Caenorhabditis elegans ftn-1 and ftn-2, which encode the iron-storage protein ferritin, are transcriptionally inhibited during iron deficiency in intestine. Intestinal specific transcription is dependent on binding of ELT-2 to GATA binding sites in an iron-dependent enhancer (IDE) located in ftn-1 and ftn-2 promoters, but the mechanism for iron regulation is unknown. Here, we identify HIF-1 (hypoxia-inducible factor -1) as a negative regulator of ferritin transcription. HIF-1 binds to hypoxia-response elements (HREs) in the IDE in vitro and in vivo. Depletion of hif-1 by RNA interference blocks transcriptional inhibition of ftn-1 and ftn-2 reporters, and ftn-1 and ftn-2 mRNAs are not regulated in a hif-1 null strain during iron deficiency. An IDE is also present in smf-3 encoding a protein homologous to mammalian divalent metal transporter-1. Unlike the ftn-1 IDE, the smf-3 IDE is required for HIF-1–dependent transcriptional activation of smf-3 during iron deficiency. We show that hif-1 null worms grown under iron limiting conditions are developmentally delayed and that depletion of FTN-1 and FTN-2 rescues this phenotype. These data show that HIF-1 regulates intestinal iron homeostasis during iron deficiency by activating and inhibiting genes involved in iron uptake and storage. Caenorhabditis elegans as a model organism to study mechanisms of iron regulation. Our previous studies show that the iron-storage protein ferritin  is transcriptionally inhibited in intestine during iron deficiency, but the mechanisms regulating iron regulation are not known. Here, we find that hypoxia-inducible factor 1 (HIF-1) transcriptionally inhibits ftn-1 and ftn-2 during iron deficiency. We also show that HIF-1 activates the iron uptake gene smf-3. Transcriptional activation and inhibition by HIF-1 is dependent on an iron enhancer in the promoters of these genes. HIF-1 is a known transcriptional activator, but its role in transcriptional inhibition is not well understood. Our data show that HIF-1 regulates iron homeostasis by activating and inhibiting iron uptake and storage genes, and they provide insight into HIF-1 transcriptional inhibition.Due to its presence in proteins involved in hemoglobin synthesis, DNA synthesis, and mitochondrial respiration, eukaryotic cells require iron for survival. Excess iron can lead to oxidative damage, while iron deficiency reduces cell growth and causes cell death. Dysregulation of iron homeostasis in humans caused by iron deficiency or excess leads to anemia, diabetes, and neurodegenerative disorders. All organisms have thus developed mechanisms to sense, acquire, and store iron. We use  Iron is essential due to its presence in proteins involved in DNA synthesis, mitochondrial respiration and oxygen transport. Regulation of cellular iron content is crucial: excess cellular iron catalyzes the generation of reactive oxygen species that damage DNA and proteins, while cellular iron deficiency causes cell cycle arrest and cell death. Dysregulation of iron homeostasis caused by iron deficiency or iron excess leads to hematological, neurodegenerative and metabolic diseases in humans. Iron must therefore be maintained within a narrow range to avoid the adverse consequences of iron depletion or excess.3+ is reduced by membrane bound ferric reductases  before transport across the enterocyte apical membrane by divalent metal transporter-1  3+ by membrane and soluble multicopper oxidases where it is incorporated into transferrin for delivery to tissues. When body iron stores are high, cytosolic iron is not exported into blood, and is instead sequestered into ferritin Maintaining iron content within this physiological range requires precise mechanisms for regulating its uptake, storage and export  mediated expression of DMT1 and DCYTB Caenorhabditis elegans. The HIF-1 pathway in C. elegans consists of hif-1, aha-1, vhl-1 and egl-9, which are orthologous to genes encoding mammalian HIF-1α, HIF-1β, VHL and PHD C. elegans express a single hif gene, which encodes a protein homologous to vertebrate HIF-1α and HIF-2α C. elegans HIF-1 regulates target genes involved in metabolism, extracellular remodeling hif-1 mutant animals display increased embryonic and larval lethality in oxygen concentrations less than 1%, demonstrating the importance of HIF-1 for survival during hypoxia Oxygen and iron homeostasis pathways are conserved in C. elegans express genes homologous to ferritin (ftn-1 and ftn-2), DMT1  and ferroportin . Vertebrate ferritin is a mixture of 24 light-(L) and heavy-(H) subunits that form a shell that can accommodate up to 4500 iron atoms. The H-subunits exhibit ferroxidase activity and facilitate the oxidation of iron, whereas the L-subunits function with the H-subunits in iron nucleation C. elegans FTN-1 and FTN-2 display greater homology to the human H-subunit (55% and 60%) than to the L-subunit (46% and 50%), and notably both proteins contain ferroxidase active-site residues. ftn-1 and ftn-2 genes are transcriptionally repressed during iron deficiency, which is dependent on an iron-dependent enhancer (IDE) located in the ftn-1 and ftn-2 promoters ftn-1 and ftn-2 transcription, but the mechanism regulating iron-dependent transcriptional repression is unknown. Unlike ftn-1 and ftn-2, vertebrate ferritin-H and -L subunit mRNAs are translationally repressed by iron-regulatory proteins 1 and 2 (IRP1 and IRP2) during iron deficiency  ftn-1 or ftn-2 promoter sequences, including the IDE, fused to the initiator ATG of nuclear-localized GFP-histone ftn-1::GFP-his and ftn-2::GFP-his in worms fed control (empty vector L4400) RNAi by 60% and 80%, respectively, compared to worms grown on NGM  and vhl-1(ok161). vhl-1(ok161) mutant animals lack VHL required for HIF-1 ubiquitination and proteasomal degradation, leading to constitutive expression of HIF-1 hif-1(ia04) mutant animals and increased HIF-1 levels in vhl-1(ok161) mutant and in N2 wildtype animals cultured in NGM-BP  mutant animals, and notably hif-1(ia04) animals cultured in NGM express higher amounts of ftn-1 mRNA compared to N2 wildtype animals (vhl-1(ok161) mutant animals, ftn-1 and ftn-2 mRNAs are reduced to levels found in N2 wildtype animals cultured in NGM-BP. Taken together, these data show that ftn-1 and ftn-2 are transcriptionally inhibited by HIF-1 during iron deficiency.We next determined whether endogenous n NGM-BP . BP redu animals . In vhl-Electrophoretic mobility shift assays were used to determine whether HIF-1 binds to the HREs in the IDE. Radiolabeled wildtype IDE or an IDE containing mutations in the three HRE sites (HRE3m) was incubated with reticulocyte lysate-synthesized HIF-1 and AHA-1 . Complexin vivo, ChIP was performed on chromatin isolated from vhl-1(ok161) and hif-1(ia04) mutant animals. The binding of HIF-1 to the IDE or to the ftn-1 coding region used as a negative control was determined by ChIP using HIF-1 antibody. IDE DNA was enriched 4-fold in vhl-1(ok161) immunoprecipitates as compared to hif-1(ia04) immunoprecipitates  mutant animals cultured in NGM or NGM-BP. BP increases smf-3 mRNA levels 2-fold as compared to untreated N2 wildtype animals, but has no effect on smf-1 or smf-2 mRNA levels (hif-1(ia04) mutant animals, smf-3 mRNA levels are reduced by 50% as compared to N2 wildtype animals, and are not increased by BP  or NGM-BP. FAC reduces GFP expression, whereas BP increases GFP expression as compared to untreated worms ::GFP-his expression (hif-1 RNAi completely blocks BP-induced increased GFP expression (smf-3(-1500)::GFP-his reporter containing 250 nt of upstream sequences containing the IDE is increased by BP in control RNAi fed worms and is reduced by BP in hif-1 RNAi fed worms  mutant animals. We found that ftn-1 mRNA levels, which are positively correlated with cellular iron levels (smf-3(ok1035) mutants as compared to N2 wildtype animals, but not in smf-1(ok1748) or smf-2(gk133) mutant animals (smf-3(ok1035) mutant animals is 45% of N2 wildtype animals consistent with reduced ftn-1 mRNA levels in these animals (smf-1(ok1748) and smf-2(gk133) mutant animals is not significantly different as compared to N2 wildtype animals. The total Mn content in smf-3(ok1035) mutant animals is 60% of N2 wildtype controls (smf-1(ok1748) and smf-2(gk133) strains tended to be lower as compared to N2 wildtype controls, but did not reach significance. These results are consistent with a role for SMF-3 in intestinal iron transport.The localization of SMF-3 to the apical membrane of intestinal cells n levels , are red animals . Quantif animals . The totcontrols  in agreehif-1(ia04) mutant animals have no overt phenotype in normoxia, but display reduced viability in oxygen concentrations less than 1% hif-1(ia04) mutant animals develop normally under normoxic conditions, but are developmentally delayed when cultured under iron deficient normoxic conditions (NGM plus 20 µM BP) as compared to N2 wildtype animals (smf-3(ok1035) mutant animals and smf-3 expression is reduced in hif-1(ia04) animals, these data suggest that total iron content might also be reduced in hif-1(ia04) animals. ICP analyses show that the total iron content in hif-1(ia04) mutant animals is 60% of N2 wildtype animals (hif-1(ia04) mutant animals. animals . As tota animals . The tothif-1(ia04) mutant animals cultured in BP can be rescued by reducing FTN-1 and FTN-2 expression, which would lead to an increase in the cellular labile iron pool. This pool contains chelatable redox-active iron that constitutes less than 5% of total cellular iron ftn-1 and ftn-2 by RNAi in N2 wildtype and in hif-1(ia04) mutant animals cultured on NGM or NGM plus 20 µM BP, and the number of worms reaching L4 stage was measured. ftn-1/ftn-2 RNAi increases the number of hif-1(ia04) mutant animals reaching L4 stage from 28% in untreated animals to 78% in ftn-1/ftn-2 RNAi-fed animals (hif-1(ia04) mutant animals during iron deficiency can be partially rescued by reducing FTN-1 and FTN-2 levels.We next questioned whether the developmental delay observed in  animals . These dftn-1 and ftn-2 transcription in intestine is activated by iron and inhibited by iron deficiency ftn-1 and ftn-2 transcription during iron sufficiency, but how transcription is repressed by iron deficiency was not understood. Here, we identify HIF-1 as a negative regulator of ftn-1 and ftn-2 transcription during iron deficiency. We also show that smf-3 is regulated by HIF-1 during iron deficiency, but unlike ftn-1 and ftn-2, HIF-1 activates smf-3 transcription. The activation of smf-3 and inhibition of ftn-1 and ftn-2 by HIF-1 provides a mechanism to increase iron uptake and decrease iron storage during iron deficiency.Previous studies showed that ftn-1 and ftn-2 in C. elegans, vertebrate ferritin H- and -L subunit mRNAs are translationally repressed during iron deficiency by the binding of IRP1 and IRP2 to an iron-responsive element (IRE) in ferritin mRNAs C. elegans express an IRP1 homolog (ACO-1), it lacks RNA-binding activity and functions solely as a cytosolic aconitase ftn-1and ftn-2 is essential as depletion of FTN-1 and FTN-2 rescues the growth delay observed in hif-1 null worms grown under iron limiting conditions. Recent studies show that constitutive expression of ferritin-H and -L subunits in mice with intestinal specific deletion of IRP1 and IRP2 have reduced survival rates In mice, increased iron uptake by DMT1 during iron deficiency is mediated by HIF-2α ftn-1and ftn-2 genes by HIF-1 during iron deficiency was unexpected. HIF-1 is a potent transcriptional activator of hundreds of HRE target genes The transcriptional inhibition of ftn-1, ftn-2 and smf-3 IDEs differ in number, spacing and orientation of the GATA and HIF-1 binding sites. It is likely that architecture of the IDE dictates physical interactions of ELT-2, HIF-1, coactivators and other transcription factors to activate or repress transcription. Further studies are needed to determine how the structure of the IDE affects HIF-1 transcriptional responses.One question is how HIF-1 mediates transcriptional activation and inhibition through the IDE. The ftn-1, ftn-2 and smf-3 are essential for intestinal expression because mutations of these sites or depletion of ELT-2 reduces intestinal expression of ftn-1 and smf-3 GFP transcriptional reporters HREs can be flanked by transcription factor binding sites, and it is the cooperation of HIF with these transcription factors that enhance transcription or direct cell specific expression ftn-1 and ftn-2. Both GATA binding sites are required for ftn-1 expression as deletion of either site abolished expression of an ftn-1 GFP transcriptional reporter ftn-1 IDE abolished GFP reporter expression, suggesting that a transcriptional activator may bind to the HREs enhancing ELT-2 function smf-3 transcription is reduced during iron sufficiency due to decreased HIF-1α levels. Our data also show that a small amount of HIF-1 is expressed during normal growth conditions that can interact with HREs in ftn-1 and smf-3. During iron deficiency, HIF-1 is stabilized and transported to the nucleus where it dimerizes with AHA-1. We propose that HIF-1/AHA-1 competes with a transcriptional activator for binding to the ftn-1 and ftn-2 HREs, inhibiting ftn-1 and ftn-2 transcription. HIF-1/AHA-1 binds to the smf-3 HREs, recruits coactivators and cooperates with ELT-2 to activate transcription.Based on our findings, a model for HIF-1 regulation of intestinal iron homeostasis is proposed . During 2+ homeostasis and sensitivity smf-3(ok1035) mutant animals indicate a role for SMF-3 is intestinal iron transport in C. elegans. The regulation of iron homeostasis by HIF-1 provides a mechanism to ensure that C. elegans maintain sufficient iron stores for growth and survival when iron is limiting.SMF-1, SMF-2 and SMF-3 have been characterized with regard to their role in MnEscherichia coli OP50 at 20–22°C. For iron chelation experiments, synchronized larvae were grown for 24 h on NGM plates then transferred to NGM plates supplemented with 100 uM 2,2′-dipyridyl (BP) for 18 h unless indicated. For iron experiments, larvae were grown for 18 h on NGM plates supplemented with 6.6 mg/ml ferric ammonium citrate (FAC). The pH of FAC-supplemented NGM agar was adjusted to pH 7.0.Strains were cultivated on nematode growth medium (NGM) agar plates seeded with C. elegans Genetics Center are: wild-type Bristol N2, vhl-1 (ok161) hif-1 (ia04) smf-1(ok1748), VC171 smf-2(gk133) and RB1074 smf-3(ok1035). XA6900 pha-1(e2123ts) III; qaEx1  and XA6901 lin-15(n765ts) X; qaEx2  were previously described . Strains generated in this study are: XA6904 pha-1 (e2123ts) III; qaEx04  and XA6905 pha-1(e2123ts) III; qaEx05 .The strains provided by smf-3(-1500)::GFP-his and smf-3(-250)::GFP-his were generated by PCR amplification of sequences 1500 nt or 250 nt, respectively, upstream from the initiation ATG of smf-3 (Y69A2AR.4) using primers containing SalI and NheI restriction sites on the 5′ and 3′ termini, respectively. The PCR products were cloned into TOPO Zeroblunt (Invitrogen) followed by digestion and insertion into the SalI and NheI sites of pAP.10. Transgenic strains were made according to standard microinjection procedures. Plasmids B696 (smf-3(-1500)::GFP-his) and B698 (smf-3(-250)::GFP-his) were each co-injected (20 ng/ul) with selection plasmid pBX-1(100 ng/ul). Transgenic worms were recovered after growth at 20–22°C.ftn-1 (C54F6.14) and ftn-2 (D1037.3) were from the ORFeome-based RNAi library hif-1 (F38A6.3) was from the Ahringer feeding library ftn-1and ftn-2, hif-1 or L4400.RNAi clones against hif-1(ia04) animals containing gravid adults were transferred to RNAi plates seeded with bacteria expressing ftn-1 and ftn-2 dsRNA or empty vector control (L4400) for 24 h at 20–22°C. Synchronized larval stage (L1) worms were obtained by treating RNAi fed gravid adults with alkaline hypochlorite and allowing eggs to hatch overnight in sterile S-basal media. L1 larvae were spotted on ftn-1 and ftn-2 or L4400 RNAi plates supplemented with or without 20 µM BP and incubated at 20–22°C for 48 h prior to scoring for larval stage using a Leica MZ9.5 stereomicroscope.Mixed-stage populations of N2 wildtype and ftn-1::GFP-his, ftn-2::GFP-his, smf-3(-1500)::GFP-his and smf-3(-250)::GFP-his reporter lines were synchronized by treating gravid adults with alkaline hypochlorite followed by hatching eggs in S-basal medium  overnight. Synchronized L1 worms were grown on control or hif-1 RNAi plates for 32 h. Worms were then rinsed from the plates and washed in M9 buffer  and spotted onto fresh 10 cm control or hif-1 RNAi plates supplemented with 100 µM BP and grown for 18 h. Worms were rinsed from plates and washed with MT buffer (M9 buffer containing 0.1% Triton X-100) by repeated rounds of centrifugation until free of debris. Worms were analyzed using the COPAS Biosort  as described 2 or 100 uM BP plus 100 uM FAC. Worms were grown for an additional 18 h prior to data acquisition as performed above.Images of GFP expression were captured using an Axio Imager  outfitted with the Zeiss filter set 38HE  and an AxioCam HRm camera  using AxioVision software. Following acquisition, images were rotated, cropped and sized using Adobe Photoshop.hif-1(ia04) and vhl-1(ok161) animals were transferred from NGM plates to fresh NGM and 100 uM BP supplemented plates for 16 hr prior to harvesting. Worms were collected, washed with ddH2O and resuspended in 200 µl lysis buffer . Worms were sonicated twice using a Misonix 3000 , centrifuged and the protein concentration of the clarified lysate was determined using Coomassie Plus protein assay reagent (Thermo). Samples (20 ug) were fractionated on a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and western blotting was carried out using rabbit CeHIF-1 antibody at  Mixed-stage populations of N2, Synchronized L1 larvae were grown on NGM plates seeded with OP50 for 48 h prior to harvest. For iron chelation experiments, synchronized worms were transferred to new NGM plates or NGM plates supplemented with 100 uM BP after 32 h and harvested at 48 h. At harvest, worms were washed from plates and cleaned by repeated rounds of centrifugation and resuspension in ddH2O. Pelleted worms were resuspended in TRIZOL (Invitrogen) and total RNA was purified according to manufacturer's protocol. cDNA was synthesized using 1 ug of purified total RNA using SuperscriptIII Supermix for qRT-PCR (Invitrogen). Semiquantitative RT-PCR (sqRT-PCR) was performed using Recombinant Taq Polymerase (Invitrogen). PCR products were run on ethidium bromide stained 1.2% agarose gels. Images were captured on a FluorChem IS-8900 (Alpha Innotech) and analyzed using ImageQuaNT (Molecular Dynamics) and ImageJ. Primer sequences are shown in vhl-1 (ok161) and hif-1 (ia04) worms were grown on two 150 mm NGM plates and harvested. Worm pellets were homogenized in crosslinking buffer  and incubated for 20 min at room temperature. The reactions were quenched with glycine for 20 min and snap frozen. The frozen pellets were resuspended in HLB buffer  containing protease inhibitor cocktail , sonicated and the supernatants were precleared using 30 ul salmon sperm DNA/protein A agarose beads. After centrifugation, 10% of each supernatant was kept as input, while the remaining supernatants were incubated with CeHIF antibody (4 ul) overnight at 4°C. DNA-protein antibody complexes were incubated with Protein A agarose (30 ul slurry) for 2 h at 4°C, and the beads were then resuspended in proteinase K buffer (20 mg/ml proteinase K) and incubated at 45°C for 2 h. The samples were further incubated at 65°C overnight to reverse crosslinks. DNA was purified using a QIAquick PCR purification kit (Qiagen) according to manufacturer's protocol. Quantitative (q) PCR was then performed using Sybr Green mix (Invitrogen) run on an ABI 7000 Sequence Detection System and additionally visualized by 2% ethidium bromide stained agarose gels. Primers flanking the ftn-1 IDE and control primers within the ftn-1 coding region are shown in ChIP assays were performed as previously described CACGTAGC>ACATGCTA) were excised from TopoZero blunt (Invitrogen) with EcoRI and gel purified. Wildtype IDE was radiolabeled with 50 µCi of 32P[α-dATP] using Klenow DNA polymerase. HIF-1 and AHA-1 were synthesized in TNT SP6 Quick Coupled Transcription/Translation system (Promega) using hif-1 (pSP64-HIF-1) and aha-1 (pJ343) hif-1 cDNA from pR33 2, 1 mM DTT) containing 32P-labeled wildtype or HRE3m probes (0.4–1 ng), poly dI-dC (80 ng) and HIF-1 and/or AHA-1 (1–4 µl) at room temperature for 30 min. CeHIF-1 antibody (2 ul) was added during the last 5 min of the binding reactions. For competition experiments, unlabeled IDE or HRE3m DNA (10–100× molar amounts) was added to the reactions 5 min before addition of 32P-labeled probes. Samples were fractionated on a 5% non-denaturing polyacrylamide gel (37.5∶1 acrylamide∶bis).Wildtype IDE and IDE containing mutations in the three HRE binding sites (HRE3m)  and heated to 95°C for 2 min in a heating block. Solubilized samples were diluted to a final nitric acid concentration of 10% with ddH2O and measured on an Optima 3000 XL ICP-OES (Perkin Elmer). Serial dilutions of commercially available mixed metal standards were used to calibrate the instrument. Results were normalized to the simultaneously acquired signal for sulfur for each sample p<0.05.Data are presented as the means ± SEM. Two-tailed unpaired Student's t test were used for statistical analysis. Data are considered statistically significant at Figure S1ftn-1::GFP-his and smf-3(-1500)::GFP-his expression are regulated by iron, but not by MnCl2. MnCl2 has been shown to regulate transcriptional expression of smf-32 regulates expression of smf-3(-1500)::GFP-his, L1 stage worms were cultured on NGM, NGM-100 uM BP, NGM-100 uM BP plus 100 uM FAC and NGM-100 uM BP plus 100 µM MnCl2 for 18 hr. Worms were harvested and GFP expression was quantified by the COPAS Biosort. Dot plots of sorted worms are shown where each dot represents one worm. Mean GFP fluorescence (± SEM) is expressed as a percentage of worms grown on NGM (n = 1000 worms/treatment), *p<0.0001. (A) BP increases smf-3(-1500)::GFP-his expression, which is blocked by addition of equimolar amount of FAC plus BP. By contrast, GFP expression is not reduced when MnCl2 is present with BP. (B) BP reduces ftn-1::GFP-his expression and is reversed when FAC is present with BP. Addition of MnCl2 with BP did not increase GFP expression.(EPS)Click here for additional data file.Figure S2smf-3(-250)::GFP-his expression is regulated by HIF-1. GFP-his reporters were constructed containing 250 nt of smf-3 promoter sequences 5′ to the initiator ATG (smf-3 IDE. To determine whether smf-3(-250)::GFP-his was regulated by HIF-1, L1 stage worms carrying smf-3(-250)::GFP-his were spotted on hif-1 or control (empty vector L4400) RNAi plates supplemented with or without 100 uM BP. GFP fluorescence was quantified by the COPAS Biosort after 18 h. Dot plots of sorted worms are shown where each dot represents one worm. Mean GFP fluorescence (± SEM) is reported as a percentage of control RNAi fed worms on NGM (n = 650 worms/treatment), *p<0.0001. BP increases GFP expression in control RNAi fed worms after growth on NGM-BP as compared to NGM-control fed RNAi. By contrast, BP-induced increased GFP expression is inhibited in worms fed hif-1 RNAi.ator ATG . This re(EPS)Click here for additional data file.Table S1Sequences of primers used in this study.(DOC)Click here for additional data file.


Flaviviridae viruses such as hepatitis C virus (HCV), whose assembly and pathogenesis depend on interaction with lipid droplets (LDs). One such master regulator of cholesterol metabolic pathways is the host subtilisin/kexin-isozyme-1 (SKI-1) – or site-1 protease (S1P). SKI-1/S1P plays a critical role in the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which control expression of the key enzymes of cholesterol and fatty-acid biosynthesis. Here we report the development of a SKI-1/S1P-specific protein-based inhibitor and its application to blocking the SREBP signaling cascade. We demonstrate that SKI-1/S1P inhibition effectively blocks HCV from establishing infection in hepatoma cells. The inhibitory mechanism is associated with a dramatic reduction in the abundance of neutral lipids, LDs, and the LD marker: adipose differentiation-related protein (ADRP)/perilipin 2. Reduction of LD formation inhibits virus assembly from infected cells. Importantly, we confirm that SKI-1/S1P is a key host factor for HCV infection by using a specific active, site-directed, small-molecule inhibitor of SKI-1/S1P: PF-429242. Our studies identify SKI-1/S1P as both a novel regulator of the HCV lifecycle and as a potential host-directed therapeutic target against HCV infection and liver steatosis. With identification of an increasing number of human viruses that use host LDs for infection, our results suggest that SKI-1/S1P inhibitors may allow development of novel broad-spectrum biopharmaceuticals that could lead to novel indirect-acting antiviral options with the current standard of care.HCV infection is a major risk factor for liver cancer and liver transplantation worldwide. Overstimulation of host lipid metabolism in the liver by HCV-encoded proteins during viral infection creates a favorable environment for virus propagation and pathogenesis. In this study, we hypothesize that targeting cellular enzymes acting as master regulators of lipid homeostasis could represent a powerful approach to developing a novel class of broad-spectrum antivirals against infection associated with human  Chronic hepatitis C virus (HCV) infection is one of the leading causes of liver cancer and liver transplantation worldwide. No vaccine is available for preventing the spread of HCV, and the current therapeutic regimen is only moderately effective and causes serious side effects. New antiviral agents are required to treat HCV infection, but the high mutation rate of HCV hinders the effectiveness of virus-specific inhibitors. Targeting the host enzymes required for HCV to replicate offers a promising new direction for antiviral therapy. During infection, HCV promotes excessive fat accumulation in the liver, which benefits the virus as this promotes formation of lipid droplets, a cellular organelle essential for assembly of new HCV infectious viral particles. Here, we report the development of a specific inhibitor targeting SKI-1/S1P, a host enzyme required for lipid production in human cells. We show that inhibiting SKI-1/S1P activity in human liver cells effectively blocks lipid droplet formation and HCV infection. Many prevalent human viruses, such as dengue, rotavirus, and hepatitis B virus, hijack host lipid metabolic pathways similar to those targeted by HCV to complete their lifecycle. Thus, we propose that cellular SKI-1/S1P is a potential target for developing desperately needed novel broad-spectrum antiviral drugs. Hijacking of host lipids and their biosynthetic pathways is a common strategy for microbial infection. Human enveloped viruses including hepatitis C virus (HCV) and human immunodeficiency virus (HIV)-1 use cholesterol-rich lipid rafts for entry Flaviviridae family and an enveloped virus, is encoded by a single-stranded positive-sense RNA genome HCV is a globally important human pathogen afflicting more than 170 million people worldwide Several host metabolic pathways tightly control cellular lipid synthesis. Targeted disruption of these pathways Overstimulation of host lipid metabolism by HCV during infection is achieved through a variety of molecular mechanisms , also widely known as subtilisin/kexin-isozyme-1 (SKI-1), is a membrane-bound subtilisin-related serine endoprotease that belongs to a group of nine mammalian proprotein convertases (PCs) in family S08 serine protease inhibitors (serpins) to develop effective and selective PC inhibitors Given that SKI-1/S1P-dependent proteolytic cleavage of SREBPs is a master molecular switch for controlling host cell cholesterol homeostasis Drosophila serpin, Spn4A, as a prototype macromolecular inhibitor scaffold. The identification of Spn4A by our group as the most potent natural serpin inhibitor of the human PC furin  resulted in the development of Spn4A-RRLL, a novel, selective, and effective serpin-based inhibitor of SKI-1/S1P. We demonstrated the anti-proteolytic and anti-HCV activities of our new recombinant adenovirus-expressing Spn4A-RRLL “secreted” (s) variant directed at the secretory pathway SKI-1/S1P. Expression of Spn4A.RRLL(s) in Huh-7.5.1 cells results in a strong inhibition of the SKI-1/S1P-mediated activation of SREBP-1 and down-regulation of SREBP target gene products. As hypothesized, inhibiting SKI-1/S1P activity robustly blocked HCV infection of Huh-7.5.1 cells in a dose-dependent manner. We found that specific inhibition of SKI-1/S1P activity by Spn4A.RRLL(s) dramatically reduced the abundance of LDs in hepatoma cells. Use of the specific active site-directed small-molecule inhibitor of SKI-1/S1P, PF-429242, confirmed the results of our studies with the protein-based therapeutic Spn4A.RRLL(s), with a robust inhibition of HCV infection.We selected a Ki,13 pM  providesThe results of our studies contribute to our understanding of the HCV lifecycle and HCV-associated steatogenesis and to efforts in developing novel host-directed antiviral therapeutic agents against HCV. In addition, with the finding that an increasing number of human enveloped viruses employ host LDs for infection Drosophila melanogaster serpin scaffold to engineer a novel protein-based inhibitor directed at the PC SKI-1/S1P. First, we cloned our pre-His/FLAG-tagged Spn4A construct P4-ArgP3-LysP2-ArgP1↓ were substituted to generate ArgP4-ArgP3-LeuP2-LeuP1↓, thereby mimicking the Lassa virus glycoprotein precursor GP-C cleavage site Our previous studies have demonstrated that Spn4A architecture can inhibit two evolutionary divergent members of the PC family (furin and PC2) in cellulo, recombinant adenoviruses (Ad) expressing the Spn4A constructs Ad-Spn4A.RRLL(r) and Ad-Spn4A.RRLL(s) were produced as described previously in celluloTo test the serpin-like properties and antiviral activities of our Spn4A variants The level of expression of Spn4A variants was first optimized by infecting human hepatoma Huh-7.5.1 cells (highly permissive for HCV JFH-1 infection) with different multiplicity of infection (moi) of recombinant adenovirus expressing intracellularly retained Spn4A.RRLL(r). Cellomics HCS analysis revealed that over 90% of these Huh-7.5.1 cells expressed Spn4A.RRLL(r) at a moi of 50 . CellomiThe serpin-secretion profile in Huh-7.5.1 cells was examined by Western blotting of cell lysates and extracellular media. As expected, a prominent 45-kDa band was detected in Ad-Spn4A.RRLL(r)-infected and Ad-Spn4A.RRLL(s)-infected cell lysates using anti-FLAG antibody , lane 2.A stable, acyl-enzyme complex is formed between a protease and a functional inhibitory serpin following RCL cleavage. This allows for detection of the high molecular weight, heat-stable, and SDS-stable enzyme-inhibitor (EI) complex by standard SDS-PAGE and Western blot in vitro and in vivo for its anti-lipidemic properties including efficient inhibition of nuclear SREBP accumulation. As previously described To confirm that expression of Spn4A.RRLL(s) in Huh-7.5.1 cells inhibits endogenous SKI-1/S1P-mediated cleavage of SREBP molecules, we examined nuclear SREBP-1 protein levels in cells infected with adenovirus-expressed Spn4A variants. As a positive control, we also treated cells with the selective, reversible, and competitive small-molecule inhibitor of SKI-1/S1P: PF-429242 Next, to determine whether the Spn4A.RRLL(s)-mediated reduction in nuclear SREBPs was associated with a concomitant decrease in the protein levels in SREBP-target genes, we examined the fate of three SREBP-dependent gene products, SREBP-2, LDLR, and PCSK9. We investigated these host cell proteins because of their proposed contribution to HCV entry (LDLR and PCSK9) and propagation (SREBP-2) A critical function of the SREBP pathway and the genes that it regulates is to control lipid homeostasis LDs are dynamic intracellular lipid storage compartments made up of triglyceride and cholesterol esters, surrounded by a phospholipid membrane and associated with specific marker proteins including adipose differentiation-related protein (ADRP), also known as perlipin 2 Since the SREBP signaling pathway is induced by HCV-encoded proteins during infection The anti-HCV properties of Spn4A.RRLL(s) were confirmed and extended by further virological studies on cells pre-treated with our serpin-based inhibitors for 48 hours prior to 72 hours of HCV infection . First, To determine if a decrease in intracellular HCV RNA in Spn4A.RRLL(s)-treated cells  is due tThe impact of Spn4A.RRLL(s) treatment on HCV replication was further investigated using HCV subgenomic replicons 50 6.4±1.3 µM)  . A near ±1.3 µM) .50 concentration of 1.03±0.3 µM. Interestingly, a single prophylactic pre-treatment of Huh-7.5.1 cells with 40 µM PF-429242 was sufficient to maintain a 10-fold decrease of intracellular HCV core production 72 hours post-infection   . These r50 of 6.0±3.5 µM)  . These rBecause PF-429242 and Spn4A.RRLL(s) decrease the abundance of LD components, we hypothesized that HCV assembly, rather than HCV replication, can also blocked by SKI-1/S1P inhibition. To support this hypothesis, Huh.8 and Huh.2 cells were treated with 10 µM PF-429242 for 72 hours. No significant changes in HCV subgenomic RNA levels in either cell line  treated It is now well established that hijacking of host-cell biosynthetic pathways by human enveloped viruses is a shared molecular event essential for the viral lifecycle To investigate the biological consequences of inhibiting SKI-1/S1P endoprotease activity on biochemical pathways of lipid homeostasis hijacked by HCV, we first employed a protein-based inhibitor strategy. We engineered and developed a novel, recombinant adenovirus expressing an effective and specific secretory pathway, SKI-1/S1P-directed serpin, Spn4A.RRLL(s). We showed that Spn4A.RRLL(s) forms a kinetically trapped heat- and SDS-stable complex with SKI-1/S1P molecules characteristic of other physiological serpin-protease pairs. We then demonstrated that it blocks the SKI-1/S1P-mediated cleavage of endogenous SREBP-1 and the expression of SREBP downstream effector gene products . The SREBP target gene products identified in Spn4A.RRLL(s)-treated cells are involved in lipid homeostasis and reported to participate in HCV-host interactions. SREBP-2, along with the other SREBP isoforms SREBP-1a and -1c, are activated by HCV-encoded proteins or during HCV infection In addition to blocking SREBP-mediated up-regulation of hepatic genes during HCV infection, we hypothesized that blocking SKI-1/S1P endoprotease activity would also compromise cellular lipid storage. Analysis of intracellular lipid content and cytoplasmic LD abundance in Spn4A.RRLL(s)-expressing cells confirmed our hypothesis. In addition, we observed a decrease in ADRP/perilipin 2 abundance in Spn4A.RRLL(s)-expressing cells compared to control-treated cells. The physiological role of ADRP has yet to be fully established but it plays an important role in LD structure and formation. Interestingly, ADRP is degraded through the proteasome-dependent pathway during regression of lipid-storing cells, indicating that when ADRP is not bound to LDs, such as in SKI-1/S1P-inhibited cells, it will be susceptible to rapid proteasomal degradation As hypothesized, inhibiting SKI-1/S1P-mediated SREBP endoproteolytic cleavage events using Spn4A.RRLL(s) resulted in a dose-dependent decrease in HCV infection. We demonstrated that HCV core expression and HCV RNA levels are reduced in Spn4A.RRLL(s)-expressing cells following HCV infection, leading to a robust reduction in extracellular infectious HCV particle release. Western blotting also confirmed that HCV core protein post-translational processing was unaltered by serpin expression because the size of the protein was unaltered. Supplementing Spn4A.RRLL(s)-expressing cells with compounds such as mevalonate, oleate, and cholesterol resulted in an incomplete rescue of HCV infection, suggesting that the antiviral activity of our protein-based inhibitor cannot be explained solely by the decreased availability of lipids in these cells. This also supports our hypothesis that reduced LDLR levels may compromise HCV entry into Spn4A.RRLL(s)-treated hepatocytes. LDLR, a well-established SREBP-regulated gene, has been repeatedly shown to support HCV entry into hepatocytes To gain further insight into the different stages of the viral lifecycle targeted by our SKI-1/S1P inhibitor, we used an active-site-directed small-molecule inhibitor of SKI-1/S1P, PF-429242. This pharmacologic inhibitor of SKI-1/S1P has recently been characterized for its effectiveness in blocking cleavage of SREBP-2, for blocking expression of SREBP-activated genes, and also for inhibiting arenavirus glycoprotein processing We first confirmed a reduction in abundance of neutral lipids and ADRP/perilipin 2 expression in PF-429242-treated cells. Then, we confirmed that inhibition of SKI-1/S1P using PF-429242 blocks HCV infection and extracellular infectious virus production in a dose-dependent manner. The anti-HCV activity of PF-429242 is very robust and particularly striking. A single 24-hour pre-treatment with the compound was sufficient to block HCV infection, and the antiviral effect of PF-429242 was still apparent 72 hours post-treatment. Importantly, pharmacological treatment of already infected HCV cells resulted in a 90% reduction of HCV virus production. Similar to Spn4A.RRLL(s) treatment, PF-429242 did not reduce HCV RNA levels in the two stable subgenomic replicon cell lines that were examined. This, in combination with the observed reduction in abundance of a central organelle (LD) involved in HCV assembly, and the reduction in HCV particle secretion in cells treated 24 hours after HCV inoculation, supports PF-429242 as an inhibitor of late stages of the HCV lifecycle, i.e., during assembly or egress. Taken all together, these results indicate that inhibiting SKI-1/S1P can interrupt the HCV lifecycle at multiple stages of viral infection both preventing naïve cells from becoming infected and preventing virus release from already infected cell populations. Thus, developing more effective active-site-directed SKI-1/S1P small-molecule inhibitors (< nM range) with better pharmacokinetic properties In conclusion, pharmacologic inhibition of SKI-1/S1P offers a very promising avenue for the development of novel anti-HCV therapeutics . On one Flaviviridae viruses such as dengue virus, whose replication and pathogenesis also depend on the interaction with lipid droplets Finally, our results reveal that targeting host LD biogenesis by inhibiting SKI-1/S1P endoproteolytic activity may have far-reaching applications in the therapeutic treatment of other important human Human hepatoma Huh-7.5.1 cells were kindly provided by Dr. Francis Chisari  A mouse anti-core antibody  was employed for detecting HCV infection. Antibodies used to detect cellular proteins included rabbit anti-human proprotein convertase subtilisin/kexin type 9 (PCSK9) , rabbit anti-LDLR , mouse anti-SREBP-2 , rabbit anti-adipose differentiation-related protein (ADRP) , mouse anti-ADRP , rabbit anti-β-tubulin , mouse anti-β-tubulin , mouse anti-GM130 , rabbit anti-SREBP-1, clone H160 , and mouse anti-fibrillarin . Tagged proteins were detected using mouse anti-FLAG M2 , rabbit anti-FLAG , and mouse anti-His antibodies . Secondary antibodies used for immunofluorescence were Alexa Fluor-488-conjugated or Alexa Fluor-568-conjugated donkey anti-mouse or donkey anti-rabbit antibodies . Secondary antibodies used for Western blot were IRDye 680-conjugated (red bands) or 800-conjugated (green bands) donkey anti-mouse or goat anti-rabbit antibodies . Hoechst 33258 (10 µg/ml) and BODIPY  493/503  were used for detection of nuclei and LDs A plasmid containing the cDNA of an HCV consensus clone isolated from a Japanese patient with fulminant hepatitis (JFH-1) (GenBank accession number AB047639) Purified HCV RNA was used to transfect Huh-7.5.1 cells as a means of studying HCV infection independently of receptor-mediated entry. Five micrograms of purified RNA was incubated with 10 µl of lipofectamine 2000 (Invitrogen) in minimal essential media (MEM) for 30 minutes. The RNA-lipid complexes were added to cells in MEM for 16 hours; then cells were washed with phosphate-buffered saline (PBS) and complete media was added for the remainder of the experiment.4 Huh-7.5.1 cells were plated in each well of a 96-well plate and infected with 10-fold serial dilutions of HCV-infected cell media. At 72 hours post-infection, cells were fixed and probed as described in the ArrayScan Quantification methods section. An ArrayScan VTI High Content Screening (HCS) Reader (Thermo Scientific) was used to acquire images of the entire group of infected wells. Titers were determined by manually counting foci (fluorescence forming units (FFU)) in the lowest dilutions with positive signal.The amount of infectious HCV particles generated for viral stocks or in the described experiments was determined using a modified, previously described protocol 1-AT) followed by a 6-His tag (HHHHHH) and FLAG-tag (DYKDDDDK) sequence (HF) P4-Arg-Lys-ArgP1↓ (RRKR) in the reactive center loop (RCL). The ER “retained” (r) variant of the furin-directed serpin, designated Spn4A.RRKR(r) (P4-Arg-Leu-LeuP1↓ (RRLL) Spn4A  , presentDrosophila melanogaster Spn4B sequence (GeneBank Accession number gi|24586105|ref|NP_524955.2) exhibits 34% sequence homology with the human neuroserpin (hNS), for which a crystal structure is available The HHHHHHHHSGS) were expressed in Sf9 insect cells as previously described The first 997 amino acids of human SKI-1/S1P lacking the C-terminal transmembrane domain but containing a C-terminal 8-His-tag (PGDDDDKCultured cells were washed with ice-cold PBS and re-suspended in cold radioimmunoprecipitation assay (RIPA) buffer . Whole cell extracts were vortexed and then clarified by centrifugation at 12000 x g for 15 minutes. Soluble extracts mixed with 2 X sample buffer  were electrophoresed on 8–15% SDS polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked in Odyssey blocking buffer (LI-COR Biosciences) for one hour, and proteins of interest were detected by probing with the appropriate primary and secondary antibodies diluted in Odyssey blocking buffer containing 0.1% Tween 20. Protein bands were detected and quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences). All immunoblots were scanned at a wavelength of 700 nm for detecting IRDye 680 labeled antibodies and at a wavelength of 800 nm for IRDye 800CW conjugated antibodies 5 cells per well in a 6-well plate and infected after 24 hours with Ad-Spn4A.RRLL(s), Ad-Spn4A.RRLL(r), Ad-Spn4A.RRKR(s), or Ad-Empty at moi 50. After 72 hours, media and cell extracts  were mixed 1∶1 with enzyme reaction buffer , cells were plated  and infected as described in the methods below. Following infection, cells were fixed in 4% formaldehyde v/v diluted in PBS and blocked in PBS containing 3% BSA, 0.3% Triton X-100, and 10% FBS. Cells were first probed with HCV anti-core antibody (1∶500) in PBS containing 3% BSA and 0.3% Triton X-100 (Binding Buffer), then incubated with Alexa Fluor-568-conjugated donkey anti-mouse secondary antibody (1∶1000) and 10 µg/ml Hoechst dye. Cells were analyzed by a quantitative, high-throughput, fluorescence microscope system called the Cellomics ArrayScan VTI High Content Screening (HCS) Reader (Thermo Scientific) using the software Target Activation BioApplication (TABA). TABA was used to count the total number of cells (Hoechst-stained nuclei) and the percentage of those cells that were expressing HCV core .6 Huh-7.5.1 cells were plated into 100 mm plates. On Day 2, cells were infected with recombinant adenoviruses expressing Spn4A.RRLL(r) or Spn4A.RRLL(s), or with Ad- Empty (moi 50). On Day 3, separate cells were treated with the inhibitor PF-429242 or the control DMSO. On Day 5, all samples (except the untreated control) were treated for 1 hour with 25 µM α-N-acetyl-Leu-Leu-Nle-CHO ((2S)-2-[[(2S)-2-acetamido-4-methylpentanoyl]amino]-4-methyl-N-[(2S)-1-oxohexan-2-yl]pentanamide) (Sigma-Aldrich Corp.). Nuclear fractionation of cell lysates was performed at 4°C as described previously with modifications 2, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose) containing 1 X complete protease inhibitor cocktail (Roche). To shear DNA, the cells were passed 20 times through a 23-gauge needle. The lysate was centrifuged at 1100 x g for 7 minutes and the resulting supernatant was centrifuged again at 25000 x g for 60 minutes to obtain the membrane pellet. The pellet containing membrane-bound SREBP-1 was re-suspended in 75 µl of SDS-lysis buffer . The pellet from the 1100 x g spin was re-suspended in 100 µl buffer D  containing 1 X complete protease inhibitor cocktail. Nuclear pellets were rocked for 1 hour, after which the samples were centrifuged at 25000 x g for 60 minutes to obtain the clarified supernatant containing the nuclear fraction.On Day 1, 2.5×10After Huh-7.5.1 cells were seeded onto coverslips for 24 hours, they were infected with adenovirus (moi 50) for 72 hours. Cells were fixed in 4% v/v formaldehyde in PBS, then permeabilized and blocked in PBS containing 0.05% saponin (wash buffer) and 1% BSA (binding buffer). Blocking of cells stained with BODIPY 493/503 was done in the presence of 0.2 M glycine to reduce background fluorescence. Cells were probed with primary antibodies in binding buffer, then incubated with a secondary antibody, Hoechst dye (10.0 µg/ml), and BODIPY 493/503  diluted in PBS. Cells were mounted onto slides with an anti-fade solution and sealed with clear nail polish. The slides were then imaged using a Leica TCS SP5 confocal microscope  or an Olympus Fluoview FV1000 laser scanning confocal microscope  Huh-7.5.1 cells were infected with recombinant adenovirus at different moi in complete media with or without exogenous sterols . After 48 hours, the cells were infected with HCV moi 0.1 or transfected with purified HCV genomic RNA for 72 hours, and then cells were analyzed by Cellomics ArrayScan HCS, or total RNA was isolated from cell extracts using the RNeasy plus kit  including on-column DNase digestion. Media from treated and infected cells were harvested for HCV titer determination as described above. To examine the Spn4A.RRLL(s) mediated block in PCSK9, LDLR, and SREBP-2 expression, Huh-7.5.1 cells were grown in media supplemented with LPDS for 24 hours, infected with adenovirus variants, and harvested 72 hours later.5′-CATGGCGTTAGTATGAGTGTCG-3′) and reverse primer (5′-GGTTCCGCAGACCACTAT-3′) were used in combination with 200 nM of probe #75 from the Human Probe Library (Roche). HCV RNA levels were relatively quantified across samples and normalized to beta-actin RNA levels using 500 nM primers (forward: 5′- GCC CTG AGG CAC TCT TCC and reverse: 5′ GGA TGT CCA CGT CAC ACT TC-3′) and 250 nM probe (5′AC TCC ATG CCC AGG AAG GAA GGC-3′ with a 5′ Cy5 fluorophore and 3′ black hole quencher).Purified total RNA was reverse transcribed to cDNA using TaqMan reverse transcription reagents . Real-time quantitative PCR was carried out using Brilliant II Fast QPCR reagents  according to the manufacturer's instructions on an Mx3005P QPCR system (Stratagene). Online ProbeFinder software (Roche Applied Science) was used to find primers that would allow amplification of the HCV RNA 5′ end in combination with the Human Universal Probe Library from Roche (Roche Applied Science). For amplification of the HCV RNA 5′ region, 400 nM of both forward primer . This assay employs a tetrazolium compound , which is bio-reduced by cells into a colored formazan product that can be detected in tissue culture media at 490 nm wavelength PF-429242, an active site-directed small-molecule inhibitor of SKI-1/S1P 2O (for triglyceride assay). To extract triglycerides, samples were slowly heated to 90°C and brought to room temperature, twice. Total cholesterol, cholesterol esters , phospholipids, and triglycerides  were quantified using commercially available kits. Lipid levels were normalized to cellular protein content .To measure the level of intracellular lipids following 72-hour recombinant adenovirus expression or 48-hour PF-429242 treatment, cellular extracts were harvested in 1% triton-X 100 in PBS  or 5% triton-X 100 in H50 values. The reported EC50 values are the average of the values calculated from three independent experiments plus or minus the standard deviation. The student's t-test (unpaired) was used to calculate significance, which is represented in the figures by the following notation: * denotes p<0.05, ** denotes p<0.01, and *** denotes p<0.005.The sigmoidal fit function in Igor Pro software  was used for fitting HCV and PF-429242 inhibition curves and for determining ECFigure S1Optimization of adenovirus-expressed Spn4A.RRLL(r) expression in Huh-7.5.1 cells. Huh-7.5.1 cells were infected with moi 1, 12.5, 25, and 50 of the intracellularly retained serpin Ad-Spn4A.RRLL(r). Treated cells were fixed 48 hours post-infection and probed for serpin expression using mouse anti-FLAG antibody. Cell nuclei were stained with Hoechst dye to determine the total cell number. The percentage of Spn4A.RRLL(r)-expressing cells was quantified using Cellomics HCS. Results (mean ± SEM) from 2 independent experiments are shown.(TIF)Click here for additional data file.Figure S2Effect of Spn4A variant treatment and HCV infection on Huh-7.5.1 cell growth. Huh-7.5.1 cells were infected with various moi (1 – 50) of Ad-Empty, Ad-Spn4A.RRLL(r), or Ad-Spn4A.RRLL(s) for 48 hours in complete media. Treated cells were infected with HCV (moi 0.1) and fixed 72 hours post-infection. Fixed cells were probed with Hoechst dye to stain for cell nuclei, which were then quantified using Cellomics HCS to determine the relative number of cells in each well under the varying conditions. All values are expressed as relative cell number in serpin-treated cells compared to cells infected with Ad-Empty, which is set to 1. Results (mean ± SEM) from 3 independent experiments are shown.(TIF)Click here for additional data file.Figure S3Serpin-like properties of recombinant adenovirus-expressed Spn4A variants expressed in Huh-7.5.1 cells. Huh-7.5.1 cells were infected with recombinant adenovirus expressing the His- and FLAG-tagged Spn4A variants indicated or the Ad-Empty control for 72 hours. Media alone (upper panels) or cell extracts (lower panels) lysed in RIPA buffer were combined with recombinant His-tagged SKI-1/S1P (TIF)Click here for additional data file.Figure S4Time course analysis of LDLR expression in Spn4A.RRLL(s)-treated cells. Huh-7.5.1 cells were grown in complete media for 24 hours and then infected with Ad-Empty (control) or Ad-Spn4A.RRLL(s). Cell extracts were harvested for Western blot 24, 48, and 72 hours post-infection, and lysates were then subjected to Western blot. Anti-LDLR antibody was used to detect protein-expression levels in control- and Ad-Spn4A.RRLL(s)-treated cells, and β-tubulin was probed for normalizing LDLR expression. Values are plotted relative to LDLR expression in control (Ad-Empty)-treated cells, which was set to 1. The representative results of 2 independent experiments are shown.(TIF)Click here for additional data file.Figure S5Spn4A.RRLL(s) does not block HCV core production post-transfection of HCV RNA. Huh-7.5.1 cells were infected with Ad-Empty (control), Ad-Spn4A.RRLL(r), or Ad-Spn4A.RRLL(s) (moi 50) for 48 hours in complete media and then transfected with genomic HCV RNA for 72 hours. Relative HCV-core expression  in serpin-treated cells compared to control-treated cells was quantified by examining total cell lysates using Western blot analysis. Results (mean ± SEM) from 2 independent experiments are shown. A representative Western blot is shown to the right of the graph.(TIF)Click here for additional data file.Figure S6The effect of PF-429242 on cell viability. Huh-7.5.1 cells were treated with DMSO (control) or various concentrations of PF-429242 for 24 hours before the inhibitor was removed, and fresh complete media was added to the cells for an additional 48 hours. The relative cytotoxicity of the compound was then determined using an MTS-based cell viability assay. The absorbance measured at 490 nm is proportional to the number of living cultured cells. Results (mean ± SEM) from 3 independent experiments are shown. Statistical significance was calculated for PF-429242-treated cells compared to DMSO-treated cells.(TIF)Click here for additional data file.


A novel anti-apoptotic alpha-adrenergic agonist preserves left ventricular ejection fraction in mice following myocardial infarction. This therapeutic effect is able to be detected and quantified non-invasively, using T2* signal loss assessment of an Annexin-SPIO molecular apoptosis probe.Myocardial infarction (MI) leads to cardiomyopathy through a combination of programmed cell death (re: apoptosis) and necrotic cell death. The relative contribution of apoptosis to ischemic cardiomyopathy and the effect of targeting apoptosis specifically to prevent MI-induced damage is unknown. Our laboratory previously developed and validated an in vivo, MRI-detectable apoptosis probe. Annexin-V (ANX), which binds to cells in the earliest stages of apoptosis, was conjugated to superparamagnetic iron oxide (SPIO) nanoparticles, allowing for the non-invasive detection of early apoptotic cell populations (ANX-SPIO r1: 8.6 ± 0.61 mM-1 s-1 and r2: 326 ± 16 mM-1 s-1). To test the effect of apoptosis reversal in an MI model, we employed A61603 (A6), an α1-adrenergic receptor agonist, which was previously shown to rescue cardiac cells from Doxorubicin-induced cardiac apoptosis through activation of the cardio-protective ERK pathway. Our hypothesis is that A6 therapy will protect against MI-induced cardiomyopathy, and that cardiac MRI of systemic ANX-SPIO will detect and longitudinally monitor this therapeutic effect in vivo.Mice underwent MI (via LAD ligation) along with a subcutaneous pump implant that delivered A6 or vehicle (VEH) solution at a rate of 10 ng/kg/day over a course of two weeks. Cardiac MRI (CMR) was performed at 2 days, 1 week, and 2 weeks post MI. ANX-SPIO was delivered via tail vein one day prior to CMR to simultaneously assess left ventricular function and apoptosis  in vivo.Although A6-treated  and VEH-treated 38±10%, n=6) mice exhibited identical ejection fractions (EFs) 2 days post-MI, A6-treated mice exhibited significantly (p<0.05) higher ejection fractions (EFs) versus their VEH-treated counterparts at both 1 week  and 2 weeks  post-MI , R01 (PY).


Here, we report reproducible iPS derivation of epidermal keratinocytes (HK) from elderly T2D patients. Transduced with human OCT4, SOX2, KLF4 and c-MYC stemness factors under serum-free and feeder-free conditions, reprogrammed cells underwent dedifferentiation with mitochondrial restructuring, induction of endogenous pluripotency genes - including NANOG, LIN28, and TERT, and down-regulation of cytoskeletal, MHC class I- and apoptosis-related genes. Notably, derived iPS clones acquired a rejuvenated state, characterized by elongated telomeres and suppressed senescence-related p15INK4b/p16INK4a gene expression and oxidative stress signaling. Stepwise guidance with lineage-specifying factors, including Indolactam V and GLP-1, redifferentiated HK-derived iPS clones into insulin-producing islet-like progeny. Thus, in elderly T2D patients, reprogramming of keratinocytes ensures a senescence-privileged status yielding iPS cells proficient for regenerative applications.Nuclear reprogramming enables patient-specific derivation of induced pluripotent stem (iPS) cells from adult tissue. Yet, iPS generation from patients with  Over 200 million people worldwide, ranging from 20 to 79 years in age, suffer from diabetes mellitus, typically the late onset type 2 diabetes (T2D) , 2. By 2Competent for multilineage differentiation, embryonic stem (ES) cells are regarded potentially promising for regenerative applications. Differentiation of ES cells into transplantable tissues could lead to repair therapies for severe degenerative diseases, including diabetes. In preclinical studies, human ES cells have been differentiated into insulin-producing cells -5, whichHere, we examined the feasibility of iPS derivation from epidermal keratinocytes from elderly T2D patients, analyzed molecular and cellular events associated with nuclear reprogramming, and determined differentiation propensities of derived pluripotent cells. Keratinocytes from T2D patients offered a reproducible source for patient-specific iPS generation, proficient in yielding insulin-producing islet-like progeny, through suppressed senescence-related pathways.5 transduced cells with maintained pluripotent markers and absence of replicative crisis even at 7 months post-initial vector infection (up to passage 60).Lentiviral vectors encoding human OCT4, SOX2, KLF4 and c-MYC, at an approximate multiplicity of infection of 5 each, transduced early passage human keratinocytes (HK cells) derived from 56 to 78 year-old individuals with or without T2D. Under serum-free and feeder-free conditions, within 1 to 2 weeks after viral vector infection, small reprogrammed colonies, characterized by a sharp-edged, flat, tightly-packed morphology, were apparent Figure . Individin vitro into cells of all three germ layers within embryoid body (EB) formations  individuals were enzymatically processed. Using sterile techniques, skin samples were incubated overnight at 4°C in dispase (25 U/ml) to cleave epidermis from dermis. The epidermal layer was then placed into a recombinant trypsin/EDTA solution  and incubated for 30 min at 37°C. Trypsin/EDTA was neutralized with a trypsin inhibitor  and epidermal pieces pipetted to release epidermal cells. The suspension was then passed through a 70 μm cell strainer and pelleted. Cell viability was determined by the trypan blue exclusion method. Cells were seeded in a plate coated with an animal component-free (ACF) coating matrix (Invitrogen). Selective trypsinization removed fibroblasts at ~6 min, while human keratinocytes (HK) were dissociated at ~20 min. HK cell populations were then grown in EpiLife Medium and S7 growth supplement  in 5% COin vitro in ACF EpiLife Medium in a matrix-coated plate. Cultures were transduced overnight with human OCT4, SOX2, KLF4 and cMYC expressing lentiviral vectors [HEScGRO medium  supplemented with mTeSR-1 maintenance media  [Lentiviral vectors, pSIN-OCT4, pSIN-SOX2, pSIN-KLF4 and pSIN-cMYC were manufactured as previously described to express pluripotency factors from an internal spleen focus-forming virus (SFFV) promoter . HIV vec Canada) . One to  Canada) . At thretrans Retinoic Acid (RA) (Sigma) in high glucose DMEM (Invitrogen) supplemented with 20% advanced RPMI, Pen/Strep, 1x B27 supplement (Invitrogen) for 4 days. Cells were then cultured in 50 ng/ml FGF10, 300 nM ILV (Axxora) and 55 nM GLP-1 (Sigma) in DMEM (high glucose) supplemented with 20% advanced RPMI and 1x B27 for 4 days. Differentiation medium including 10 μM DAPT (Sigma) and 55 nM GLP-1 in DMEM (high glucose) with 20% advanced-RPMI and 1x B27 was used to culture cells for the next 6 days. Finally, cells were cultured in 50 ng/mL hepatocyte growth factor (HGF) (R&D systems), 50 ng/ml insulin-like growth factor 1 (IGF-1) (R&D systems) and 55 nM GLP-1 in CMRL-1066 medium (Invitrogen) with 1 × B27 for 8 days.iPS clones were treated with 25 ng/ml Wnt3a (R&D systems) and 100 ng/ml activin A (Peprotech) in advanced RPMI (Invitrogen) with Pen/Strep for 1 day, followed by treatment with 100 ng/ml activin A in advanced RPMI supplemented with 0.2% fetal calf serum (FCS) (Invitrogen) for two days. Next, cells were cultured in high glucose DMEM (Invitrogen), supplemented with 20% (v/v) advanced RPMI medium containing 50 ng/ml FGF10 (R&D systems), 0.25 μM KAAD-cyclopamine (CYC), and 2% FCS for 2 days. Cells were then treated with 50 ng/ml FGF10, 0.25 μM CYC, and 2 μM all-For immunostaining, iPS cells were fixed for 20 min at room temperature (RT) in 4% paraformaldehyde (PFA), washed in PBS and blocked for 30 min in 5% FBS PBST (PBS with 0.1% Tween-20 (Sigma) and 5% FBS). Cells were stained with primary antibodies overnight at 4°C, rinsed by PBS, and incubated with secondary antibodies for 1 h at room temperature. Separately, cells at different stages of differentiation were fixed and stained with primary and secondary antibodies. Primary and secondary antibodies used for characterization were: SSEA-1, SSEA-4, TRA-1–60 TRA-1-81 (Millipore #SCR001), OCT4 , SOX2 , KLF4 (Abcam #ab26648) and NANOG (Abcam #ab21624), anti-SOX17 (R&D Systems #MAB1924), anti-HNF3 beta/FOXA2 (Millipore #07-633), anti-PDX1 (Santa Cruz Biotechnology#sc-25403 and Abcam #ab47267), and anti-insulin (Sigma # I2018). Texas Red-conjugated anti-rabbit IgG (Jackson Laboratories # 711-075-152), Texas Red-conjugated anti-mouse IgG (Jackson Laboratories # 715-075-151), FITC-conjugated anti-rabbit IgG (Jackson Laboratories # 711-095-152), and FITC-conjugated anti-mouse IgG (Jackson Laboratories # 715-095-151) were used as secondary antibodies. DAPI was used to counter-stain nuclei. Stained cells were analyzed using confocal laser-scanning microscopy . Alkaline phosphatase staining was performed with an Alkaline Phosphatase Detection Kit (Millipore). Antibodies FOXA2 for endoderm, beta III tubulin (Abcam # ab41489) for ectoderm and CD31 (Santa Cruz Biotechnology # sc-1506) for mesoderm were used to immunostain embryoid body-derived cells.SCID-beige mice were anesthetized, and the kidney exposed for iPS transplantation under the kidney capsule. To this end, a small incision was made in the kidney capsule and a blunt needle was used to create a pocket under the kidney capsule. Following iPS cell injection, the kidney was placed back into the abdomen, and the incision closed. Mice were maintained for 4 weeks and sacrificed for harvesting normal and iPS-transplanted kidneys. OTC-embedded frozen tissues were cryo-sectioned for H&E staining.For amplification of mitochondrial DNA, mitochondria-specific primer pairs  were used. For RT-PCR analysis, total RNA was isolated using TRIzol (Invitrogen) and reverse transcription was performed with oligo (dT) primer using RNA to cDNA EcoDry (Clontech). Platinum Taq DNA polymerase (Invitrogen) and primer pairs for TERT , INS , SST , GCG , GLUT2  and α-tubulin  were used for the reaction. Separately, total RNA was isolated using TRIzol (Invitrogen) and further purified using RNeasy Plus spin columns (QIAGEN). Turbo DNA-free DNase  was used to digest all genomic DNA that could lead to false positive gene expression results. RNA quantity and purity were measured with a Nanodrop spectrophotometer  and RNA integrity was determined using the Agilent 2100 Bioanalyzer . Microarray analysis was performed using the Affymetrix HG-U133 Plus2 GeneChip Array platform . Data were preprocessed using MicroArray Pre-Processing workflow and hierarchical clustering was performed by Pearson Dissimilarity. For comparison of transcriptome data between pre- and post-reprogramming, the data set of parental HK cells from three patients  were compared with those of three iPS clones from the same patients . Student's t-test was performed to assess significance (p<0.05) in normalized gene expression levels between HK and HK-derived iPS clones. The Heatmap Builder software  was used to generate the heatmap for the transcriptome data set. Enrichment analysis was also performed to match gene IDs in functional ontologies. The registered GEO transcriptome information  was used as reference.i)/Σ(ODi/L), where ODi and L were the optical density and length of fragment, respectively.Total genomic DNA was isolated from patient-derived HK and iPS cells using QIAGEN DNeasy Blood & Tissue Kit. Telomere length was determined using TeloTAGGG telomere length assay (Roche). Genomic DNA digestion, Southern blotting and chemiluminescence detection was performed as per established protocols. Densitometric analysis was performed on Adobe Photoshop and terminal restriction fragment lengths determined by Σ Relative cytochrome B (CYTB) and NADH mitochondrial DNA (mtDNA) copy numbers before  and after (iPS) reprogramming. mtDNA copy numbers were normalized to total genomic DNA and represented as a percentage of parental cell mtDNA copy number. (B) Immunocytochemistry analysis of iPS clone SW4 #N1 with mitochondrial marker AIF and (C) iPS clones SW4 #N1 and SW10 #5P with MitoTracker (Molecular Probes) staining. (D) Heatmap demonstrating up (red) and down-regulation (green) of genes involved in mitochondrial biogenesis upon reprogramming. No statistically significant change was observed in any of the genes listed. (E) Heatmap of expression profiles for genes involved in glycolysis, anaerobic glycolysis and citric acid cycle were compared between parental HK and HK-derived iPS cells. Statistically significant changes are indicated by asterisks (p<0.05). (F) RNA expression profiles of genes involved in the mitochondrial/oxidative stress response pathway between parental HK and iPS cells are shown. Statistically significant changes are indicated by asterisks (p<0.05).(


Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4+ T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4+ T-cells ex vivo. Furthermore, levels of gene-marked CD4+ T-cells in peripheral blood increased despite systemic infection with either CXCR4- or CCR5- tropic HIV-1 in vivo. These results demonstrate that transplantation of HSPCs engineered with our combination shRNA vector may be a potential therapy against HIV disease.Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT) mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC) transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA). However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR). Here, we report that human CD4 Acquired immune deficiency syndrome (AIDS) caused by human immunodeficiency virus type 1 (HIV-1) infection remains one of the most important global public health threats HIV-1 utilizes the CD4 receptor and, primarily, chemokine receptors CXCR4 and CCR5, as co-receptors that are crucial for viral entry into host cells in vitro. Importantly, we found it necessary to screen extensively for shRNAs that maintain potency at low expression levels to avoid cytotoxic effects associated with shRNA overexpression RNA interference (RNAi) is a highly evolutionarily conserved mechanism of post-transcriptional gene silencing that can be triggered by small interfering RNAs (siRNAs) in vivo efficacy of various CCR5-targeted HIV-1 therapies. While several humanized mouse model studies have focused on systemically delivered methods such as CCR5-specific RNAi inducers, coupled with aptamers in vivo. While others have employed zinc-finger nuclease-mediated genome editing in vivo AIDS gene therapy model.Use of various humanized small animal models, where immunocompromised mice are reconstituted with either HSPCs or differentiated T-cells to confer susceptibility to HIV-1 infection, have facilitated preclinical assessments of + (FL-CD34+) cells transduced with the sh1005-encoding vector and differentiation into CCR5-down-regulated T-cells and monocytes/macrophages in peripheral blood and systemic lymphoid organs The hu-BLT mouse model provides robust peripheral reconstitution of human T-cells, B-cells, and macrophages and importantly, unlike other models, efficient repopulation of many lymphoid tissue compartments including highly CCR5-expressing bone marrow and gut-associated lymphoid tissue (GALT), a primary target site of CCR5-tropic HIV-1 infection in vitro, we evaluated the in vivo reconstitution and stability of HSPCs engineered with our novel sh1005/sh516 combination vector and assessed conferred anti-viral potency of transplanted HSPC-derived T lymphocytes. Here, we report that transplantation of sh1005/sh516-transduced HSPCs resulted in efficient engraftment, stable marking in resultant hematopoietic lineages and potent inhibition of HIV-1-mediated depletion of modified CD4+ T-cells in vivo. This work describes a novel and safe combination vector which may provide both potent anti-viral inhibition and high reconstitution efficiencies for effective control of HIV infection.To confer protection against HIV-1 strains unimpeded by sh1005-mediated CCR5 down-regulation, we evaluated the anti-HIV-1 effects of selected previously published shRNAs targeting conserved regions of the HIV-1 genomic sequence. After screening for candidates with high anti-viral effects at low shRNA expression levels, we selected sh516, which targets the long terminal repeat (LTR) R region of HIV-1. Following extensive vector characterization NL4-3-specific 19-mer sequences and ranked 96 target sequences based on their degree of conservation, specificity, and suppressive activities in vitroNL4-3 reporter viruses that express either firefly luciferase or murine heat stable antigen (HSA) as a marker gene . Based on suppressive activities, potential to down-regulate multiple HIV-1 genes, and high conservation among known HIV-1 strains, we considered sh516 a candidate for anti-HIV-1 combination therapy.Importantly, the sh516 target sequence resides within the R-region of the HIV-1 LTR, specifically at the poly(A) hairpin which contains the polyadenylation hexamer motif. As both the 5′ and 3′ LTRs of HIV-1 possess this region, all HIV-1 transcripts, including all spliced transcripts Next, we generated a lentiviral vector capable of co-expressing shRNAs targeting CCR5 and HIV-1 to inhibit replication of both R5- and X4- tropic HIV-1. We applied a tandem shRNA expression cassette architecture to our existing CCR5 shRNA-expressing FG12-based lentiviral vector construct (FG12-sh1005) used in previous gene therapy studies NL4-3 reporter virus pseudotyped with G glycoprotein of the vesicular stomatitis virus (VSVG) (NL4-3 EGFP R-E-) To assess the suppressive activities of our resultant combination vector, we challenged vector-transduced CCR5-expressing CEM cells (CEM-NKR.CCR5) Lentivirally expressed shRNAs might recognize target sequences residing within packaging or transgene-expressing vector constructs, resulting in RNAi-mediated down-regulation of lentiviral genes or delivered transgenes NL4-3NFNSX SL9NL4-3, and the HIV-1-permissive human CCR5-expressing MOLT4/CCR5 T-cell line NFNSX SL9 or HIV-1NL4-3 and virus replication was measured by HIV-1 p24 antigen ELISA of culture supernatants. Expression of sh1005 and sh516 individually (Mono sh1005 and Mono sh516) or combined  inhibited replication of HIV-1NFNSX SL9 11.2–14.0 fold relative to vector alone. As expected, Mono sh1005 transduction rendered no significant effect on replication of HIV-1NL4-3, whereas sh516 expression inhibited replication by 12.5 and 6.9 fold relative to Vector alone in Mono sh516- and Dual sh1005/sh516- transduced cells, respectively  allows for a more practical evaluation of viral inhibition. We performed viral challenge experiments using replication competent X4-tropic HIV-1ectively . These r+ cells in untransduced and transduced populations) while no effect on CCR5 expression was observed in cells transduced with either Vector alone or Mono sh516 vectors . shRNA quantitation via real-time quantitative reverse transcription PCR (qRT-PCR) showed that sh516 expression did not have significant effects on sh1005 expression in Dual sh1005/sh516-transduced cells . Mono sh vectors . FACS-soor alone . These rWe examined the persistence of the integrated vector in Dual sh1005/sh516-transduced MOLT4/CCR5 cells as well as PBMCs. We found previously that shRNA expression driven by the U6 promoter in PBMCs resulted in a rapid decline in transduced cells over time TM Cytotoxicity Assay (Promega). Expression of sh1005, sh516, or both concurrently had no major effect on the dead:total cell number ratio (We next determined if sh1005/sh516 co-expression had adverse effects on cell viability using PBMCs. We and other groups have shown that high levels of shRNA expression can decrease cell viability er ratio . To deteer ratio . Therefo+ (mPB-CD34+) cells to differentiate into the various hematopoietic lineages, we next determined if sh1005/sh516 co-expression had adverse effects on HSPC differentiation potential. Vector-transduced mPB-CD34+ cells were subjected to a semi-solid methylcellulose-based colony forming cell assay in vitro  and control vector- (mCherry+) transduced cells, allowing comparative assessment of stability, engraftment, and specificity of CCR5 reduction within the same animal. We transplanted NOD.Cg-scid Il2rgtm1WjlPrkdc/SzJ (NSG) mice with either Mono sh1005- or Dual sh1005/sh516- transduced HSPC to allow side-by-side evaluation of therapeutic efficacies of sh1005/sh516 co-expression and previously demonstrated sh1005 expression. FL-CD34+ cells isolated from three independent donors were transduced with mCherry-marked control vector, EGFP-marked Mono sh1005, or EGFP-marked Dual sh1005/sh516 with a multiplicity of infection (MOI) of three. Mean vector transduction efficiencies in FL-CD34+ cells used for transplantation were 79.1%, 69.4%, and 63.4% for Mono sh1005, Dual sh1005/sh516, and control vectors, respectively. The increased transduction efficiencies were used to improve in vivo repopulation of marked cells. Busulfan-conditioned NSG mice received a 50∶50 mixture of control vector- and either Mono sh1005- or Dual sh1005/sh516- transduced FL-CD34+ cells transplanted with Matrigel under the kidney capsule with a thymus segment as well as IV injection of transduced FL-CD34+ cells  cells were detected in peripheral blood isolated from all Mono sh1005-  and Dual sh1005/sh516-  transplanted mice. Within a lymphocyte-gated population, proportions of CD3+CD45+  and CD19+CD45+  cells were similar between Mono sh1005- and Dual sh1005/sh516- transplanted mice, suggesting sh516 expression does not skew repopulation of engineered HSPC. Reconstitution of hematopoietic lineages from Dual sh1005/sh516-transduced HSPCs was observed in all transplanted mice, yielding marking in lymphocyte (8.9–40.1% EGFP+), T-cell (7.5–40.1% EGFP+) and B-cell (13.4–43.0% EGFP+) populations within a lymphocyte-gated population (e.g. HIV-1 infection). Comparable levels of EGFP and mCherry marking in CD45+, CD3+CD45+, and CD19+CD45+ populations within a lymphocyte-gated population as well as similar CD4/CD8 ratios were detected between Mono sh1005- and Dual sh1005/sh516- transplanted mice  or HIV-1NFNSX+ T-cells as percentages within total peripheral blood CD3+ cells for 10–12 weeks. Levels of control vector-transduced CD4+ T-cells began to decline after week 4 and week 2 post-viral challenge with HIV-1NFNSX and HIV-1NL4-3, respectively. Therefore, comparisons of CD4+ T-cell levels were assessed 4–12 weeks post-HIV-1NFNSX infection and 2–10 weeks post-HIV-1NL4-3 infection. As mCherry+ CD4+ T-cell levels declined in all infected mice (n = 9) over time (−19 to −92% with R5-tropic HIV-1 and −37 to −53% with X4-tropic HIV-1), EGFP+ CD4+ T-cell levels increased in five out of five HIV-1NFNSX-infected (16 to 217%) and three out of four HIV-1NL4-3-infected (16 to 92%) hu-BLT mice. However with the remaining HIV-1NL4-3-infected mouse, the decrease in EGFP+ CD4+ T-cell levels (−1%) was less than observed with mCherry+ CD4+ T-cells (−41%) , but decreased with mCherry+ cells , with statistically significant differences in relative fold changes between EGFP- and mCherry- marked CD4+ T-cell levels appearing as early as week 8 post-R5-tropic HIV-1 infection and week 4 post-X4-tropic HIV-1 infection  , suggestnfection . These rin vitro, recent HSPC-based approaches observed reduced vector titers possibly due to high complexity of vector constructs and/or inclusion of shRNAs which target both vector and HIV-1 genes in vivo efficacies observed with previous HSPC-based gene therapy strategies may be due to lack of a small animal model system to support HIV-1 replication to aid preclinical assessment. The hu-BLT mouse model affords high T-cell reconstitution efficiencies, human thymus-mediated T-cell development, and engraftment in clinically relevant systemic lymphoid organs such as GALT, a primary site of HIV-1 infection in vivo via CCR5 shRNA-transduced HSPC transplantation into hu-BLT mice, thus pioneering the application of the hu-BLT system for AIDS gene therapy development.Transplantation of engineered HSPCs holds great promise for stably controlling HIV-1 infection. Unlike current antiretroviral therapies, HSPC-based gene therapy may be potentially curative, reversing the virus's damage inflicted on the immune system, with a single or few administrations. While high viral inhibition can be achieved 8–1×109 transduction units per mL after 200-fold concentration) can be routinely achieved after minor vector LTR modification to a vector LTR region, which is suggested by previous mutagenesis studies of the HIV-1 poly(A) hairpin to tolerate minor changes In vitro characterization of sh1005/sh516 co-expression revealed effective inhibition of both R5- and X4- tropic HIV-1 replication with no obvious adverse effects on cell viability or hematopoietic differentiation.To improve anti-viral efficacy and to address potential drawbacks associated with a gene therapy strategy based solely on CCR5 down-regulation, such as viral escape mutagenesis and ineffectiveness against X4-tropic HIV-1 infection, we have incorporated an HIV-1 transcript-targeting shRNA into our current sh1005-expressing vector. Our screening for anti-HIV-1 shRNAs recognizing highly conserved regions and retaining potency at low expression levels revealed a candidate that targets the HIV-1 LTR. Although the sh516 target site is involved in a secondary RNA structure which may dampen RNAi knockdown efficiencies, the high potency of sh516 may be due to the stability of its target site structure  in vivo CCR5 down-regulation studies, we assessed the engraftment and reconstitution properties of Dual sh1005/sh516-transduced HSPC in the hu-BLT mouse model. We observed successful engraftment of engineered HSPCs in all transplanted animals. Dual sh1005/sh516-transduced HSPC successfully differentiated into hematopoietic lineages in all transplanted mice. Increased levels of reconstitution from Dual sh1005/sh516-transduced HSPC may be expected with exclusion of control vector-transduced HSPCs from the transplant. Marker gene expression and efficient CCR5 down-regulation were observed in Dual sh1005/sh516-transduced HSPC-derived CD4+ T-cells isolated from peripheral blood, spleen, bone marrow, and GALT, suggesting potential anti-viral protection in various systemic lymphoid tissues. Furthermore, sh1005/sh516 expression effectively inhibited both R5- and X4-tropic HIV-1 replication in spleen-derived T-cells ex vivo. Lastly, Dual sh1005/sh516-transduced HSPC-derived CD4+ T-cells resisted depletion by both R5- and X4- tropic HIV-1 infection in 5/5 and 3/4 transplanted hu-BLT mice, respectively. The observed difference in effectiveness against R5- and X4- tropic viruses may be due to sh1005/sh516 co-expression providing two anti-R5 virus reagents (sh1005 and sh516), but only one anti-X4 virus reagent (sh516). To our knowledge, this is the first demonstration of an shRNA-based HSPC gene therapy that confers resistance against HIV-1 strains utilizing either CCR5 or CXCR4 co-receptors to CD4+ T-cells in the hu-BLT model.Similar to our Our current study demonstrates the potential therapeutic efficacy of our combinatorial shRNA-based gene therapy strategy. Even so, any anti-HIV-1 therapy is potentially susceptible to emergence of therapy-resistant viral strains, necessitating the concurrent use of multiple therapeutic reagents. Co-expression of two therapeutic genes potentially has a lower chance for viral resistance than with a monotherapy. Moreover, shRNA efficacy can be diminished with minor mutagenesis of the targeted region Human PBMCs were obtained without identifying information from the UCLA Center for AIDS Research (CFAR) Virology Core Laboratory in accordance with UCLA Institutional Review Board (IRB) approved protocols along with an IRB-approved written consent form. Human fetal tissues were obtained without identifying information from the UCLA CFAR Gene and Cellular Therapy Core Laboratory and Advanced Biosciences Resources  under federal and state regulations and their use did not require IRB approval. Animal research described in the study was conducted under UCLA's Chancellor's Animal Research Committee  Protocol Number 2007–092 in accordance with guidelines for housing and care of laboratory animals of the National Institutes of Health (NIH) and the Association for the Assessment and Accreditation of Laboratory Animal Care (AALAC) International. All efforts were made to minimize pain and discomfort for the animals.For initial shRNA screening, H1-promoter-driven shRNA expression cassettes were PCR-amplified from pBS hH1-3 HEK-293T cells were cultured as previously described + cells (9.8×104), provided by AllCells , were pre-stimulated with SCF, TPO, Flt-3L, and IL-3  at 50 ng/mL each in X-VIVO 10 medium , 1x GlutaMax (Life Technologies), 0.5x Penn-Strep  for 24 hours then transduced with lentiviral vectors at MOI 10 for 21 hours.Transductions of HEK-293T, CEM.NKR-CCR5, and MOLT4/CCR5 were performed with indicated cell numbers and multiplicities of infections (MOIs) for 2 hours in the presence of 8 μg/ml of polybrene. Transductions of PBMCs were performed as previously described NL4-3, HIV-1NFNSX, HIV-1NFNSX SL9, and HIV-1JRCSF as well as VSVG-pseudotyped reporter viruses NL4-3.Luc.R-E-, NL4-3.HSA.R+.E-, and NL4-3 EGFP R-E- were produced by calcium phosphate transfection of HEK-293T cells as previously described pNL4-3, pNL4-3.Luc.R-E-, and pNL4-3.HSA.R+.E- were obtained from the AIDS Research and Reference Reagent Program of the National Institutes of Health. HIV-1+ and FL-CD34− cells, vector transduction of FL-CD34+ cells, and preparation of hu-BLT mice were prepared as previously described scid Il2rgtm1WjlPrkdc/SzJ (NSG) mice were purchased from the UCLA Defined Flora Mice Facility and maintained at UCLA animal facilities. Six- to nine- week-old female NSG mice were administered Busulfan (35 mg/kg) intraperitoneally. 24 hrs later, the mice were implanted with a portion of human fetal thymus combined with FL-CD34− cells and a 50∶50 mixture of control vector- and therapeutic vector- transduced FL-CD34+ cells solidified in Matrigel (BD Biosciences) under the kidney capsule and also transplanted with the 50∶50 vector-transduced autologous FL-CD34+ cell mixture via retro-orbital vein injection. To achieve high efficiencies of FL-CD34+ cell transduction, lentiviral vectors with minimum titers of 5×108 TU/mL (in HEK-293T) were employed and desired transduction efficiencies in FL-CD34+ were pre-determined by transductions with titrated amounts of vector. Single-cell suspensions were prepared from peripheral blood, bone marrow, spleen, thy/liv implant, and GALT as previously described NFNSX or HIV-1NL4-3, (200 ng/p24) via retro-orbital vein injection.Isolation of thymus segments and FL-CD34TM Cell Analyzer (BD Biosciences) and Cytomic FC500 .Cell staining with monoclonal antibodies and flow cytometry analysis were performed as previously described Cytotox-Glo Cytotoxicity Assays  were performed according to manufacturer's instructions. To assess induction of interferon response, total RNA from vector-transduced PBMCs was isolated using the mirVana miRNA Isolation Kit (Life Technologies). Ten ng of total RNA were subjected to OAS1 mRNA detection using the Interferon Response Detection Kit , the iScript One-Step RT-PCR Kit with SYBR Green , and the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories) per manufacturers' instructions. OAS1 mRNA levels relative to β-globin were calculated by the ΔΔCt method. Cytotoxicity and interferon response assays were performed in triplicate. Luciferase luminescence assays were performed using the Luciferase Assay System (Promega) and the FLUOstar Optima microplate reader  according to manufacturers' instructions.TM Supermix (Bio-Rad Laboratories), the CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories), and the following PCR parameters: 95°C, 10 min, 1 cycle; 95°C, 15 sec and 60°C 1 min, 40 cycles. siRNA-specific RT primers and Taqman® probes were provided by Custom Taqman® Small RNA Assays (Life Technologies) generated with the following submitted target sequences:Total RNA from PBMCs harvested four days post-transduction or from mouse tissue-derived lymphocytes isolated eleven weeks post transplantation were isolated using the mirVana miRNA Isolation Kit (Life Technologies). cDNA synthesis was performed using 20 ng (PBMC-derived) or 10 ng (tissue-derived) of total RNA, target-specific reverse transcription primers, and the Taqman® MicroRNA Reverse Transcription Kit (Life Technologies) per manufacturer's instructions. qRT-PCR was then performed using ∼1.33 ng (PBMC-derived) or ∼0.67 ng (tissue-derived) of cDNA, target-specific FAM-based Taqman® probes, iQ5′-GGUGUAAACUGAGCUUGCUCUU-3′ for si1005 and 5′-GCUUUAUUGAGGCUUAAGCUU-3′ for si516. RNU38B levels were assessed by RNU38B-specific TaqMan® MicroRNA Assay (Life Technologies) for normalization of siRNA copies. Complementary pairs of synthetic RNAs  (GR25-GR27), Brilliant® II QPRC Master Mix (Agilent Technologies), the Stratagene Mx300P/Mx3005P QPCR System Instrument (Agilent Technologies), and the following PCR parameters: 50°C, 2 min, 1 cycle; 95°C, 10 min, 1 cycle; 95°C, 15 sec then 60°C, 1 min, 40 cycles. Serial dilutions of FG12 plasmid, FG12 Mono sh1005 plasmid, and PBMC-derived genomic DNA were used to generate quantitation standards for WPRE, cPPT-H1, and β-globin copies, respectively. Sequences of primers and probes used for qPCR analysis can be found in Genomic DNA from PBMCs harvested seven days post-transduction or from mouse tissue-derived lymphocytes isolated eleven weeks post-transplantation were isolated using the PureLink Genomic DNA Mini Kit (Life Technologies). Quantitative real-time DNA PCR detecting WPRE (for PBMCs), cPPT-H1 (for tissue-derived lymphocytes), and β-globin was performed using GR19-GR24 forward/reverse primers, target-specific PrimeTime+ cells were resuspended in MethoCult H4034  and 500 cells were plated per replicate. Twelve to fourteen days later, CFUs were counted and scored using a Nikon Eclipse TS100  microscope.Vector-transduced mPB-CD34Figure S1A. Schematic of potential RNAi-mediated attenuation of sh1005-sh516 vector anti-viral activity. The sh516 target sequence (black box) resides within the LTR R region as well as the sh516 expression cassette. The sh1005 expression cassette possesses an sh1005 target sequence (striped box). sh516 may target vector LTRs in packaging cell as well as vector–derived mRNA, reducing vector titer and protein expression. B. Schematic of the sh516 target sequence and vector LTR mutation. C. Vector titers of unconcentrated vector stocks were calculated by transduction of HEK-293T cells. Titers were normalized by p24 concentration of vector preparation. D. Marker gene expression in HEK-293T cells transduced with lentiviral vectors. mCherry MFI in mCherry+ cells was assessed by flow cytometry analysis. Parental –sh1005/sh516 co-expressing vector. LTR modified – sh1005/sh516 co-expressing vector with modified vector LTRs. (C–D) Error bars – mean + SD. Statistical significance calculated by Student t test. ns  =  not statistically significant. ***  =  p value ≤0.0002.Optimization of sh516-expressing vectors via mutagenesis of vector LTRs. (TIF)Click here for additional data file.Table S1siRNA expression in vector-transduced hematopoietic cells.(DOCX)Click here for additional data file.Table S2+ T-cell levels in peripheral blood of HIV-1-infected Dual sh1005/sh516-transduced hu-BLT mice.Gene-marked CD4(DOCX)Click here for additional data file.Table S3Sequences of DNA and RNA oligos used in this study.(DOCX)Click here for additional data file.


The nonstructural protein 1 (NSP1) of rotavirus has been reported to block interferon (IFN) signaling by mediating proteasome-dependent degradation of IFN-regulatory factors (IRFs) and (or) the β-transducin repeat containing protein (β-TrCP). However, in addition to these targets, NSP1 may subvert innate immune responses via other mechanisms.The NSP1 of rotavirus OSU strain as well as the IRF3 binding domain truncated NSP1 of rotavirus SA11 strain are unable to degrade IRFs, but can still inhibit host IFN response, indicating that NSP1 may target alternative host factor(s) other than IRFs. Overexpression of NSP1 can block IFN-β promoter activation induced by the retinoic acid inducible gene I (RIG-I), but does not inhibit IFN-β activation induced by the mitochondrial antiviral-signaling protein (MAVS), indicating that NSP1 may target RIG-I. Immunoprecipitation experiments show that NSP1 interacts with RIG-I independent of IRF3 binding domain. In addition, NSP1 induces down-regulation of RIG-I in a proteasome-independent way.Our findings demonstrate that inhibition of RIG-I mediated type I IFN responses by NSP1 may contribute to the immune evasion of rotavirus. Rotavirus is a major cause of acute diarrhea in children under 5 years old, leading to approximately 600,000 annual deaths in the world . AlthougThe type I interferon (IFN) mediated immune response constitutes the first line of host defense against virus infection . Host ceReoviridae family, are non-enveloped icosahedra viruses containing 11 segments of a double stranded RNA (dsRNA) genome within a triple-layered particle. The rotavirus genome encodes six structural proteins (VPs) and six nonstructural proteins (NSPs). The structural proteins  form the virion. The NSPs (NSP1-6) function in dsRNA replication, transcription and translation of viral mRNA, and maturation of viral particles . In experiments examining the effect of the proteasome inhibitor MG132 , the culture medium was removed at different time points post-transfection (p.t.) and replaced with fresh medium containing 25 μM MG132. Cells were harvested at 36 or 48 h p.t. by lysis in RIPA buffer. For RIG-I mRNA detection, total cellular RNA was extracted using Trizol reagent (Invitrogen) at different time points after transfection.5 MA104 cells in 6-well plates were infected at a multiplicity of infection (MOI) of 0.1 with activated rotavirus SA11. Virus was added to the cells for adsorption for 45 min at 37°C and then washed with media to remove unbound virus. At different time points post-infection (p.i.), cells were lysed in RIPA buffer and assayed for protein content by immunoblot analysis. Total cellular RNA was extracted simultaneously.The rotavirus SA11 strain was activated by incubation with acetylated trypsin (10 μg/ml) for 30 min at 37°C prior to infection. Approximately 2.5 × 105 cells per well. Twelve hours later, cells were transfected with a control plasmid or plasmids expressing RIG-I, MDA5, or MAVS, and OSU NSP1 or SA11 NSP1ΔIRF3 BD along with pGL3-IFN-β-Luc and pRL-SV40 using Lipofectamine 2000. The total amount of DNA was kept consistent for all constructs by adding vector plasmids without inserts. Each sample was collected in triplicate, and each experiment was performed three times. At 48 h after transfection, cells were harvested, and cell lysates were used to determine luciferase activities using the Dual Luciferase Reporter Assay Kit . Luciferase activity was normalized using Renilla luciferase as an internal control, and the fold induction of luciferase activity above control was calculated.For the luciferase reporter assays, 293FT cells were cultured in 24-well plates at a cell density of 1.5 × 10Cells were collected at 48 h after cotransfection and then lysed with 25 mM Tris-HCl buffer (pH 7.4) containing 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, and proteinase inhibitor cocktail . Cell lysates were incubated overnight at 4°C with monoclonal antibodies against Myc  in the presence of protein A/G agarose beads . Immunocomplexes captured on the affinity gel or protein A/G agarose beads were extensively washed with lysis buffer and eluted with SDS loading buffer by boiling for 5 min. Then the samples were subjected to SDS-PAGE and Western blot analysis.RNA samples were treated with DNase I , and reverse transcription was carried out using a Superscript cDNA Synthesis Kit (Invitrogen) according to the manufacturer's instructions. cDNA samples were subjected to PCR amplification and electrophoresis to detect RIG-I and NSP1 expression. Specific primers for RIG-I (RIG-I-F-5'-CTCCCGGCACAGAAGTGT-3' RIG-I-R-5'-CCTCTGCCTCTGGTTTGG-3'), SA11-NSP1 (NSP1-F-5'-CATCTAATCACCCAGGCAATG-3 'NSP1-R-5'-TCACGAATCCGCCAATCA-3'), SA11-VP6 (VP6-F-5'-GACCAGTCTTTCCACCAGG-3' VP6-R-5'-GCCACTGTAAATATGCGTTG-3') and GAPDH (F-5'-GAAGGTGAAGGTCGGAGTC'-3' R-5'-GAAGATGGTGATGGGATTTC-3') were used.Cells were pelleted by centrifugation and lysed in RIPA buffer. Aliquots of cell lysates were resolved on 12% SDS-PAGE and transferred to a nitrocellulose membrane . The membranes were blocked with 5% nonfat milk and then probed with anti-GFP monoclonal antibody (Sigma), anti-Myc monoclonal antibody (Sigma), anti-IRF3 monoclonal antibody (Santa Cruz Biotechnology), anti-RIG-I monoclonal antibody (Santa Cruz Biotechnology), anti-rotavirus VP6 monoclonal antibody (prepared in our laboratory) and anti-β-actin monoclonal antibody (Sigma) primary antibodies at 4°C overnight, respectively. This was followed by incubation with the corresponding IRD Fluor 800-labeled IgG or IRD Fluor 680-labeled IgG secondary antibody . After washing, the membranes were scanned using an Odyssey Infrared Imaging System (Li-Cor) at a wavelength of 700-800 nm and analyzed with Odyssey software. The molecular sizes of the developed proteins were determined by comparison with prestained protein markers .NSP1: nonstructural protein 1; IFN: interferon; IRF: IFN-regulatory factor; β-TrCP: β-transducin repeat containing protein; RIG-I: retinoic acid inducible gene I; MAVS: mitochondrial antiviral-signaling protein; JAK: Janus Kinase; STAT: Signal Transducer and Activator of Transcription; RSV: respiratory syncytial virus; HPV: human papilloma virus; dsRNA: double stranded RNA; NF-κB: nuclear factor-κB; NSP1ΔIRF3BD: an IRF3 binding domain truncated NSP1; MDA5: melanoma differentiation-associated gene 5; cDCs: conventional dendritic cells; VSV: vesicular stomatitis virus; ISRE: interferon stimulated response element; TLR3: toll-like receptor 3; PKR: protein kinase R; SeV: Sendai virus; ORF: open reading frame; MOI: multiplicity of infection; RT-PCR: reverse transcription-polymerase chain reaction.The authors declare that they have no competing interests.LQ, LR, ZZ, XL, LC, QX and ZS carried out the experiments. LQ, LR, ZZ, JW and TH designed the research, wrote the manuscript and reviewed the drafts. All authors read and approved the final manuscript.


TLR7 3'-UTR SNP (rs3853839) associated with increased TLR7 transcripts, a more pronounced IFN signature and risk for SLE in 9,274 Eastern Asians (Pcombined = 6.5 × 10-10) [We identified the G allele of TLR7-TLR8 in European Americans (EA), African Americans (AA) and Hispanics enriched for the Amerindian-European admixture (HS). Haplotype-based conditional testing was conducted to distinguish independent association signals. Mantel-Haenszel testing was used in transancestral meta-analysis. Association of genotypes with TLR7 expression was examined using RT-PCR, flow cytometry and reporter assays. Pyrosequencing was used to measure allelic variations in TLR7 transcript levels.We conducted genotyping, imputation and association for 98 to 116 SNPs (varying among different ancestries) covering 80 kb of TLR7-TLR8 exhibiting consistent and independent association with SLE in our transancestral fine-mapping  = 1.24 (1.18 to 1.34)) in 13,339 subjects of EA , AA  and HS  ancestries. PBMCs from normal G-allele carriers exhibited elevated levels of TLR7 mRNA (P = 0.01 in men and P = 0.02 in women) and protein (P = 0.009 in men and P = 0.038 in women). PBMCs from heterozygotes exhibited higher G/C allele ratios of TLR7 transcripts 4 hours after incubation with actinomycin D (inhibitor of transcription initiation) (P = 0.04), indicating slower degradation of G allele-containing transcript. The nonrisk allele, but not the risk allele, was predicted to match microRNA-3148 (miR-3148) at the second base in the binding site. Transcript levels of miR-3148 and TLR7 were inversely correlated in PBMCs from 16 SLE patients and 21 controls , suggesting miR-3148 modulating TLR7 expression. Overexpression of miR-3148 via transfection into HEK 293 cells led to a more than twofold reduction in luciferase activity driven by the TLR7 3'-UTR segment containing the nonrisk allele than that containing the risk allele (P = 0.001).The rs3853839 was confirmed as the only variant within Pmeta = 6.4 × 10-19, OR (95% CI) = 1.26 (1.20 to 1.32)). Reduced modulation by miR-3148 confers slower degradation of the risk allele containing TLR7 transcript, resulting in elevated levels of gene products and a more robust type I IFN signature.We identified and confirmed a genome-wide significant association between rs3853839 and SLE susceptibility in 22,613 subjects of Eastern Asian, EA, AA and HS ancestries (