. The resulting high-quality solution NMR structure of CV_2116 revealed a novel α + β fold containing two anti-parallel β-sheets in the N-terminal two-thirds of the protein and one α-helix in the C-terminal third of the protein. CV_2116 does not belong to any known protein sequence family and a Dali search indicated that no similar structures exist in the protein data bank. Although no function of CV_2116 could be derived from either sequence or structural similarity searches, the neighboring genes of CV_2116 encode various proteins annotated as similar to bacteriophage tail assembly proteins. Interestingly, C. violaceum exhibits an extensive network of bacteriophage tail-like structures that likely result from lateral gene transfer by incorporation of viral DNA into its genome (prophages) due to bacteriophage infection. Indeed, C. violaceum has been shown to contain four prophage elements and CV_2116 resides in the fourth of these elements. Analysis of the putative operon in which CV_2116 resides indicates that CV_2116 might be a component of the bacteriophage tail-like assembly that occurs in C. violaceum.CV_2116 is a small hypothetical protein of 82 amino acids from the Gram-negative coccobacillus  Chromobacterium violaceum is a Gram-negative non-sporulating coccobacillus. It produces a natural antibiotic called violacein, which may be useful for treatment of colon and other cancers [C. violaceum, which was published in 2003 [CV2114 to CV2150, including CV_2116, which encodes a hypothetical protein of 82 residues (UniProtKB/TrEMBL ID Q7NW74_CHRVO). To date, CV_2116 does not belong to any known protein families  and is simply annotated as a putative uncharacterized protein [ cancers . The comCupriavidus basilensis OR16  with sequence identity of 43% over all 82 residues. Likewise, a PSI-BLAST search using OR16_04617 as the query identified CV_2116 as the only significant sequence match. Gramella forsetti (UniProtKB/TrEMBL ID A0LXN2_GRAFK) [Bacillus coagulans (UniProtKB/TrEMBL ID G2TJM8_BACCO) [A sequence similarity search against CV_2116 using PSI-BLAST identified only one hit, a hypothetical protein OR16_04617 from 2_GRAFK)  with seq1H-15N-HSQC spectrum  of 6.2 ± 0.5 ns derived from T1 and T2 measurements confirmed its monomeric state in solution based on a curve of molecular weight versus τc from other proteins characterized in our laboratory with molecular weights under 20 kDa as shown in A high quality solution structure of CV_2116 was determined by NMR spectroscopy. CV_2116 exhibited an excellent spectrum  indicatiN-terminal two-thirds of the protein and one α helix in the C-terminal third of the protein  and one between β6 and α-helix (E52-D72), and the C-terminal tail (R73-G82) were flexible and disordered in the NMR structure. Structural statistics for CV_2116 are presented in Two β-strands  compose the second anti-parallel β sheet. The 21-residue helix (E52-D72) in the N-acetylmuramate-alanine ligase (PDB ID: 1P31-A), and hyponastic leave 1 (PDB ID: 2L2N-A), had the highest Z scores of 3.7, despite the fact that they had low sequence identities of 8%, 8% and 5%, respectively. No biochemical or biological functions could be derived from the structural comparisons with the known structures in the PDB.CV_2116 adopted an apparently novel fold without any current matches in the CATH  structurThe electrostatic surface diagram of CV_2116 is shown in CV_2116 gene from C. violaceum is sandwiched between several neighboring genes that encode for apparent bacteriophage tail-like assembly proteins. In fact, C. violaceum has been observed to exhibit an unusual and extensive organization of bacteriophage tail-like particles [CV_2116, namely CV_2114, encodes a probable pyocin R2_PP tail fiber protein. R-type pyocins are a component of the non-flexible tail structure of bacteriophage [CV_2116, namely CV_2115, encodes a protein homologous to other bacteriophage J tail proteins belonging to the protein family PF04865, which are involved in baseplate assembly. Among the genes immediately following CV_2116, CV_2117 encodes a protein homologous to other bacteriophage V proteins (PF04717), which is also involved in baseplate assembly [CV_2118 is homologous to several members of PF05954, which are phage late control gene D proteins. Hence, it is reasonable to speculate that CV_2116 is a component of a bacteriophage-like tail assembly specific to C. violaceum.Although the function of the hypothetical CV_2116 protein could not be predicted by either sequence or structure similarity searches, the articles . The locriophage  The geneC. violaceum genome has shown that it contains four prophages denoted CvP1 through CvP4 [CV_2114 to CV_2150) and is thought to be a chimeric prophage containing ORFs with similarity to different phages. Specifically, it has been suggested that the tail and tail fiber genes, which include CV_2114-CV_2118, are similar to P2-like phage genes [Salmonella enterica subsp. enterica serovar Typhi, which are the so-called CT18 phages [S. enterica into the C. violaceum genome. A PSI-BLAST search of CV_2117 indicated ~100 hits to proteins with >30% identity, including NP_456044 from S. enterica subsp. enterica serovar Typhi str. CT18, which had 41% identity with 96% coverage and E = 9e−40. Examination of the genes surrounding NP_456044 indicated a putative operon containing 10 consecutive genes spanning NP_456044 to NP_456053, all encoding proteins involved in bacteriophage tail-like assembly. Examination of the strongest hit PSI-BLAST hit  against CV_2117, namely YP_003745638 from Ralstonia solanacearum CFBP2957, which is an aerobic non-sporing, Gram-negative plant pathogenic bacterium, also revealed a putative operon with a collection of genes spanning YP_003745640 to YP_003745645 that are all annotated as bacteriophage tail-like assembly proteins. These comparisons further support the speculation that CV_2116 is likely a novel gene of prophage origin that plays a structural or functional role in bacteriophage tail-like assembly in C. violaceum.Previous analysis of the ugh CvP4 . The lat8 phages ,18. CollC. violaceum was cloned into the expression plasmid p15TvLic including an additional N-terminal hexa-histidine tag and TEV cleavage site , and the plasmid was transformed into E. coli BL21 (DE3)-RIPL (Stratagene). E. coli cells were grown in 0.5 L of 2 × M9 minimal medium containing 15NH4Cl and 13C-glucose as the sole nitrogen and carbon sources, and supplemented with ZnSO4, thiamine, and biotin. Cells were grown at 37 °C to an OD600 of 1.0, followed by addition of 1 mM isopropyl β-d-thiogalactoside. The temperature was reduced to 15 °C, and cells grew overnight before harvesting. Cell pellets were lysed by sonication in a buffer containing 500 mM NaCl, 20 mM Tris and 5 mM imidazole (pH 8.0). The proteins were isolated from the lysate by nickel affinity chromatography (Qiagen) after washing the beads three times with five column volumes of 500 mM NaCl, 20 mM Tris (pH 8.0), 30 mM imidazole. Protein was eluted with five column volumes of 500 mM imidazole added to the washing buffer. For NMR structure determination, the hexahistidine tag of CV_2116 was cleaved with TEV protease and the mixture passed through a nickel affinity column. The purified 13C/15N labeled protein (referred to as the NC sample) was concentrated, and buffer was exchanged by ultrafiltration and dilution/reconcentration into the NMR buffer containing 10 mM Tris (pH 7.0), 300 mM NaCl, 10 mM DTT, 1 mM benzamidine, 0.01% NaN3, 1× inhibitor cocktail (Roche Applied Science), 95% H2O/5% D2O. Following the same procedure, a uniformly 100% 15N-labeled and 7% biosynthetically directed 13C (NC7) CV_2116 sample was also prepared for stereospecific assignments of isopropyl methyl groups of Val and Leu residues.The full-length gene for CV_2116 from 15N-HSQC CA, HNCACB, CBCA(CO)NH, C(CO)NH, HC(CO)NH, HBHA(CO)NH, HCCH-COSY, HCCH-TOCSY and (H)CCH-TOCSY spectra. Stereospecific assignments of isopropyl methyl groups of Val and Leu residues were determined from characteristic 1H-13C coupling patterns in a high resolution 1H-13C HSQC of the NC7 sample [15N-edited NOESY-HSQC (mixing time τm = 70 ms) and two 13C-edited NOESY-HSQC (τm = 70 ms) optimized for either aliphatic or aromatic carbon detection. Additional NOEs were assigned from a 4D 13C-13C-HMQC-NOESY-HMQC (τm = 70 ms) recorded after lyophilization and dissolution into pure D2O. Spectra were processed by NMRPipe and analyzed with Sparky 3.110 [NMR spectra were acquired on 250 μL NC and NC7 samples in 5 mm Shigemi NMR tubes at 293 K on Varian Inova 600 and 750 MHz NMR spectrometers. Backbone and sidechain resonance assignments were obtained from the analysis of 15N-HSQC , 13C-HSQ7 sample . NOE disky 3.110 . Nearly The solution NMR structure of CV_2116 was initially calculated using both AutoStructure . Inputs C. violaceum is not highly conserved among known protein families. The three-dimensional structure of CV_2116 was determined using solution state NMR spectroscopy and analysis of the structure indicated that CV_2116 adopted a novel fold containing two anti-parallel β-sheets and one α-helix. Despite determination of a high quality three-dimensional structure, no biochemical or biological function of CV_2116 could be predicted based on sequence similarity or structural alignment searches. However, analysis of the genes neighboring CV_2116 indicated that it resides in a putative operon that encodes for five proteins that are likely involved in bacteriophage tail-like assembly, and that the gene and its associated operon is of prophage origin, thereby giving C. violaceum distinctly bacteriophage-like characteristics that include the occurrence of a bacteriophage tail-like assembly. In closing, the analysis of CV_2116 from C. violaceum, which is both a hypothetical protein and a singleton, is typical of the challenge that is often encountered when researchers attempt to construct a hypothesis regarding the biochemical or cellular function of a hypothetical and/or singleton protein exclusively from sequence and structural information when little other knowledge about the protein is available.In conclusion, protein sequence analysis indicated that the hypothetical protein CV_2116 from