key: cord-0001530-33y74q1k
authors: Lyn, Rodney K.; Singaravelu, Ragunath; Kargman, Stacia; O'Hara, Shifawn; Chan, Helen; Oballa, Renata; Huang, Zheng; Jones, Daniel M.; Ridsdale, Andrew; Russell, Rodney S.; Partridge, Anthony W.; Pezacki, John Paul
title: Stearoyl-CoA desaturase inhibition blocks formation of hepatitis C virus-induced specialized membranes
date: 2014-04-01
journal: Sci Rep
DOI: 10.1038/srep04549
sha: 6b86206d67ce695a7a523a0702921b742f9f5250
doc_id: 1530
cord_uid: 33y74q1k

Hepatitis C virus (HCV) replication is dependent on the formation of specialized membrane structures; however, the host factor requirements for the formation of these HCV complexes remain unclear. Herein, we demonstrate that inhibition of stearoyl-CoA desaturase 1 (SCD-1) halts the biosynthesis of unsaturated fatty acids, such as oleic acid, and negatively modulates HCV replication. Unsaturated fatty acids play key roles in membrane curvature and fluidity. Mechanistically, we demonstrate that SCD-1 inhibition disrupts the integrity of membranous HCV replication complexes and renders HCV RNA susceptible to nuclease-mediated degradation. Our work establishes a novel function for unsaturated fatty acids in HCV replication.

position of long-chain acyl-CoAs, with greater selectivity for palmitoyl-and stearoyl-CoA 15 . The monounsaturated fatty acid (MUFA) products of SCD-1 enzymatic activity are shuttled as substrates for the synthesis of membrane phospholipid fatty-acyl chains, triglyceride biogenesis, and cholesterol esterification ( Fig. 1) 12, 17, 18 . A variety of small molecule inhibitors have been used to show that inhibiting lipogenesis negatively affects HCV replication 19 . To determine whether HCV replication is dependent on SCD-1 activity, we treated human hepatoma cells (Huh7) stably expressing an HCV replicon with the SCD-1 inhibitor A 20 (Fig. 2) . Dose dependent reduction of viral RNA replication was observed following 96 hr treatments with inhibitor A (EC 50 structural classes [20] [21] [22] [23] [24] , were also tested against genotype 1a and 1b HCV replicons, with EC 50 values for inhibition of viral replication measured as low as 0.74 nM (Supplementary Table S1 ). Inhibition by the SCD-1 inhibitors compared well with the direct-acting antiviral (DAA) inhibitor B 25 that inhibits HCV NS3 protease with an EC 50 value of 8.3 nM (Fig. 2e) . In some cases SCD-1 inhibitors (Supplementary Table S1 ) blocked HCV replication to a low level but did not abolish all replication as seen in DAA treatments, indicating a different mechanism of action for the SCD-1 inhibitors as demonstrated by a lack of inhibitory effect on in vitro NS3 protease and NS5B polymerase activity (Supplementary Table S2 ). Similar levels of inhibition of HCV replication and virus production were observed in a full-length genotype 2a (JFH-1 T ) 26 model (Fig. 3) . These results suggest that SCD-1 activity is highly advantageous full-length virus (JFH-1 T ) used in this study. All replicons encode for a neomycin (neo) resistance gene used as a selectable marker, and expression of HCV polyprotein is driven by encephalomyocarditis (EMCV) internal ribosomal entry site (IRES). All replicons are flanked by the HCV 59 untranslated region (UTR) and 39UTR. Both SGRs encode for NS3-NS5B. HCV SGR-Luc/Ubi (genotypes 1A and 1B) also encodes a luciferase reporter tag (luc) followed by a ubiquitin (ubi) gene. (B) Chemical structure of SCD-1 thiazole analogue inhibitor A. (C) Huh7 cells stably expressing HCV-SGR (genotype 1B) were treated with varying concentrations of the SCD inhibitor A. Quantitative real-time PCR (qRT-PCR) performed on RNA isolates 96 hours post-treatment demonstrates a decrease in HCV RNA levels induced by inhibiting SCD-1 in a dose-dependent manner. The error bars represent standard error of the mean (n $ 3). (D) Chemical structure of NS3 protease inhibitor B. (E) A dose response curve of HCV inhibition by inhibitor B after a 96 hour treatment in Huh7.5 cells stably expressing HCV-FGR (genotype 1B). RNA levels were analyzed as in (C). for HCV replication and pharmacological inhibition of SCD-1 leads to an antiviral effect similar to DAAs.

To confirm the specificity of the effect, we utilized a 14 C isotope method to directly measure SCD-1 inhibitory effect of our panel of SCD inhibitors in Huh7 cells harboring HCV replicons (Supplementary Fig. S2 ). We observed strong correlation between HCV inhibition and SCD-1 inhibition within these cells suggesting that the antiviral properties of our SCD inhibitors are due to on-target inhibition of the SCD-1 enzyme (Fig. 4) . The specificity was further demonstrated by the ability of OA addition to rescue the antiviral effects of the SCD inhibitors (Supplementary Table S1 ). Finally, SCD inhibitors had no observed effect on HIV replication/spread in a cellular assay. Taken together, these data suggest that inhibitor A acts to block HCV replication by reducing OA levels and that this effect is specific for HCV.

To investigate how SCD inhibitors block HCV replication, we initially focused on their effects on LDs since neutral lipids that form triglycerides and cholesterol esters are the major components of LDs 27 . Because MUFAs such as OA are highly incorporated into triglycerides, it is conceivable that there would be a reduction in storage of these neutral lipids in LDs upon SCD-1 inhibition 15 . Thus, we treated Huh7 cells, with and without a HCV subgenomic replicon, with the SCD-1 inhibitor A at concentrations ranging from 1 nM to 1 mM. We then performed cellular imaging using coherent anti-Stokes Raman scattering (CARS) microscopy, a label-free method for LD detection 28, 29 and voxel analysis, which quantifies lipid volumes from the threshold CARS signal specific for LDs 30 . We detected a small change in LD density when Huh7 cells stably expressing an HCV subgenomic replicon were treated with concentrations well above the EC 50 value for inhibitor A (Fig. 5) . The LD size and cellular distribution did not change in cells treated with inhibitor A below 250 nM. This indicates that there is no appreciable reduction in LD size or density at concentrations of SCD-1 inhibitor that can cause inhibition of HCV replication. At concentrations above 250 nM, we observed a significant reduction in cellular lipid content.

Our observations were specific for SCD-1 inhibitor A 20 as treatment with inhibitor B 25 , which targets the NS3 protease, produced no discernible changes in LDs across all concentrations of inhibitor ( Supplementary Fig. S3 ).

As our CARS imaging data suggested that inhibitor A mediated repression of HCV occurs prior to any detectable effect on LDs, we considered alternative mechanisms. Specifically, we postulated that the pharmacological blockade of SCD-1 would alter membrane composition, fluidity and/or the curvature required for functional HCV replication complexes. To test this hypothesis, we assessed the cellular localization of double-stranded (ds) HCV RNA, a species normally localized to intact replication complexes, upon treatment with varying concentrations of SCD inhibitor A (Fig. 6 ). Using an antibody specific for dsRNA 30 and Huh7 cells stably transfected with HCV replicons, confocal microscopy revealed that in untreated cells, ds-HCV RNA exhibits a punctate staining pattern within the cytoplasmic space of the cell (Fig. 6a ). Under mock treatment, a similar punctate pattern was also observed, as expected (Fig. 6b) . However, upon addition of 100 nM of SCD-1 inhibitor A, we detected a diffuse dispersion pattern of ds-HCV RNA intermediates that are consistent with a significant change in localization (Fig. 6c) . We reasoned that the exclusion of ds-HCV RNA from HCV-modified membranes caused the down-regulation of HCV replication since the RNA is no longer protected from endogenous nucleases and is likely no longer associated with host and viral proteins that mediate replication, as previously reported 30 . This is consistent with SCD inhibitor A affecting HCV modified membranes.

Next, we sought to further examine the possibility that inhibitor A is disrupting HCV-induced modified membranes through SCD-1 inhibition and the depletion of intracellular MUFAs. Previous studies demonstrated that HCV RNA in replication complexes is nuclease resistant 31 . On the molecular level, the replication complex membranes have high curvature that is likely induced at least in part by oleic acid-derived phospholipids. These membranes are characterized by their detergent and nuclease resistant properties 9,31,32 . Therefore, inhibiting SCD-1 may modulate the properties of the replication complex, rendering HCV RNA susceptible to the endogenous nucleases that are employed by the cell as a cellular defense mechanism.

To examine this possibility, we performed an in situ RNAse protection assay (RPA) based on a previously published methodology 9,31 . Miyanari et al. showed previously that HCV RNA in replication complexes remains intact in the presence of a membrane permeabilizing detergent (digitonin) and exogenous nucleases 9,31 . However, upon disruption of the ER membrane integrity using an additional non-ionic detergent (NP-40), HCV RNA became susceptible to nuclease-mediated degradation. We sought to investigate whether inhibitor A exerts a similar effect on the structural integrity of HCV altered membranes as NP-40, by inhibiting SCD-1 and lowering oleate levels. This is consistent also with the ability of oleic acid to rescue the effect (Supplementary Table S1 ). We performed comparative PCR for HCV RNA levels followed by densitometry to quantify the magnitudes of HCV RNA degradation after probing for nuclease accessibility to HCV RNA (Fig. 7 , and Supplementary  Fig. S4 ). The RPA was performed during mock ( Fig. 7a ,b lanes 1-6) and SCD-1 inhibitor A (Fig. 7a ,b lanes 7-11) treatments to examine the influence of SCD-1 on the nuclease and detergent resistant properties of viral RNA at replication complexes. Cells were treated at a concentration yielding ,50% reduction of HCV RNA by PCR (n 5 5) (Fig. 7b ,c and Supplementary Fig. S4 ). This was performed to ensure that we would be able to observe additional reduction of HCV RNA levels by endogenous and exogenously added nucleases. 28S and 18S ribosomal RNAs are susceptible to degradation by cytoplasmic nucleases, and their degradation is a positive control for both cell membrane permeabilization and nuclease activity ( Fig. 7a,b) . As expected, in treatments containing both nuclease and digitonin, 28S and 18S rRNA are readily degraded (Fig. 7a ,b, lanes 3 and 9), whereas HCV RNA is not. In the absence of digitonin, the exogenous nuclease fails to enter the cytoplasm, and as expected, no degradation is observed (Fig. 7a ,b, lane 6). Additionally, when NP-40 was added in the absence of nuclease and digitonin, ribosomal RNAs remained intact ( Fig. 7a ,b, lanes 4 and 10). SCD-1 inhibition alone has no effect on the integrity of ribosomal RNA bands ( Fig. 7a,b, lane 7) . Interestingly, we observed that for digitonin and nuclease treated cells, inhibitor A ( Fig. 7a ,b, lane 9) reduced HCV RNA levels similar to the effects of NP-40 ( Fig. 7a ,b, lane 5). This suggests that the SCD-1 inhibitor disrupts the structural integrity of HCV modified membranes to a similar extent to that of NP-40, which allows exogenous nucleases access to HCV RNA. For inhibitor A as well as digitonin plus nucleases treated cells, the HCV RNA levels showed above 50% reduction in HCV RNA levels (See Fig. 7a ,b, lane 8 vs 9). This further supports the notion that SCD-1 inhibitor treated HCV replicon containing cells are more susceptible to nucleases where SCD-1 inhibitor-induced membrane perturbation allows nuclease access into the replication complexes to degrade HCV RNA to a greater extent than mock treated cells. After repeating the exogenous nuclease protection assay (n 5 5), we observed that HCV RNA levels in digitonin, nuclease, and inhibitor A treated HCV replicon containing cells showed no statistically significant difference in comparison to digitonin, nuclease, and NP-40 treatment, which further confirms that the effects of inhibitor A mimic NP-40 treatment in disrupting the protective environment of the membranous webs (Fig. 7c) . As expected, no significant degradation of HCV RNA was observed in cells treated with digitonin only (Fig. 7a,b, lane 1) , NP-40 only (Fig. 7a,b, lane 4) , and nuclease only (Fig. 7a ,b, lane 6), in both mock and SCD-1 inhibitor A (Fig. 7a ,b, lanes 8 and 10) treated groups. Ultimately, the RPA demonstrates that reduced oleate from SCD-1 inhibitor treatment caused a change in the nuclease resistant HCV replication complexes, which then become accessible to endogenous or exogenously added nucleases.

Finally, we performed electron microscopy (EM) of Huh7 SGR cells treated with inhibitor A. We found that at a dose of 100 nM there was a significant decrease in the numbers of membranous webs, consistent with HCV knockdown, and a dramatic change in the structure of the membranous webs (Fig. 8) . Specifically, double membrane vesicles, a proposed site of HCV replication, displayed a distorted morphology (Fig. 8c,f) and increased prevalence of small vesicles was observed. Collectively, these observations are consistent with SCD inhibition altering and ultimately blocking the formation HCV-induced specialized membranes. EM imaging of Huh7 SGR cells at a dose of 500 nM showed almost no membranous webs and drastically fewer LDs compared with control cells ( Supplementary  Fig. S5 ), consistent with our observations using CARS microscopy (Fig. 5) . Table S1 ) via luciferase assays. In parallel, the cell-based enzymatic activity of SCD-1 was measured, as described in Materials and Methods. (A-B) For each inhibitor, the EC 50 of HCV inhibition is plotted against the EC 50 of SCD-1 inhibition. (C) HepG2 cells were similarly treated with each SCD-1 inhibitor, and EC 50 of SCD-1 inhibition in HepG2 cells is plotted against the respective EC 50 of SCD-1 inhibition in Huh7 SGR-Luc/Ubi cells. Discussion HCV, like other viruses, partitions its genome into membranous compartments in order to increase viral replication kinetics and shield the viral genome 33, 34 . However, our current understanding of the biogenesis of HCV-associated membranous webs is limited. While it is well established that HCV hijacks host lipid homeostasis to facilitate its pathogenesis 19, 35, 36 , there is an emerging body of evidence that these lipid pathways play an important role in HCV's formation of membranous replication complexes. This includes the modulation of phosphoinositide signaling and cholesterol synthesis to induce the formation of the HCV replication compartments 2,37 . Previous work analyzing the role of fatty acid biosynthesis in HCV pathogenesis has had a central focus on its relevance in HCVinduced hepatic lipid accumulation. Our study demonstrates an important role for unsaturated fatty acids in the formation of HCV-induced membranous compartments.

Our findings provide a new context for previous research investigating fatty acids' role in HCV pathogenesis. Earlier work demonstrated that HCV activates SREBP-1C and PPAR-c activity to activate fatty acid biogenesis 16, 38, 39 . Separate studies report HCV inhibits PPAR-a activity to decrease fatty acid catabolism [40] [41] [42] . This HCV-induced increase in cellular fatty acid levels may serve as a source of unsaturated fatty acids for formation of the viral replication complex. Our proposed pro-viral role of SCD-1 is consistent with recent studies demonstrating HCV activates SCD expression and activity 38, 43, 44 . Conversely, our data highlights an additional potential anti-viral mechanism of action for inhibitors of fatty acid synthesis previously reported to repress the HCV viral life cycle [45] [46] [47] , through downstream modulation of unsaturated fatty acid levels, such as is the case for inhibitor A.

Overall, we have established inhibitor A as a probe for oleic aciddependent membrane environments. We have shown that inhibiting SCD-1 decreases HCV RNA replication, through compromising the integrity of the membrane environment at replication complexes. Also, we demonstrated that SCD-1 inhibition increases the susceptibility of HCV RNA to exogenous nucleases in permeabilized cells suggesting that SCD-1 inhibition disrupts oleic acid-induced negative membrane curvature. To the best of our knowledge, this is the first direct evidence of unsaturated fatty acids playing a role in the formation of HCV replication complexes. Finally, given the recent development of liver-targeted SCD inhibitors 48 , SCD-1 inhibition represents a novel host-targeted therapeutic strategy for the treatment of HCV infection.

Cell culture and reagents. Human hepatoma cells (Huh7) cells harboring the pFK-I389neo/NS3-39/5.1 subgenomic replicon (Huh7 SGR; genotype 1b) and Huh7.5 cells stably expressing the full-length HCV genotype 1b replicon with a S2204I adaptive mutation in NS5A (Huh7.5 FGR) were maintained in DMEM medium supplemented with 100 nM nonessential amino acids, 50 U/mL penicillin, 50 mg/mL streptomycin, and 10% FBS (CANSERA, Rexdale, ON), and 250 mg/mL G418 Geneticin (GIBCO-BRL, Burlington, ON). Huh7 cells co-expressing genotype 1a or 1b and luciferase reporter were grown in complete DMEM media containing 0.25 mg/ml G418 antibiotic and supplemented with 10% fetal bovine serum, non-essential amino acids, penicillin/streptomycin, L-Glutamine, and Sodium pyruvate.

Huh7 cells harboring the genotype 1a or 1b replicon and the neomycin resistance gene were grown in complete DMEM media containing 0.25 mg/ml G418 antibiotic and supplemented with 10% fetal bovine serum, non-essential amino acids, penicillin/streptomycin, and L-Glutamine.

The pFK-I389neo/NS3-39/5.1 subgenomic replicon was kindly provided by Ralf Bartenschlager (Institute of Hygiene, University of Heidelberg, Germany). The Huh7. 5 strain was derived from the cell culture-adapted JFH-1 strain JFH-AM1, as previously described 26 .

SYBR Green-based quantitative RT-PCR. Dose response curves were performed using Huh7-SGR cells for inhibitor A and Huh7.5-FGR cells for inhibitor B. At cell confluency between 20-30% in 6-well plates, cells were treated with serial dilutions of inhibitor A (1 mM-10 nM) or inhibitor B (1 mM-10 nM) or DMSO (mock) and incubated for 96 hours. RNA isolation from hepatocytes was performed using TriZol (Invitrogen) as per the manufacturer's protocol. RNA integrity was confirmed by electrophoresis on 0.8% agarose gel in 13 TBE (Ambion, Austin, TX). 250 ng of total RNA was reverse transcribed into cDNA using the Superscript II Reverse Transcriptase kit (Invitrogen, Carlsbad CA). Quantitative PCR (qPCR) of HCV and 18S rRNA levels was performed on an iCycler (Bio-Rad, Hercules, CA) using iQ SYBR Green Supermix (Bio-Rad,), as per manufacturer's protocol. The primer sequences are described in Supplementary Table S3 . A 20 mL reaction was assembled according to the manufacturer's protocol. For data analysis, the 2 2DDCt method was used, and mean fold changes in expression are shown relative to mock or control transfected samples 49 .

Luciferase assay. HCV replication was measured in Huh7 cells expressing an HCV replicon and a luciferase reporter essentially as described previously 50 . Cells were harvested by trypsinization, quenched with complete media with G418, filtered through a 70 mm cell strainer, and then resuspended in complete FBS media (without G418) or complete media supplemented with 50% normal human serum (NHS, without G418). Cells were plated at 30 ml/well and at 2000 cells per well (FBS media) or 4000 cells per well (NHS media) in white 384 wells plates (Costar, 3570) using a Biomek FX with a 384 well head. Plates were left at room temperature for 30 min at which point 150 nL compound at various concentrations or DMSO, as a control, was added using the Echo 555 acoustic liquid handler followed by incubation at 37uC, 5% CO 2 . After 72 hours, cells were allowed to equilibrate to room temperature prior to addition of Bright-Glo luciferase substrate (Promega E220). Following a 5 minute incubation at room temperature in the dark, luminescence (correlated with RNA replication) was detected using an Envision Multilabel plate reader (Perkin Elmer). For rescue experiments, media was supplemented with palmitoleic or oleic acid. Prior to cell treatment, stocks of oleic or palmitoleic acid in 100% ethanol were added to DMEM media (final concentration of palmitoleic/oleic acid of 100 mM, final ethanol concentration of 0.1 to 0.2%) and were sonicated in a water bath for 5 min at room temperature. Rescue experiments were performed for 72 hours in the presence of SCD inhibitors.

Taqman-based quantitative RT-PCR. Viral replication was measured in Huh7 cells harboring the genotype 1a or 1b replicon and the neomycin resistance gene as described previously 51 . Cells were harvested using 0.25% trypsin-EDTA, and resuspended in complete FBS media (without G418) or complete media supplemented with 50% normal human serum (NHS). Replicon cells were plated at 8000 cells per well in a 96-well plate in 100 ml of Complete Media (-G418) and incubated at 37uC with 5% CO2. Twenty-four hours later, 25 ml of compound or DMSO control in complete media (without G418) containing 1% DMSO was added to each well and the plates were incubated at 37uC, 5% CO 2 . Seventy-two hour later, replicon RNA was isolated using RNeasy 96 well kit (Qiagen, Mississauga, Ontario) according to the manufacturer's instructions, and quantitated using real-time polymerase chain reaction (qRT-PCR) with the EZ RT-PCR kit (Applied Biosystems, Foster City, CA). The following primer/probes were used as a measure of HCV replication: Neo Forward Cell-based HIV replication assay. HIV replication was measured using a multi-cycle HIV replication assay and wild-type virus. Viral replication was determined by measuring HIV-1p24 gag antigen using the AlphaLISA technology (Perkin Elmer) after lysis of cells and viruses. MT-4 cells were maintained at a density of 0.1-3 million cells/mL in RMPI media supplemented with 10% FBS, L-glutamine, and Pen/Strep (10,000 IU/mL Pen and 10,000 ug/mL Strep). Virus was diluted in RPMI containing 10% FBS. MT-4 cells were washed in serum free medium, resuspended in medium containing virus at 250,000 cells/ml and incubated for 24 hours at 37uC and 5% CO 2 for 24 hours. Bulk-infected cells were washed in serum free media, resuspended in complete medium at 400,000 cells/ml and added to 384 TC-treated clear polystyrene plates (Costar 3701) at 30 ml per well. 30 ml of complete media containing inhibitor or DMSO were added the assay plates (final DMSO concentration of 0.25%) and incubated at 37uC and 5% CO 2 . After 72 hours, cells were lysed, and incubated with acceptor (Anti-HIV p24 gag AlphaLISA Acceptor beads (5 mg/mL) (Perkin Elmer custom preparation CUSM73518000EA; Ref# 73518) and donor beads (Perkin Elmer 6760002) according to the manufacturer's instructions. Plates were incubated for 60 minutes at room temperature in the dark followed by signal detection using a Pherastar plate reader.

NS3 protease activity -time-resolved fluorescence assay. NS3 protease activity was measured essentially as described previously 52 using the full length NS3/4A enzyme in reactions catalyzing the cleavage of a substrate peptide in a time-resolved fluorescence format. Briefly, the assay was performed in a final volume of 100 ml in assay buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 15% glycerol, 0.15% Triton X-100, 10 mM DTT, and 0.1% PEG 8000. NS3 protease was pre-incubated with various concentrations of inhibitors in DMSO or DMSO control for 30 min at room temperature. The reaction was initiated by adding peptide substrate (final concentration 100 nM). The reaction was quenched after 1 hour at room temperature with 100 ml of 500 mM MES, pH 5.5. Product fluorescence was detected in time resolved mode with excitation at 340 nm and emission at 615 nm.

NS5B polymerase activity assay. RNA polymerase activity was assessed essentially as described in Carroll et al. 53 except that polymerase activity was determined in reactions catalyzed by NS5BD21 (genotype 1b). The activity assay involved measuring the incorporation of 3 H-UTP homopolymeric PolyA/OligoU template/ primer, and reaction conditions included 5 nM NS5BD21 containing 20 mM Tris pH 7.5, 10 mM NaCl, 2 mM MgCl 2 , 0.01% Triton, 1 mM DTT, 1 mg/0.5 mM PolyA/ Oligo U 18 , and 10 mM 3 H-UTP.

SCD enzymatic activity assay. HepG2 and Huh7 cells as well as Huh7 cells coexpressing genotype 1b and luciferase were used for SCD activity assays. Cells were harvested with trypsinization and subsequently quenched with complete media and then passed through a 70 mm cell strainer. Cells were centrifuged at 800 rpm for 5 minutes and then resuspended in fresh media to 200,000 cells/ml. Cells were transferred at 80 ml per well to 96-well plates (Costar 3595) and incubated at 37uC and 5% CO 2 . After overnight incubation, 10 ml of compound or DMSO control in DMEM media/5% DMSO was transferred to each well and the plates were incubated at 37uC and 5% CO 2 . Fifteen minutes later, 10 ml of a sonicated media containing 14 C-stearic acid (final concentration 0.4 mCi/ml) was added to each well and plates were incubated for 4 hours at 37uC and 5% CO 2 . Cells monolayers were washed with PBS and subsequently lysed by incubation at 65uC for 1 h with 2N NaOH. Cell lysates were acidified with phosphoric acid, and lipids were extracted with acetonitrile. [1] [2] [3] [4] [5] [6] or inhibitor A (Lanes 7-11) at a concentration yielding a 50% reduction in HCV RNA levels. 96 hours post-treatment, samples were treated with a combination of digitonin (Lanes 2, 3, 5, 8, 9, 11) micrococcal nuclease (Lanes 3, 5, 6, 9, 11) or NP-40 (Lanes 4, 5, 10, 11). A representative integrity gel is shown (n 5 5) illustrating 18S and 28S ribosomal RNA integrity after digitonin, micrococcal nuclease, and NP-40 treatments. Degradation of 18S and 28S rRNA bands is observed only upon permeabilizing the plasma membrane with digitonin, and treatment with exogenous micrococcal nuclease (Lanes 3, 5, 9, 11) . Comparative RT-PCR was performed on HCV RNA to illustrate the relative HCV RNA abundance (cropped image below the RNA integrity gel) in the presence and absence of inhibitor A after treatments with digitonin, micrococcal nuclease, and NP-40. (B) Average HCV RNA abundance is shown from comparative PCR detecting HCV RNA. Densitometry was performed to calculate the HCV RNA abundance. All values were normalized by Lane 1 (no treatment). (C) Schematic model for SCD-1 inhibition-mediated disruption of HCV replication. Inhibition of SCD-1 alters HCV RNA's susceptibility to exogenously added nucleases by disrupting membrane HCV-associated membranes at the replication complexes. Multiple models are depicted for HCV RNA's susceptibility to endogenous nucleases (En nuc) and exogenous nucleases (Ex nuc). The size of the red arrow represents the magnitude of HCV RNA degradation by endogenous and exogenous nucleases under the different treatments including digitonin, micrococcal nucleases, and/or NP- 40. www.nature.com/scientificreports SCIENTIFIC REPORTS | 4 : 4549 | DOI: 10.1038/srep04549

Radiolabeled oleic and stearic acid were quantified by HPLC using a C18-reverse phase column (Zorbax extended C18 (4.6 mm 3 75 mm), eluting with a 3% water (0.1% formic acid)/acetonitrile to 97% at a flow rate of 2 mL/min in 4 min, with detection via a Packard Flow scintillation analyzer.

Coherent anti-Stokes Raman scattering (CARS) imaging and quantitative voxel analysis. The CARS microscopy system uses a single femtosecond Ti:sapphire oscillator as the excitation source, as previously described 28, 30 . At confluency between 20-30%, Huh7 and Huh7 SGR cells (described above) were treated with a serial dilution of inhibitor A (1 mM-10 nM) and inhibitor B (1 mM-10 nM) for a 96 hour incubation period. A total volume of media used was 2 mL for each Lab-Tek chamber (VWR, Radnor, PA, USA) for CARS imaging. Quantitative data from the CARS images was determined using a voxel counting routine in ImageJ as previously described 30, 54 . After collecting multiple images for the treated samples, the average lipid volume based on voxel counting was analyzed from several cells within the same field of view.

Immunofluorescence. Huh7 cells harboring the HCV subgenomic replicon (Huh7 SGR) were seeded at 8.0 3 10 4 cells/well in DMEM on coverslips in a 12-well plate. After 24 h, at a confluency of 25%-30%, cells were treated with inhibitor A for 96 h. After inhibitor A treatments, cells were washed once with PBS pH 7.4 and fixed with precooled 100% methanol for 10 min at220uC. Cells were washed three times with 13 PBS and incubated for 1 h at room temperature with a mouse monoclonal antibody specific for dsRNA (J2, 15300 dilution in PBS, Scicons, Hungary). After three more washes with PBS, cells were incubated with Cy2-labeled donkey antimouse IgG secondary antibody (151000 dilution in PBS, Jackson ImmunoResearch Laboratories, Inc., Westgrove, PA) for 1 h at room temperature. Following three more washes with 13 PBS, cells were rinsed in H2O before being mounted onto slides with 50% glycerol in PBS. Cells images were captured using an Olympus IX81 inverted microscope (Olympus America Inc.) and fluorescence was detected through a 1003 NA 1.40 oil objective. The images were analyzed using ImagePro software (MediaCybernatics) and ImageJ.

RNAse protection assay. The RNAse protection assay was adapted from Miyanari et al. 31 . After 96 hours of mock or inhibitor A treatment, Huh7 SGR cells in 60 mm cell culture plates (maintained as described above) were washed once with cold buffer B (20 mM HEPES-KOH (pH 7.7), 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA, and 2 mM dithiothreitol). For selected samples undergoing digitonin (Sigma-Aldrich, Oakville, ON) treatment, buffer B containing 50 mg/ml of digitonin was added to cells for 5 min at 27uC. The reaction was stopped by washing twice with cold buffer B. For samples treated with micrococcal nuclease (MJS Biolynx, Brockville, ON) and/or NP-40 substitute (octyl I phenoxypolyethoxyethanol; Bioshop Canada Inc., Burlington, ON), the cells were washed twice with buffer D ((20 mM HEPES-KOH (pH 7.7), 110 mM potassium acetate, 2 mM magnesium acetate, 2 mM dithiothreitol, and 1 mM CaCl 2 )) and treated with buffer D containing 0.1 unit/mL micrococcal nuclease, with or without, 0.45% NP-40 substitute for 15 min at 37uC. Samples treated with 0.45% NP-40 substitute only were incubated for 10 min at 37uC.

Total RNA was extracted using the RNeasy Mini Kits (Qiagen, Germantown, MD). The RNA integrity was assessed by electrophoresis on a 0.8% agarose, 13 TBE gel (Ambion, Austin, TX). 250 ng of total RNA was reverse transcribed into cDNA using the Superscript II Reverse Transcriptase kit (Invitrogen). Hexamers and dNTPs were purchased from Applied Biosystems. Using equal amounts of cDNA, 18S rRNA and HCV were amplified on an iCycler (Bio-Rad) using iQ SYBR Green Supermix (Bio-Rad), as per the manufacturer's protocol. The primer sequences for 18S rRNA are described in the Supplementary Table S3 . Various cycle numbers were assessed to determine the cycling conditions for sub-saturating levels of the amplicon to ensure exponential amplification. The resulting amplified genes were visualized by electrophoresis on a 2% agarose 13 TAE gel. Densitometry was calculated using Image J 1.45S 55 .

HCV infection and titering. JFH-1 T infection of Huh7.5 cells were performed in 6well plates. In brief, cells were seeded with 2 3 10 5 cells per well. 24 hours later, cells were either infected with JFH-1 T (MOI 5 0.1) or mock infected. 4 hrs post-infection (or mock infection), medium was removed and replaced with fresh medium (the normal DMEM 1 10% FCS). Cells were harvested for RNA isolation 72 hours postinfection. To calculate infectious titres, supernatants were passed through a Millex-HV 45-mm filter (Millipore) before being serially diluted 10-fold in DMEM. One day prior to infection, 8-well Lab-Tek chamber slides were seeded with 5 3 10 4 Huh7.5 cells/well and incubated overnight. 24 hrs later, 100 ml of each infectious dilution was used to infect chamber slides for 4 hrs before the supernatants were removed and replaced with fresh DMEM. 72 hrs post-infection, chamber slides were fixed with acetone and cells were stained using an antibody directed against core (B2; Anogen, Mississauga, Canada).

Statistical analysis. Statistical significance of difference in HCV expression levels was assessed using the paired student t-test using GraphPad Prism 4.01 (GraphPad Software Inc., La Jolla, CA).

Electron microscopy. Huh7 SGR cells were treated with vehicle (DMSO), 100 nM or 500 nM inhibitor A in antibiotic-free media for 96 h. Cells were fixed with 2.5% glutaraldehyde in PBS at pH 7.4. The cell pellet was resuspended in 22% BSA in PBS, recentrifuged and the resulting pellet was cut into 1 mm pieces. Samples were postfixed in 1% osmium tetroxide in 0.1 M Na cacodylate buffer, en bloc stained in 3% aqueous uranyl acetate, dehydrated in ascending ethanol and embedded in Spurr epoxy resin. Ultra-thin sections were cut on a Leica EM UC6 ultramicrotome. Sections were then stained with 1% lead citrate. Digital images were taken using a JEOL 1230 TEM adapted with a 2000 by 2000 pixel bottom mount CCD digital camera (Hamamatsu, Japan) and AMT software.

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Biological activity and preclinical efficacy of azetidinyl pyridazines as potent systemically-distributed stearoyl-CoA desaturase inhibitors

Synthesis and biological activity of a potent and orally bioavailable SCD inhibitor (MF-438)

Nicotinic acids: Liver-targeted SCD inhibitors with preclinical anti-diabetic efficacy

Development of a Liver-Targeted Stearoyl-CoA Desaturase (SCD) Inhibitor (MK-8245) to Establish a Therapeutic Window for the Treatment of Diabetes and Dyslipidemia

Pharmacodynamic Analysis of a Serine Protease Inhibitor, MK-4519, against Hepatitis C Virus Using a Novel In Vitro Pharmacodynamic System

Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus

Lipid droplets: a unified view of a dynamic organelle

Optimally chirped multimodal CARS microscopy based on a single Ti:sapphire oscillator

Chemical contrast for imaging living systems: molecular vibrations drive CARS microscopy

Direct imaging of the disruption of hepatitis C virus replication complexes by inhibitors of lipid metabolism

Hepatitis C virus non-structural proteins in the probable membranous compartment function in viral genome replication

Hepatitis C virus RNA replication occurs on a detergent-resistant membrane that cofractionates with caveolin-2

Architecture and biogenesis of plus-strand RNA virus replication factories

Replication Vesicles are Load-and Choke-Points in the Hepatitis C Virus Lifecycle

Hepatitis c virus and host cell lipids: An intimate connection

Unique ties between hepatitis C virus replication and intracellular lipids

Morphological and Biochemical Characterization of the Membranous Hepatitis C Virus Replication Compartment

Hepatitis C virus induces proteolytic cleavage of sterol regulatory element binding proteins and stimulates their phosphorylation via oxidative stress

HCV core protein induces hepatic lipid accumulation by activating SREBP1 and PPARc

Hepatitis C virus induced up-regulation of microRNA-27: A novel mechanism for hepatic steatosis

Impaired expression of the peroxisome proliferator-activated receptor alpha during hepatitis C virus infection

Hepatitis C virus core protein modulates fatty acid metabolism and thereby causes lipid accumulation in the liver

Hepatitis C Virus Proteins Induce Lipogenesis and Defective Triglyceride Secretion in Transgenic Mice

Pathogenesis of lipid metabolism disorder in hepatitis C: Polyunsaturated fatty acids counteract lipid alterations induced by the core protein

Genomic analysis of the host response to hepatitis C virus infection

Fatty acid synthase is up-regulated during hepatitis C virus infection and regulates hepatitis C virus entry and production

Modulation of Fatty Acid Synthase Enzyme Activity and Expression during Hepatitis C Virus Replication

Discovery of potent and liver-targeted stearoyl-CoA desaturase (SCD) inhibitors in a bispyrrolidine series

Analysis of relative gene expression data using realtime quantitative PCR and the 2 2DDCt Method

A replicon-based bioassay for the measurement of interferons in patients with chronic hepatitis C

Identification and Biological Characterization of Heterocyclic Inhibitors of the Hepatitis C Virus RNA-dependent RNA Polymerase

A time-resolved, internally quenched fluorescence assay to characterize inhibition of hepatitis C virus nonstructural protein 3-4A protease at low enzyme concentrations

Inhibition of Hepatitis C Virus RNA Replication by 29-Modified Nucleoside Analogs

Dynamics of lipid droplets induced by the hepatitis C virus core protein

NIH Image to ImageJ: 25 years of image analysis

RS thanks NSERC for a Vanier Graduate Scholarship and the CIHR National Canadian Research Training Program in Hepatitis C for additional support and training

J.P.P. and A.P. contributed in conception and design, data analysis and interpretation, and manuscript writing. R.K.L. contributed in conception and design, collection and assembly of data, data analysis and interpretation, and aided in the preparation of the manuscript. R.S., S.K., S.O., H.C., R.O., Z.H., A.R., R.S.R. and D.M.J. contributed in collection and assembly of the data, data analysis and interpretation, and aided in the preparation of the manuscript. if the image is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the image. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/