Systems Virology Identifies a Mitochondrial Fatty Acid OxidationEnzyme, Dodecenoyl Coenzyme A Delta Isomerase, Required for
Hepatitis C Virus Replication and Likely Pathogenesis�†Angela L. Rasmussen,1 Deborah L. Diamond,1 Jason E. McDermott,2 Xiaoli Gao,3 Thomas O. Metz,3
Melissa M. Matzke,2 Victoria S. Carter,1 Sarah E. Belisle,1 Marcus J. Korth,1 Katrina M. Waters,2Richard D. Smith,3 and Michael G. Katze1*
Department of Microbiology, University of Washington School of Medicine, Seattle, Washington 981951; Computational Biology &Bioinformatics, Pacific Northwest National Laboratory, Richland, Washington 993522; and Biological Sciences Division,
Pacific Northwest National Laboratory, Richland, Washington 993523
Received 4 July 2011/Accepted 25 August 2011
We previously employed systems biology approaches to identify the mitochondrial fatty acid oxidationenzyme dodecenoyl coenzyme A delta isomerase (DCI) as a bottleneck protein controlling host metabolicreprogramming during hepatitis C virus (HCV) infection. Here we present the results of studies confirming theimportance of DCI to HCV pathogenesis. Computational models incorporating proteomic data from HCVpatient liver biopsy specimens recapitulated our original predictions regarding DCI and link HCV-associatedalterations in cellular metabolism and liver disease progression. HCV growth and RNA replication in hepa-toma cell lines stably expressing DCI-targeting short hairpin RNA (shRNA) were abrogated, indicating thatDCI is required for productive infection. Pharmacologic inhibition of fatty acid oxidation also blocked HCVreplication. Production of infectious HCV was restored by overexpression of an shRNA-resistant DCI allele.These findings demonstrate the utility of systems biology approaches to gain novel insight into the biology ofHCV infection and identify novel, translationally relevant therapeutic targets.
Lipids play a role in numerous steps of the hepatitis C virus(HCV) replication cycle, including RNA replication associatedwith the lipid droplet (6, 35, 48), virus uptake, assembly, andsecretion in association with cellular apolipoproteins (2, 9, 16,23, 33, 37, 44), fusion with host membranes during virus entry(13), endocytic trafficking (3, 8), and reorganization of cellularmembranes associated with replication and assembly (1, 4, 49,51). Furthermore, patients with HCV often exhibit hepaticsteatosis and the upregulation of a number of genes involved inhepatic lipid metabolism (11, 52).
Recently, using integrated modeling efforts combining pro-teomic and lipidomic data, we identified two enzymes, dode-cenoyl coenzyme A (CoA) delta isomerase (DCI) and hy-droxyacyl-CoA dehydrogenase beta subunit (HADHB), thatwere upregulated during in vitro infection and in patients withhistological evidence of fibrosis. These two enzymes were pre-dicted to be bottleneck proteins, key regulators of the HCV-induced temporal alterations in cellular metabolic homeostasisduring viral replication (11). DCI and HADHB are localized tothe inner mitochondrion and catalyze the degradation of long-chain fatty acids during fatty acid �-oxidation (7, 38, 47). Pre-viously, �-oxidation was shown to be required for measles virus(46) and dengue virus replication (14), although DCI and
HADHB were not directly implicated in this process. Severalproteins involved in lipid catabolism, including lipases, es-terases, acyl-CoA dehydrogenases, and palmitoyltransferaseshave also been identified as important host factors in HCVreplication by RNA interference-based screening (5, 8, 31, 40,45, 49). DCI and HADHB were previously identified as hostfactors producing a subtle decrease in RNA production in areplicon system, although their function in HCV replicationremains unknown (45). In this study, we evaluated the role ofDCI by using short hairpin RNA (shRNA)-expressing lentivi-ruses to stably silence DCI gene expression, rather than thetransient-knockdown approaches used in small interferingRNA (siRNA)-based screens.
Although lipid-related metabolic dysfunction has been ob-served in the context of HCV infection, no studies have directlylinked fatty acid oxidation with HCV replication or pathogen-esis. Here, we describe an extension of sophisticated modelingtechniques to clinical proteomic data confirming that DCI iscomputationally predicted to be a key cellular protein requiredfor HCV pathogenesis in vivo. Moreover, we identified addi-tional proteins predicted to further regulate cellular metabolicalterations augmenting HCV infection. We also present bio-logical validation data proving that DCI is essential to HCVreplication, confirming our computational predictions postu-lating a critical role for mitochondrial fatty acid oxidation inthe HCV life cycle. Together, these findings illustrate thepower of systems biology approaches to identify novel hostfactors that provide both biological insight into the processesof HCV replication and unique targets for the development ofantiviral therapeutics.
* Corresponding author. Mailing address: Department of Micro-biology, University of Washington School of Medicine, Box 358070,Seattle, WA 98195. Phone: (206) 732-6136. Fax: (206) 732-6056.E-mail: [email protected].
† Supplemental material for this article may be found at http://jvi.asm.org/.
Generation of a correlation network. Our clinical modeling efforts leveragedquantitative proteomic data generated from a previous study describing globalproteomic alterations accompanying liver disease progression in patients withchronic hepatitis C virus (HCV) infections (10). Briefly, quantitative data ob-tained on a total of 1,641 proteins identified in human liver biopsy tissue spec-imens were filtered for those proteins showing a minimal 1.5-fold change (P �
0.05) in at least 6 of 15 patients represented in the prior study. We created acorrelation network on these data by repeating the analysis we had previouslydescribed in the in vitro cell culture model (11). Briefly, we removed abundancevalues below 1.5 (corresponding to a similar threshold used previously), calcu-lated correlation between abundance profiles of all pairs of proteins, and thencalculated correlation between all pairs of proteins. The correlation values arebased on comparing the protein abundance profiles, and we filtered these to keeponly comparisons in which the two proteins being compared were observed in thesame 6 or more patients. We then filtered the correlation matrix to includecorrelation values above 0.9 (highly correlated profiles). Protein-protein inter-actions from the Human Interactome were integrated into the resulting network,considering interactions between observed proteins. It is important to note thatthe correlation-based edges in this network are being used only for topologicalanalysis and may not represent true interactions between the proteins. Topologywas calculated in the resulting network using the igraph library in R, and proteinswere ranked on the basis of their betweenness (a topological measure thatidentifies highly central proteins in the networks that restrict flow through thenetwork) to identify bottlenecks.
Cell culture. Huh7 cells were routinely cultured in Dulbecco modified Eaglemedium (DMEM) (Invitrogen) containing 10% fetal bovine serum, penicillin(100 IU/ml) (Invitrogen), and streptomycin (100 �g/ml) (Invitrogen). To gener-ate stable short hairpin RNA (shRNA)-expressing cell lines, SMARTvectorlentiviruses expressing shRNA targeting dodecenoyl coenzyme A delta isomer-ase (DCI) or a scrambled target sequence were obtained from Thermo Scientific.The following sequences in DCI were targeted: DCI-1 (5�-CATTCCAGACCATGCTCGA-3�), DCI-2 (5�-CCAGGGAGGTCTTAAACAA-3�), and DCI-3 (5�-AGGTACTGCATAGGACTCA-3�).
Huh7 cells were transduced at a multiplicity of infection (MOI) of 10 withshRNA-expressing lentiviruses and Polybrene (10 �g/ml). After 48-h incubationwith lentiviruses, fresh medium containing puromycin (2 �g/ml) (Sigma) wasapplied. After 3 weeks of drug selection, target protein knockdown was evaluatedby Western blotting. Stable knockdown cell lines were routinely maintained inmedium containing puromycin (2 �g/ml). HeLa S3 cells and Huh7.5 cells werealso routinely cultured in DMEM containing 10% fetal bovine serum, penicillin(100 IU/ml), and streptomycin (100 �g/ml). BHK-21 cells were routinely cul-tured in minimal essential medium (MEM) (Invitrogen) containing 10% fetalbovine serum, 1% nonessential amino acids (Invitrogen), penicillin (100 IU/ml),and streptomycin (100 �g/ml).
Western blotting. Western blotting was performed using anti-DCI antibody at3 �g/ml (Abcam) with horseradish peroxidase-conjugated secondary donkeyanti-mouse IgG diluted 1:10,000 (Jackson ImmunoLabs). Loading control blotswere performed using horseradish peroxidase-conjugated anti-GAPDH (anti-body against glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) (0.5 �g/ml)(Abcam). Immunoreactivity on the Western blots were detected using ECL PlusWestern blotting detection system (GE Biosciences). Quantitation of proteinabundance was performed by densitometry using ImageJ software (NationalInstitutes of Health) to determine DCI expression relative to wild-type Huh7 cellexpression.
Cloning and expression of shRNA-resistant DCI construct. A DCI clone(shRNA-resistant DCI-1 [SRDCI-1]) containing the following silent mutations inthe DCI-1 target site was synthesized by Blue Heron: 5�-TATCCCGGATCACGCGCGC-3�.
This clone was amplified using the following primers: forward primer, 5�-GCGCGAAGCTTATGGCGCTGGTGGCTTCTGTGCCA-3�; reverse primer, 5�-GCGGGATCCTTTAAGTTGAAAAATACC-3�. The resulting PCR productwas digested with HindIII and BamHI (New England BioLabs), ligated intoHindIII/BamHI-digested pcDNA3.1 (�)/Hygro (Hygro stands for hygromycin)using T4 DNA ligase (New England BioLabs), and transformed into electro-competent Escherichia coli DH5� (Invitrogen) using a GenePulser (Bio-Rad).Intact, properly oriented clones were selected and confirmed by DNA se-quencing.
Huh7/DCI-1 stable knockdown cells were transfected with pSRDCI-1 orempty pcDNA3.1 vector using FuGENE 6 transfection reagent (Roche) per themanufacturer’s instructions. After 48 h, hygromycin B (50 �g/ml) was applied to
the cells, and stably transfected cells were selected by 3 weeks of drug treatment.The cells were routinely maintained with drug selection.
Virus propagation. HCV genotype 2a/SJ virus stocks were generously pro-vided by Michael Gale at the University of Washington. Working stocks weregenerated by infecting Huh7.5 cells, harvesting supernatant after 5 or 6 days, andconcentrating supernatant in Centricon-70 concentrators (Millipore). Concen-trated virus stocks were titrated by a focus-forming assay. Briefly, 100-�l aliquotsof 10-fold serial dilutions of virus stock were used to infect Huh7.5 cells previ-ously seeded in triplicate in 24-well plates at a density of 1 � 105 cells/well.Following 1-h adsorption time, 0.5 ml of fresh medium was added to each well,and cultures were incubated at 37°C and 5% CO2. After 48 to 72 h of incubation,cells were fixed for 30 min in 4% paraformaldehyde. The cells were washed inphosphate-buffered saline (PBS) with 1 mM glycine, permeabilized for 15 min inPBS containing 0.2% Triton X-100, and blocked for 15 min with PBS containing10% donor equine serum. The cells were incubated overnight with pooled HCVpatient sera diluted 1:1,000 at 4°C with gentle rocking. The cells were washed andincubated with horseradish peroxidase-conjugated donkey anti-human IgG sec-ondary antibody (Jackson ImmunoLabs) for 1.5 h at room temperature withgentle rocking. The cells were washed, and focus-forming assays were developedwith VIP ImmPACT peroxidase substrate (Vector Laboratories). The virus titerwas determined by calculating the number of focus-forming units per milliliter ofvirus stock.
Plasmid pT7M bearing a full-length infectious molecular clone of poliovirustype I/Mahoney was generously provided by Vincent Racaniello at ColumbiaUniversity. pT7M was linearized with EcoRI (New England BioLabs), and viralRNA was transcribed in vitro using the Ambion MEGAscript T7 kit (AppliedBiosystems). Viral RNA was transfected into HeLa S3 cells using DEAE-dextran(Sigma). Supernatant was harvested after total cytopathic effect was apparent onthe plate, and virus was frozen and thawed three times and then centrifuged topellet debris. Clarified supernatants were titrated on HeLa S3 cells by plaqueassay. The virus titer was determined by calculating the number of PFU per mlof virus stock.
Dengue virus serotype 2/NGC was also generously provided by Michael Galeat the University of Washington. Stocks were grown at 33°C in C6/36 cells.Supernatant was harvested after the appearance of cytopathic effect, clarified bycentrifugation to pellet cell debris, and concentrated using Centricon-70 concen-trators (Millipore). Concentrated stocks were titrated on BHK-21 cells by plaqueassay.
Growth curves were set up in general by plating 2 � 104 cells per well in12-well tissue culture plates prior to inoculation. The cells were then infectedwith 100 �l of inoculum at a multiplicity of infection of 1. Following infection, theinoculum was washed off in 1 ml of infection medium, and the cells were fed with1 ml of fresh medium per well. Growth curves were titrated by harvesting andfreezing cell culture supernatants, followed by low-speed centrifugation to re-move particulate matter.
Quantitation of viral RNA. RNA was harvested from infected cells usingTRIzol reagent (Invitrogen) per the manufacturer’s instructions. RNA was re-verse transcribed using the Quantitect kit (Qiagen), and viral genomes werequantitated relative to 18S RNA by TaqMan quantitative real-time reversetranscription-PCR (RT-PCR). For quantifying positive-sense single-stranded vi-ral genomes, we used the following primers: forward primer, 5�-TCCCGGCAATTCCGGTGTAC-3�; reverse primer, 5�-TCCCGGAGAGCCATAGTG-3�. Weused the following probe: 6FAM-5�-TCT GCG GAA CCG GTG-3�-MGBNFQ(6FAM stands for 6-carboxyfluorescein, and MGBNFQ stands for moleculargroove-binding nonfluorescence quencher). We normalized the values usingprimer-probe sets for the endogenous control human 18S rRNA per the manu-facturer’s specifications (Applied Biosystems). All reactions were performedusing standard TaqMan protocols and reagents supplied by the manufacturerand run on an ABI 7500 real-time PCR instrument (Applied Biosystems).
Pharmacologic inhibition of fatty acid oxidation. Etomoxir (Sigma) was dis-solved in dimethyl sulfoxide (DMSO) (Sigma) to generate a 10 mM stock solu-tion. We performed a titration to determine the dose at which etomoxir was themost inhibitory to HCV and least cytotoxic. Twenty-four hours prior to infection,etomoxir was added to the culture medium at a final concentration of 100 �M.Mock-treated cells were treated with an equivalent volume of DMSO. The cellswere treated again at the time of infection.
Statistical analysis. All data are expressed as mean � standard error of themean. Differences between control and experimental groups were assessed byone-way analysis of variance (ANOVA), and means were compared by Student’st test with Bonferroni’s multiple test correction using JMP 9 (SAS, Inc.). P valuesless than 0.05 were considered significant.
Metabolomics data collection and statistical and functional analyses. Huh7,Huh7/NT, Huh7/DCI-1, and Huh7/DCI-3 cells were washed in ammonium bi-
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carbonate (Sigma), pelleted by centrifugation, and snap-frozen. Metabolites andlipids were extracted from six replicate cell pellets using chloroform-methanol(2:1 [vol/vol)], and the water- and lipid-soluble layers were isolated by centrifu-gation and then dried in vacuo.
Metabolites in the dried water-soluble layers were derivatized using methyoxy-amine and N-methyl-N-(trimethylsilyl)trifluoroacetamide prior to gas chroma-tography-mass spectrometry (GC-MS) analysis in duplicate. GC-MS data wereprocessed using the software program MetaboliteDetector to identify GC-MSfeatures (15), align these chromatographically across multiple data sets, and thenmatch these to the Fiehn Metabolomics RTL Library included with the AgilentGC-MS instrument (28). This software program also generates abundance foreach GC-MS peak.
Lipids in the dried lipid-soluble layers were reconstituted in 200 �l of isopro-panol and analyzed by liquid chromatography (LC)-MS. A small trapping col-umn (180 �m by 2 cm) packed with reversed-phase particles (Symmetry C18
column; 5 �m; Waters, Milford, MA) was used prior to the analytical column forfast loading of lipid samples (within 1.5 min at a flow rate of 10 �l/min), followedby washing of the column-bound lipids to remove chemical impurities and non-lipid sample components. Reconstituted lipids (4 �l) were loaded onto thetrapping column under the following isocratic conditions: 93% acetonitrile–water (40:60) containing 10 mM ammonium acetate (solvent A) and 7% aceto-nitrile–isopropanol (10:90) containing 10 mM ammonium acetate (solvent B).The lipids retained on the trapping column were then back-flushed to the ana-lytical column using 10% solvent B at a flow rate of 1 �l/min. The analyticalcolumn (150 �m by 20 cm) was slurry packed (12) with 1.8-�m particles (HSS T3;Waters) and maintained at 40°C in a column oven. Gradient elution was per-formed as follows: initial conditions, 10% solvent B; 0 to 2 min, ramp to 30%solvent B; 2 to 10 min, ramp to 40% solvent B; 10 to 20 min, ramp to 55% solventB; 20 to 40 min, ramp to 60% solvent B; 40 to 70 min, ramp to 99.5% solvent B;and 70 to 90 min, hold at 99.5% solvent B. The LC system was interfaced to anLTQ-Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA) using achemically etched electrospray ionization (ESI) emitter (25), and the ESI emitterand MS inlet capillary potentials were 2.2 kV and 12 V, respectively. Data-dependent tandem MS (MS-MS) (collision-induced dissociation [CID]) scanevents (top five ions) were performed in the ion trap using a normalized collisionenergy of 35% and were set with a maximum charge state of 2 and an isolationwidth of 2 m/z units. An activation Q value of 0.18 was used, and dynamicexclusion in the ion trap was enabled as follows: repeat count of 2, repeatduration of 30 s, exclusion list size of 200, and exclusion duration of 60 s. The fullscan mass range for negative ESI mode was 200 to 2,000 m/z, respectively. Onebiological replicate from each cell type was analyzed separately in order todetermine the appropriate loading of the LC column, and then the remaining 5biological replicates were analyzed in random order.
The PRISM Data Analysis system (26), a series of software tools freely avail-able at http://ncrr.pnl.gov/software/ and developed in-house, was used to processand analyze the LC-MS lipid data. The first step involved deisotoping of the rawMS data to give the monoisotopic mass, charge state, and intensity of the majorpeaks in each mass spectrum using Decon2LS (18). The data were next examinedin a two-dimensional (2-D) fashion using MultiAlign to identify groups of massspectral peaks that were observed in sequential spectra using an algorithm (36)that computes a Euclidean distance in n-dimensional space for combinations ofpeaks. Each group, generally ascribed to one detected species and referred to asa “feature,” has a median monoisotopic mass, central normalized elution time(NET), and abundance estimate computed by summing the intensities of the MSpeaks that comprise the entire LC-MS feature. LC-MS features were thenchromatographically aligned across all replicates for each sample using theLCMSWARP algorithm (19) in MultiAlign, and the identities of detected lipidswere determined by searching entries in the Lipid Maps database within a searchtolerance of �10 ppm mass tolerance. Tentative fatty acid identifications wereconfirmed by comparison with the retention times or ranges of authentic fattyacid standards.
The metabolite and fatty acid data were loaded into the software packageDAnTE (39), the feature abundances were transformed to log2 scale, and thedata were normalized using mean centering. ANOVA was then performed toidentify metabolites and fatty acids that differed quantitatively with q values(adjusted P values found using an optimized false discovery rate approach)of �0.05. To assist in visualization and interpretation of the heat map of signif-icantly different metabolites and fatty acids, we performed a z-score transforma-tion of the abundances for each metabolite or fatty acid. For GC-MS-based data,a G-test was also performed to identify statistically significant differences in thedata based on metabolite occurrence.
RESULTS
Clinical data support a critical role for DCI in HCV patho-genesis. To explore the role of dodecenoyl coenzyme A (CoA)delta isomerase (DCI) as a key cellular protein involved inhepatitis C virus (HCV) pathogenesis in vivo and thus define arole for DCI in clinical disease pathology, we employed acomputational strategy to model proteomic data generatedfrom core needle liver biopsy specimens taken from 15 patientschronically infected with HCV (10). Network analysis of pat-terns of protein expression was used to identify points of re-striction, bottlenecks, representing points of control for impor-tant processes related to disease progression. The abundance“profiles” of proteins, their abundance in each of the 15 patientsamples, were used to determine patterns of similar expressionusing correlation with all other proteins identified. Briefly, weremoved abundance values below 1.5 and calculated correla-tion between abundance profiles of all pairs of proteins exclud-ing correlation values based on fewer than 6 comparisonswhere a single comparison is valid if both proteins were ob-served in the same patient sample. We then filtered to retainonly highly correlated pairs of proteins (0.9 correlation).These parameters are derived from a more-extensive charac-terization of network inference from proteomic data (J. E.McDermott et al., submitted for publication), which is notfocused on DCI and describes similar network analysis inmore-general terms. Protein-protein interactions from the Hu-man Interactome were integrated into the resulting network toaccount for interactions between observed proteins. Topologywas calculated in the resulting network, and proteins wereranked on the basis of their betweenness to identify bottle-necks that are predicted to be key regulators of metabolicreprogramming in chronically infected patients (Fig. 1A).Betweenness is a topological measure that identifies highlycentral proteins in the networks that restrict flow through thenetwork. Bottlenecks are predicted to be more important tothe functional processes underlying the network because oftheir position in the network (34).
Consistent with our in vitro modeling results, DCI was thesecond highest-ranked bottleneck after histidine triad nucle-otide-binding protein 2 (HINT2) in the clinical proteomic net-work (Fig. 1B), indicating a physiologically relevant role forDCI during HCV infection in vivo. We chose to focus on DCIbecause of its role in lipid metabolism, which was highlightedin our previous work (11), and the fact that it is one of a smallnumber of bottlenecks shared between clinical and cell culturenetworks (McDermott et al., submitted). This demonstratesthe utility of a systems biology approach for identifying trans-lationally relevant host factors based both on in vitro experi-mental models and patient specimens. Our hypothesis result-ing from this analysis is that DCI represents an important pointof control for metabolic processes critical for HCV replicationand pathogenesis.
Knockdown of DCI inhibits HCV RNA replication in cul-tured hepatoma cells. To better understand DCI’s role in theHCV replication cycle, we generated Huh7 human hepatomacells stably expressing shRNAs targeting DCI. Lentivirusesexpressing three distinct shRNA constructs targeting differentregions of the DCI transcript were used to transduce Huh7cells. After puromycin selection for transduced Huh7 cells
expressing the shRNA, DCI knockdown was evaluated byWestern blotting and relative quantitation was performed us-ing ImageJ software, which demonstrated that Huh7/DCI-1cells express 18% of the amount of DCI expressed by wild-typeHuh7 cells (Fig. 2A). The DCI-1 construct demonstrated thegreatest reduction in DCI protein levels, so Huh7/DCI-1 cellswere used for all subsequent experiments. The DCI-3 con-struct did not result in observable knockdown and was used asan additional control.
Infection of the Huh7/DCI-1 cell line with the cell cultureHCV genotype 2a strain SJ resulted in a substantial reductionin virus production compared to infection of wild-type cells,cells expressing a scrambled nontargeting control shRNA, andcells expressing the DCI-3 shRNA (Fig. 2B). Huh7/DCI-1 cellsalso supported lower levels of HCV RNA replication com-pared to the controls. Quantitative PCR for viral genomesdemonstrated a substantial reduction in the DCI knockdowncells compared to the controls (Fig. 2C). Huh7/DCI-1 cells didnot support increases in positive-sense single-stranded HCVgenomes, indicating that viral replication is blocked at or priorto viral RNA production in DCI-1 knockdown cells.
Pharmacologic inhibition of fatty acid oxidation blocks HCVreplication. All fatty acid �-oxidation in cells can be abrogated
by treatment with etomoxir, an irreversible inhibitor of carni-tine palmitoyltransferase 1 (CPT-1). CPT-1 is an integral mem-brane protein localized in the outer mitochondrial membraneand transports fatty acids to the mitochondrion. BlockingCPT-1 activity with etomoxir effectively blocks the transport ofany fatty acid substrates to the mitochondrial matrix and thusinhibits activity of the �-oxidation enzymes, including DCI.Huh7 cells treated with etomoxir 24 h prior to and over thecourse of infection did not support production of infectiousHCV compared to mock-treated cells (Fig. 3). These datademonstrate that the mitochondrial fatty acid oxidation path-way is required for HCV replication.
FIG. 1. Correlation network showing metabolic bottlenecks identi-fied in proteomic data from liver biopsy specimens of patients withchronic hepatitis C virus (HCV) infections by computational modelingefforts, integrating observed proteins with known protein-protein in-teractions. (A) The full network of 103 connected proteins. Proteinsare colored by their rank according to betweenness centrality; red ishighest, and blue is lowest. Edges represent correlation associationsgreater than 0.9 or known protein-protein interactions. Colored ovalsrepresent regions of significantly (P value � 0.001) enriched biologicalprocess functions in the network. The rectangle represents the subnet-work shown in panel B. (B) Subnetwork showing the two proteins withhighest betweenness centrality, dodecenoyl coenzyme A delta isomer-ase (DCI) and histidine triad nucleotide-binding protein 2 (HINT2).
FIG. 2. DCI knockdown blocks replication in human hepatomacells. (A) Western blot showing DCI protein expression in Huh7 cellsbearing stable short hairpin RNA (shRNA)-expressing lentiviral con-structs. Western blotting for glyceraldehyde-3-phosphate dehydroge-nase (GAPDH) was performed as a loading control. (B) Huh7 cellsexpressing shRNAs were infected with HCV genotype 2a (HCV2a)/SJat a multiplicity of infection (MOI) of 1. Virus was titrated at 1, 3,and 5 days postinfection by a focus-forming assay. Values that aresignificantly different are indicated by brackets and asterisks asfollows: *, P � 0.00315; **, P � 0.0025; ***, P � 0.000067. (C) DCIknockdown inhibits replication of viral RNA. Viral RNA levelswere quantified by TaqMan real-time reverse transcription-PCR(RT-PCR) relative to cellular 18S rRNA. Values that are signifi-cantly different are indicated by brackets and asterisks as follows:*, P � 0.005; **, P � 0.0002; ***, P � 0.00007.
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DCI is not required for the replication of other positive-sense, single-stranded RNA viruses. To determine whetherDCI supports HCV replication by a mechanism that generallyaugments the assembly of viral RNA replication complex as-sembly, we tested the effect of DCI knockdown on other vi-ruses with similar biological properties. Poliovirus is an entero-virus in the family Picornaviridae, which like flaviviruses havepositive-sense, single-stranded RNA genomes. Like HCV, po-liovirus genomes replicate on specialized vesicular structuresderived from the endoplasmic reticulum (41). Furthermore,both enteroviruses and flaviviruses require a specialized lipidmicroenvironment in the membranes on which RNA replica-tion complexes assemble (17). Therefore, we hypothesized thatpoliovirus might similarly be inhibited in DCI knockdown cells.However, virus production in Huh7/DCI-1 cells infected withpoliovirus type 1/Mahoney (P1/M) was similar to that seen inP1/M-infected Huh7, Huh7/NT, and Huh7/DCI-3 control cells(Fig. 4A). We then investigated whether or not DCI is requiredfor replication of dengue virus serotype 2/New Guinea C(DENV2/NGC), which also cannot replicate in cells treatedwith etomoxir (14) and like HCV is a member of the familyFlaviviridae. DENV2/NGC production was similar in Huh7/DCI-1 knockdown cells to infected controls (Fig. 4B), indicat-ing that DCI is not required to support replication of all fla-viviruses, including ones that require �-oxidation of fatty acidsto replicate. While these data do not demonstrate a measur-able effect of DCI on poliovirus or dengue virus replication,they indicate that the requirement for DCI is highly specific toHCV infection. It also indicates that Huh7/DCI-1 cells arecompetent to support replication of viruses that are not de-pendent on DCI activity, demonstrating the specificity of DCI’smechanism of action for HCV infection.
Restoration of DCI in shRNA-expressing knockdown cellsrescues HCV propagation. To determine the specificity of DCIin regulating HCV replication, we generated Huh7/DCI-1 cellsstably transfected with a clone expressing a shRNA-resistantallele of DCI (SRDCI-1). SRDCI-1 contains silent mutationsin the region of the DCI transcript targeted by the DCI-1shRNA. This expresses a DCI protein with no changes in theamino acid sequence but contains multiple nucleotide substi-tutions in the mRNA that prevent recognition and consequentgene silencing by the mature small interfering RNA (siRNA)(Fig. 5A). Infection of Huh7/DCI-1 cells expressing SRDCI-1
restored HCV growth compared to Huh7/DCI-1 cells or Huh7/DCI-1 cells bearing an empty vector pcDNA3.1 control (Fig.5B). This clearly demonstrates that inhibition of HCV replica-tion is specific to DCI activity and not the result of off-targetgene silencing or alterations induced by constitutive shRNAexpression.
DCI deficiency causes alterations in the cellular metabo-lome. Our previous computational models predicted that DCIis an essential bottleneck protein regulating overall reprogram-ming of cellular metabolism to create a favorable environmentfor HCV replication. To gain a preliminary understanding ofthe effect of DCI on global cellular metabolism, we performedgas chromatography-mass spectrometry (GC-MS) analysis ofaqueous metabolites in uninfected Huh7/DCI-1 cells and theassociated controls. Using ANOVA (q � 0.05), we identified37 metabolites with significantly altered abundance in Huh7/DCI-1 cells (Fig. 6A; see Table S1 in the supplemental mate-rial). These data indicate that DCI has pleiotropic effects on avariety of metabolic processes, including biogenesis and ho-meostasis of amino acids, nucleotides, vitamins, and sugars.We further identified 64 metabolites with differences based onthe presence or absence in the Huh7/DCI-1 cells compared tothe controls by the G-test (P � 0.05) (Table S1), confirmingthat cells lacking DCI have a distinct metabolic profile com-pared to other cell lines expressing DCI that are permissive toHCV infection.
We also analyzed the cellular lipidome to determine the
FIG. 3. Pharmacologic inhibition of fatty acid oxidation blocksHCV replication. Huh7 cells mock treated or treated with 100 �Metomoxir were infected with HCV2a/SJ at an MOI of 1. Virus wastitrated by a focus-forming assay on Huh7.5 cells. FFU, focus-formingunit.
FIG. 4. DCI deficiency does not block growth of other RNA vi-ruses. (A) Cells were infected with poliovirus type 1/Mahoney strain(P1/M) at an MOI of 1. P1/M growth was titrated by plaque assay onHeLa S3 cells. (B) Cells were infected with dengue virus serotype2/New Guinea C strain (DENV2/NGC) at an MOI of 1. DENV2/NGCgrowth was titrated by a plaque assay on BHK-21 cells.
effect that DCI knockdown would have on fatty acid compo-sition, since its substrates are long-chain mono- and polyun-saturated fatty acids. Lipid features detected in the mass rangeof free fatty acids were matched to entries in the Lipid Mapsdatabase and subsequently identified on the basis of com-parison of retention times to those of free fatty acid stan-dards. This approach identified 26 differentially abundantfatty acid species from Huh7/DCI-1 cells compared to thecontrols (ANOVA, q � 0.05). Notably, Huh7/DCI-1 cellsshowed dramatic increases in medium- to long-chain mono-and polyunsaturated fatty acids (Fig. 6B). This is consistentwith our hypothesis that without DCI present to degrade poly-unsaturated fatty acids, these molecules accumulate in theHuh7/DCI-1 knockdown cells.
DISCUSSION
We used a combination of gene silencing and pharmacologicapproaches to validate our previous computational modelingpredictions that dodecenoyl coenzyme A (CoA) delta isomer-ase (DCI)-mediated mitochondrial fatty acid oxidation plays acritical role in hepatitis C virus (HCV) replication. Both ap-proaches confirmed the importance of DCI to the HCV rep-lication cycle, demonstrating the value of systems biology as ameans of gaining insights into HCV pathogenesis that havebeen overlooked by conventional approaches. DCI knockdowncompletely blocked viral RNA production, indicating that itsmechanism of action in augmenting HCV replication occurs ator before assembly of RNA replication complexes and initia-tion of viral transcription by the HCV RNA-dependent RNApolymerase (RdRp). Because of DCI’s essential role in thecatabolism of long-chain fatty acids and its initial identification
by a model built using proteomic and lipidomic data, we pro-pose that DCI exerts its effects on HCV replication by modu-lating lipid content in the cell.
Alterations in lipid metabolism have long been observed inboth experimental and clinical HCV infection. Specific lipidspecies undergo changes in composition and abundance thatfacilitate molecular interactions required for HCV infection.Patients with chronic HCV develop steatosis and alterations inserum lipid levels, particularly as disease progresses. ThoughDCI has previously not been implicated in mediating thesechanges, mitochondrial �-oxidation could be involved in anumber of cellular processes required for HCV replication.Numerous studies have observed changes in the lipidome ofinfected cells corresponding with known interactions betweenreplicating HCV and multiple host lipids or lipid-related ma-chinery. Lipid rafts rich in sphingomyelin are required for theassembly of RNA replication complexes and activity of theHCV RdRp (1, 51). Lipid droplets are specialized organellesdesigned for the storage of neutral lipids, which attach to HCVcore protein and facilitate assembly of viral replication com-plexes and viral particles (35). These lipid droplets are at-tached to membranes where RNA replication complexes as-semble. Both the induction of lipid droplet formation and thesubstantial organelle and membrane remodeling required forRNA replication and virion assembly at these sites necessitatesubstantial reprogramming of cellular lipid biosynthetic andmetabolic pathways, all of which may be mediated by DCI.However, despite the wealth of information concerning therole of lipids in HCV replication and the major role of fattyacid oxidation in both meeting the cellular energetic require-ments and generating building blocks for new lipid species inlipogenesis, conventional approaches have never implicatedthis pathway in playing an essential role in HCV replication.Without employing a systems-level approach, fatty acid oxida-tion would not have been identified as a key pathway regulat-ing metabolic reprogramming required for HCV replicationand pathogenesis.
Activating fatty acid oxidation pathways seems contrary tonumerous reports that lipogenesis, including fatty acid synthe-sis, is required at virtually all stages of HCV replication. DCImay be required to break down more-complex lipid species tofavor synthesis of other lipid species facilitating HCV infec-tion. Some of these lipid species might be membrane phospho-lipids, such as phosphatidylinositol-4-phosphate, which nucle-ates assembly of RNA replication complexes on membranesurfaces (17). Additionally, lipidation of host and viral proteinsis known to play an essential role in HCV infection. For ex-ample, geranylgeranylation of host proteins is required forHCV RNA replication (24, 50, 53), and the viral core andNS4B proteins are palmitoylated to facilitate interactions withmembranes and other proteins required to assemble functionalreplication complexes (32, 54). By mediating the degradationof lipids not required for HCV replication, DCI generatesenergy and materials for the biosynthesis of other lipid mole-cules required during the viral life cycle.
Another possibility is that DCI specifically reduces the quan-tity of polyunsaturated fatty acids (PUFA) in the cell, creatinga favorable environment for HCV replication. DCI specificallyisomerizes mono- and polyunsaturated fatty acids with cis dou-ble bonds at odd-numbered carbon atoms into their 2-trans-
FIG. 5. HCV replication is restored in DCI-deficient cells whencomplemented in trans by shRNA-resistant DCI-1 (SRDCI-1).(A) Western blot demonstrating DCI expression in Huh7 cells bearingstable shRNA-expressing lentiviruses with shRNA-resistant DCI alleleSRDCI-1 added in trans. (B) Growth of HCV in cells expressingSRDCI-1 infected at an MOI of 1was determined by a focus-formingassay on Huh7.5 cells.
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enoyl-CoA forms (20, 21). HCV RNA replication is inhibitedspecifically by accumulation of polyunsaturated fatty acids nor-mally degraded by DCI (30). A characteristic feature of fastingDCI knockout mice is hepatic accumulation of unsaturatedlong-chain fatty acids (21). DCI deficiency results in the accu-mulation of PUFA in Huh7/DCI-1 cells, subsequently inhibit-ing HCV replication, possibly by altering lipid rafts or mem-brane structure sufficiently as to prevent the assembly andfunction of RNA replication complexes.
Our metabolomic data provide insight into the molecularbasis for HCV inhibition in DCI-deficient Huh7 cells. Consis-tent with DCI’s function in channeling unsaturated fatty acidsinto the mitochondrial �-oxidation pathway, DCI knockdownresults in an increase in abundance of the monounsaturatedDCI substrate oleic acid. Exogenous oleic acid enhances RNAsynthesis by a full-length HCV genotype 1b (HCV1b) replicon(24), although the molecular mechanism for this enhancementand the impact of DCI-mediated catabolism during oleic acidsupplementation remain undetermined. Fatty acid oxidationmay exert pleiotropic effects on the HCV life cycle, includingpotentially significant roles in enhanced energy production andmodulating the cellular composition of lipid species with pro-or antiviral effects. Either one of these potentially significantroles could influence the global metabolic reprogrammingknown to occur during HCV infection (11). During conditionsof impaired lipid catabolism, these effects would presumablybe abrogated, forcing the cell to rely on alternative energysources. Indeed, the observed aqueous metabolic profile fur-ther revealed that Huh7/DCI-1 cells exhibited increases innumerous amino acids and exemplary intermediates of ureasynthesis. These metabolic alterations are consistent with thoseobserved in DCI knockout mice (21) and suggest an increaseddependence on amino acids for energy production.
Alternatively, increasing evidence indicates that fatty acidremodeling substantially impacts the composition, integrity,and function of biological membranes (22, 27, 29, 42, 43). Forexample, the incorporation of PUFA, and to a lesser extentoleic acid, into phosphatidylethanolamine (PE) was recentlyshown to modify the biophysical organization and fluidity oflipid rafts (43). This may be due to unfavorable interactionsbetween the disordered acyl chain of PE with sphingomyelin,resulting in PE-rich nonraft microdomains that can impactprotein conformation (22). Such membrane reorganizationsadversely impact the localization of molecules that need to beclustered in close physical proximity, such as immunologicalsynapse proteins required for antigen presentation and T cellactivation (27). The relative contribution of DCI deficiency onthe composition of lipid species comprising the various struc-tural entities supporting HCV replication remains an impor-tant unanswered question. We are actively addressing thisquestion with additional metabolomic studies to better under-stand both the full breadth of the cellular lipidome in Huh7/DCI-1 cells as well as the influence of DCI deficiency on themetabolome in the context of HCV infection.
As the phenotypic impact of impaired lipid catabolism ap-pears to be most prominent under conditions of metabolicchallenge, such as dietary fasting, it will be crucial to performadditional studies aimed at characterizing global host, gene,protein, and metabolite changes occurring in DCI-deficientcells during the cellular stress induced during HCV infection.
Comparative analyses of the molecular changes occurring inpurified subcellular structures, including lipid rafts, lipid drop-lets, viral RNA replication complexes, mitochondria, and mi-tochondrion-associated membranes (MAM) during HCV in-fection of wild-type versus DCI-deficient Huh7 cells will helpto refine our understanding of how metabolic reprogramminginfluences current models of HCV infection and pathogenesis.
Further investigation of the importance of fatty acid oxida-tion in vivo may lead to the development of novel HCV ther-apeutics targeting host factors rather than viral proteins. TheHCV therapies now in development primarily target viral en-zymes such as proteases and polymerases and must be used incombination with PEGylated alpha interferon and ribavirintherapy. These regimens are not tolerated well by patients andcan select for drug-resistant viral mutants. Targeting host fac-tors such as DCI is an approach that could potentially elimi-nate the need for multidrug regimens with severe side effects infavor of a treatment that does not select for drug-resistantviruses, may be used as monotherapy, and completely blocksHCV replication. Together, these studies demonstrate our useof systems biology to identify and evaluate a novel host factorthat both advances our understanding of the biology of HCVinfection and provides an attractive potential new target fortreating this highly prevalent and deadly disease.
ACKNOWLEDGMENTS
We thank V. Racaniello (Columbia University, New York, NY) forproviding the infectious clone of poliovirus type 1/Mahoney strain andM. Gale (University of Washington, Seattle, WA) for providing stocksof HCV genotype 2a/SJ and dengue virus serotype 2/New Guinea Cstrain.
This study was supported by National Institute on Drug Abuse grant1P30DA01562501 to M.G.K. Portions of this work were performed atthe Environmental Molecular Sciences Laboratory, a national scien-tific user facility located at the Pacific Northwest National Laboratory(PNNL) and sponsored by the U.S. Department of Energy (DOE)Office of Biological and Environmental Research. PNNL is operatedby Battelle for the DOE under contract DE-AC06-76RLO-1830.
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