Glucose-6-Phosphate Dehydrogenase Enhances Antiviral Response through Downregulation of

18 Pages·2015·3.36 MB·English

Article
Glucose-6-Phosphate Dehydrogenase Enhances
Antiviral Response through Downregulation of
NADPH Sensor HSCARG and Upregulation of
NF-κB Signaling
Yi-HsuanWu1,†,DanielTsun-YeeChiu1,2,3,†,Hsin-RuLin4,Hsiang-YuTang2,
Mei-LingCheng1,2,5andHung-YaoHo1,2,*
Received:7October2015;Accepted:10December2015;Published:17December2015
AcademicEditor:CurtHagedorn
1 DepartmentofMedicalBiotechnologyandLaboratoryScience,CollegeofMedicine,
ChangGungUniversity,Tao-yuan333,Taiwan;[email protected](Y.-H.W.);
[email protected](D.T.-Y.C.);[email protected](M.-L.C.)
2 HealthyAgingResearchCenter,ChangGungUniversity,Tao-yuan333,Taiwan;[email protected]
3 DepartmentofLaboratoryMedicine,ChangGungMemorialHospital,Lin-Kou333,Taiwan
4 MolecularMedicineResearchCenter,ChangGungUniversity,Tao-yuan333,Taiwan;
[email protected]
5 DepartmentofBiomedicalSciences,CollegeofMedicine,ChangGungUniversity,Tao-yuan333,Taiwan
* Correspondence:[email protected];Tel./Fax:[phone]
† Theseauthorscontributedequallytothiswork.
Abstract: Glucose-6-phosphate dehydrogenase (G6PD)-deficient cells are highly susceptible to
viralinfection. Thisstudyexaminedthemechanismunderlyingthisphenomenonbymeasuring
the expression of antiviral genes—tumor necrosis factor alpha (TNF-α) and GTPase myxovirus
resistance1(MX1)—inG6PD-knockdowncellsuponhumancoronavirus229E(HCoV-229E)and
enterovirus71(EV71)infection.MolecularanalysisrevealedthatthepromoteractivitiesofTNF-αand
MX1weredownregulatedinG6PD-knockdowncells,andthattheIκBdegradationandDNAbinding
activity of NF-κB were decreased. The HSCARG protein, a nicotinamide adenine dinucleotide
phosphate(NADPH)sensorandnegativeregulatorofNF-κB,wasupregulatedinG6PD-knockdown
cellswithdecreasedNADPH/NADP+ratio.TreatmentofG6PD-knockdowncellswithsiRNAagainst
HSCARGenhancedtheDNAbindingactivityofNF-κBandtheexpressionofTNF-αandMX1,but
suppressedtheexpressionofviralgenes; however, theoverexpressionofHSCARGinhibitedthe
antiviral response. Exogenous G6PD or IDH1 expression inhibited the expression of HSCARG,
resultinginincreasedexpressionofTNF-αandMX1andreducedviralgeneexpressionuponvirus
infection. OurfindingssuggestthattheincreasedsusceptibilityoftheG6PD-knockdowncellstoviral
infectionwasduetoimpairedNF-κBsignalingandantiviralresponsemediatedbyHSCARG.
Keywords: G6PD;NADPH;coronavirus;enterovirus;antiviralresponse;HSCARG
1. Introduction
Glucose-6-phosphatedehydrogenase(G6PD)deficiencyisthemostcommonenzymopathyin
humans, affecting 400 million people worldwide [1]. G6PD plays an essential role in the pentose
phosphateshuntforreducingnicotinamideadeninedinucleotidephosphate(NADP+)toNADPH.
NADPHprimarilyservestoreduceequivalentsfornumerousbiochemicalreactionssuchasreductive
biosynthesis, glutathione reduction, detoxification, and NADPH oxidase-mediated superoxide
production. Therefore, G6PD helps to maintain cellular redox homeostasis [2,3], whereas G6PD
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Viruses2015,7,6689–6706
deficiencypredisposescellstoincreasedoxidativestress. G6PD-knockdowncellsexhibitpremature
senescence, growth retardation, and increased susceptibility to stress-induced apoptosis [4–6].
Clinically, in addition to the classical association with hemolytic anemia [7,8], patients with
G6PD-deficiency have an increased risk of degenerative diseases [9–12]. G6PD-deficient cells are
more susceptible to enterovirus, coronavirus, and dengue virus infections [13–15]. These findings
suggestthattheG6PDstatus,andhencetheredoxenvironment,isariskfactorforviralinfection.The
mechanismunderlyingtheeffectoftheredoxenvironmentonviralreplicationremainselusive.
Viral replication and spread is inhibited by the antiviral defense mechanisms of the host.
Thereplicationandspreadnormallyinvolvesactivationoftheantiviralinnateimmuneresponsesand
culminatesintheproductionoftypeIinterferons(IFNs)[16]andproinflammatorycytokinessuchas
tumornecrosisfactoralpha(TNF-α)[17,18]. BothIFNsandTNF-αareantiviralcytokinesanddisplay
strongantiviralactivityinhostinnateimmunity[19]. TypeIIFNsaretheprincipalantiviralcytokines
producedduringtheinnateimmuneresponsestoviralinfections,andupregulateantiviralproteins[20–22].
More than 300 IFN-stimulated genes (ISGs), which are initiated by type I IFN signaling, have been
discovered. Some ISG-encoded proteins, such as GTPase myxovirus resistance 1 (MX1) [23], protein
kinaseR(PKR)[24],and21-51-oligoadenylatesynthetase(OAS)[25],areimplicatedintheantiviralstate.
SeveralISGsareupregulatedbyinfectionwithcoronavirusorenterovirus[26–28].TNF-αinhibitedviral
infections[19],andendogenousTNF-αinhibitedtheenhancedsusceptibilitytoinfectiousdiseases[29].
Reactive oxygen species (ROS) play crucial roles in many cellular processes including cell
proliferation,differentiation,apoptosis,andsignaltransduction[30–32]. ROShavealsobeenshown
to trigger the signaling process of innate immune responses [33,34]. Oxidative stress affects viral
replicationpartlybyalteringhostimmunity[35,36]. TheinhibitoryeffectoftheG6PDstatusonviral
replicationisunknown,whichcanbeattributedtothealterationofthehostinnateimmuneresponse
frommodulatingtheredoxhomeostasisofthehostcells.
HSCARG,alsocalledNMRAL1,isanNADPHsensor;changesintheNADPH/NADP+ratiocan
induceallostericchangeandthesubcellularredistributionofHSCARG[37,38]. HSCARGregulates
theproteolysisofRelAandthephosphorylationofIKKβ[39]andplaysanessentialroleinNF-κB
signaling [39–41]. Moreover, HSCARG suppresses the TNF-α-stimulated activation of NF-κB [40],
suggestingthattheNADPH/NADP+ratioortheredoxstatusaffectstheNF-κB-mediatedimmune
response through the modulation of HSCARG. HSCARG can inhibit TRAF3 ubiquitination and
negatively regulate the cellular antiviral response [42]. Because of the crucial role of G6PD in
maintainingcellularNADPH/NADP+,examiningtherelationshipamongtheG6PDstatus,HSCARG,
andhostinnateimmuneresponseisworthwhile.
ThisstudyaddresseshowtheG6PDstatusaffectstheinnateimmuneresponsetoviralinfection.
TheexpressionofviralgenesissubstantiallyhigherinG6PD-knockdowncellsinfectedwithhuman
coronavirus strain 229E (HCoV-229E) and EV71 than in infected control cells. The expression of
antiviral genes, such as MX1 and TNF-α, is upregulated, albeit to lower levels, in G6PD deficient
cells. HSCARG,whoseexpressionisenhancedinG6PD-deficientcells,inhibitsIκBdegradationand
theDNAbindingactivityofNF-κB.ExogenousG6PDorisocitratedehydrogenase1(IDH1),which
increases cellular NADPH/NADP+, restores NF-κB-mediated antiviral response. These findings
demonstratethatG6PDactivity,andhenceNADPH/NADP+ status,canaffectantiviralimmunity
throughthemodulationofHSCARGandtheNF-κBsignalingcascade.
2. MaterialsandMethods
2.1. Materials
Thefollowingantibodieswereused: anti-G6PD(GenesisBiotech,Taiwan),anti-Histag,anti-IκB,
anti-IDH1,anti-β-actin,anti-HSCARG,antihorseradishperoxidase(HRP)-conjugatedantimouseIgG,
andantirabbitIgG(SantaCruzBiotechnology,SantaCruz,CA,USA).TNF-αsiRNA,HSCARGsiRNA,
andnontargetingsiRNAwerepurchasedfromDharmaconRNATechnologies(Lafayette,CO,USA).
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TheELISAkitforTNF-αwaspurchasedfromR&Dsystems(Minneapolis,MN,USA).BAY11-7085
andallotherchemicalswerepurchasedfromSigma-Aldrich(St. Louis,MO,USA).
2.2. CellCulture
A549 cells (human alveolar epithelial cell carcinoma), MRC-5 cells (human lung fibroblasts),
and RD cells (human rhabdomyosarcoma) were purchased from the American Type Culture
Collection(ATCC)(Manassas,VA,USA).ThecellswereculturedinDulbeccomodifiedEaglemedium
supplementedwith10%fetalbovineserumandantibiotics(100U/mLofpenicillinand100µg/mL
of streptomycin) at 37 ˝C in a humidified 5% CO atmosphere. The cassette for expressing G6PD
2
andscrambledcontrol(Sc)shRNAhasbeendescribedpreviously[6]. Thecassettewassubclonedin
pSuperior(Oligoengine,Seattle,WA,USA).Theretroviralvectorswerepackagedintoamphotropic
virus by using PT67 cells, as previously prescribed [6]. The A549 cells were transduced with the
packagedvirusandselectedforstabletransfectantsinamediumcontainingpuromycin(2µg/mL).
2.3. G6PDActivity
G6PD activity was measured spectrophotometrically at 340 nm according to the reduction of
NADP+inthepresenceofglucose-6-phosphate,aspreviouslydescribed[43]. Inbrief,thecellswere
collected and resuspended in lysis buffer (50 mM Tris-HCl (pH 7.4), 1% Triton X-100, 0.05% SDS,
150mMNaCl,1mMEGTA,and1mMNaF).Celllysateswerecentrifuged,andthesupernatantwas
usedintheassay. G6PDactivitywasanalyzedbycombiningtheproteinandassaybuffer(50mM
Tris-HCl(pH8),50mMMgCl ,4mMNADP+,and4mMglucose6-phosphate).
2
2.4. VirusPreparationandPlaqueAssay
HCoV-229EwasprovidedbyLaiMM(AcademiaSinica,Taiwan). Thestrainwaspropagated
intheMRC-5cellsandpurifiedthroughcentrifugation. ThelungcarcinomacelllineA549wasused
fortheplaqueassay. TheviraltiterwascalculatedaccordingtotheplaqueformationontheA549
cells,asdescribedpreviously[14]. Humanenterovirus71(BrCrstrain)waspurchasedfromtheATCC
(VR784). Theviruswaspropagated,andplaqueformationwasassayedontheRDcells. Theviruswas
aliquoted,quick-frozenondryice,andstoredat´70˝Cuntilused.
2.5. Quantitative-PCR
TotalRNAwasisolatedusingTrizolreagent. cDNAwasperformedusingtheSuperScriptIII
system(Invitrogen,Carlsbad,CA,USA).Primersweredesignedaccordingtothesequencesofhuman
antiviralgenecDNAsandthoseofviralgenes. ThesequencesoftheprimersusedinRT-PCRarelisted
inTableS1. Quantitative-PCRwasperformedusingtheIQ™SyBrGreenSupermixkitonanIQ5Real
TimeThermalCycler(Bio-Rad,Hercules,CA,USA).Relativefoldexpressionvaluesweredetermined
usingthe∆∆Ctmethod.
2.6. PreparationofCellExtractsandWesternBlotAnalysis
Thecellswerewashedwithice-coldphosphate-bufferedsaline(PBS),scrapedinlysisbuffer(20mM
Tris-HCl(pH7.5),150mMNaCl,1%NonidetP-40,1mMEDTA,10µg/mLaprotinin,10µg/mLleupeptin,
1mMphenlmethylsulfonylfluoride),andcentrifugedat40,000ˆgfor30minat4˝Ctoyieldthewhole-cell
extract. Samplesweredenatured,electrophoresedon10%SDS-polyacrylamidegel,andtransferredto
polyvinylidenedifluoride(PVDF)membranes.Themembraneswereincubatedovernightat4˝Cwithan
appropriatedilutionofaprimaryantibody(1:1000)inTris-bufferedsaline(TBS)(50mMTris-HCl,150mM
NaCl,0.05%(w/v)Tween-20(pH7.4))containing5%(w/v)bovineserumalbumin(BSA).Membraneswere
incubatedwitha1:4000dilutionofantirabbitorantimouse-HRPantibodyfor1.5h.Theimmunoreactive
bandswerevisualizedbyECLreagents(GEHealthcare,LittleChalfont,Buckinghamshire,UK),withthe
signalscapturedbyexposuretoX-rayfilm.
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2.7. PlasmidConstruction
The cDNA encoding His-tagged G6PD (Accession No. NM_000402), IDH1 (Accession No.
NM_005896), and HSCARG (Accession No. NM_001305141.1) were cloned in pCI-neo plasmid
(Invitrogen). The promoter region of the TNF-α and MX1 gene was cloned from human genomic
DNA,andsubclonedinapGL3-basicvector(Promega,Mannhein,Germany). AMX1promoterregion
devoid of NF-κB binding site (´244 to ´235) was engineered. Mutant (´242 to ´239; MX1P-mut;
MX1M)anddeletion(´244to´235;MX1P-del;MX1D)formsofMX1promoterwereestablishedby
site-directedmutagenesis(Stratagene,Amsterdam,Netherlands).
2.8. TransfectionofPlasmidsorsiRNAs
TheA549cells(5ˆ105)wereseededonsix-wellplatesandtransfected24hlaterwithplasmids
using Lipofectamine 2000 (Invitrogen). During transient transfection with siRNA, the cells were
transfectedwith10nMTNF-αorHSCARGsiRNA.ThenontargetingsiRNAwasusedasacontrolfor
nonspecificeffectsoftransfectedsiRNA.Duringtransienttransfectionwithplasmid,thecellswere
transfectedaccordingtothestandardprotocol(Invitrogen,CA,USA).Thecellswereharvestedfor
analysis,orinfectedwithHCoV-229E,24haftertransfection.
2.9. ElectrophoreticMobilityShiftAssay
G6PD-knockdown(Gi)andScA549cellswereinfectedwithHCoV-229Efortheindicatedperiods.
Nuclear proteins were extracted using the Nuclear Extract kit (Active Motif, Carlsbad, CA, USA).
Electrophoreticmobilityshiftassays(EMSAs)werecarriedoutusingtheLightShiftChemiluminescent
EMSAkitaccordingtomanufacturerprotocol(ThermoScientific,Rockford,IL,USA).Inbrief,the
extract(10µg)wasincubatedfor1hat4˝Cwithbiotinend-labeledprobe(ThermoScientific,Rockford,
IL,USA)containingNF-κBDNA-bindingsites(51-AGTTGAGGGGACTTTCCCAGGC-31);theextract
wasresolvedbynondenaturingPAGEon4%polyacrylamidegel,transferredtonylonplusmembrane,
anddeterminedbychemiluminescenceaccordingtomanufacturerinstructions.
2.10. LuciferaseAssay
TheA549cellsweretransfectedwith100ngofreporterplasmidtogetherwith400ngofapRL-null
Renillaluciferase-encodingvectorbyusingLF2000(Invitrogen, Carlsbad, CA,USA).Twenty-four
hourslater,theA549cellswereinfectedwithHCoV-229Eatamultiplicityofinfection(MOI)of0.1.
ThecellularextractwasassayedforluciferaseactivityusingadualluciferaseassayandGLOMAX
luminometer (Promega, Madison, WI, USA). Firefly luciferase activity was normalized to Renilla
luciferaselevels,andisexpressedrelativetopGL3-basiclevels(RLU).
2.11. DeterminationoftheNADPH/NADP+Ratio
DeterminationofNADPHandNADP+ wasperformedaspreviouslydescribed[44]. Inbrief,
thecellswerewashedtwicewithPBSandextractedin80%methanol/10mMKOHsolution. After
centrifugation at 14,000ˆ g for 5 min, the supernatant was retained and completely dried under
nitrogen gas. The sample was analyzed using ultra performance liquid chromatography (UPLC)
equippedwithaphotodiodearraydetector. ThesamplewaschromatographedonanAcquityHSST3
reversed-phaseC18column(2.1mmˆ150mm,particlesizeof1.8mm;WatersCorp.,Milford,MA,
USA). The mobile phase was composed of 25 mM potassium monobasic phosphate buffer, pH 6
(solventA),and100%methanol(solventB).Themobilephaseconditionswereasfollows: solventA,
2min,gradientfrom0to3%;solventB,0.5min,gradientfrom3%to4%;solventB,2.5min,gradient
from4%to15%;solventB,2min,gradient15%;andsolventB,1min. Thecolumntemperaturewas
maintainedat37˝C.Theflowratewassetat0.38mL/min. Absorbancespectrawereacquiredover
thewavelengthrangefrom260to340nm.
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2.12. StatisticalAnalysis
Statistical analyses were carried out using a two-tailed Student’s t test. A p value of ď0.05
wasconsideredstatisticallysignificant. Thedatawererepresentativeofatleastthreeindependent
experiments,andthevaluesweregivenasthemeanofreplicateexperiments˘standarddeviation(SD).
3. Results
Viruses 2015, volume, page–page
3.1. G6PDDeficiencyImpairstheExpressionoftheAntiviralGenes,TNF-αandMX1,uponHCoV-229Eor
2.12. Statistical Analysis
EV71Infection
Statistical analyses were carried out using a two-tailed Student’s t test. A p value of ≤0.05 was
considered statistically significant. The data were representative of at least three independent
TheA549celelxspwerimerenetsi, nanfde tchtee vdaluwes iwtherea girveent raos tvhei rmaelanv oef cretpolircaetex epxpreerismseinntsg ± Gsta6ndPaDrd -dsepvieatcioinf i(cSD()G. i)andScshRNA.
ThegeneratedA549-GiandA549-Scwereusedtodelineatethemechanismunderlyingtheincreased
3. Results
susceptibilityofG6PD-deficientcellstoviralinfection.TheexpressionofG6PDwassignificantlyreduced
3.1. G6PD Deficiency Impairs the Expression of the Antiviral Genes, TNF-α and MX1, upon HCoV-229E or
inA549-GicellscomparedwiththeA549-Sccells(Figure1A,toppanel).TheA549-Gicellswereinfected
EV71 Infection
withtheHCoV-229EvirusataMOIof0.1.ThetiterofprogenyvirusderivedfromtheinfectedA549-Gi
The A549 cells were infected with a retroviral vector expressing G6PD-specific (Gi) and Sc
cellswassignificanshtRlNyAh. iTghhe geernecroatmed pAa54r9e-dGi wandit Ah5t4h9-eSc iwnefree cutseedd toA d5el4in9e-aStec thcee mllesch(aFniigsmu urend1erAlyi,nbg othtet ompanel).These
increased susceptibility of G6PD-deficient cells to viral infection. The expression of G6PD was
findingsareconsistentwiththetemporalchangeintheexpressionoftheviralNgene.Theexpressionof
significantly reduced in A549-Gi cells compared with the A549-Sc cells (Figure 1A, top panel). The
theNgeneincreasAe5d49w-Giit chelltsh weertei minfeecotefd iwnifthe cthtieo HnC(oFVi-2g2u9Er evi1ruBs )a,t aan MdOwI oaf s0.1h. iTghhe etitreri nof tphroegeAny5 4vi9ru-Gs icellsthaninthe
derived from the infected A549-Gi cells was significantly higher compared with the infected A549-Sc
A549-Sccells.TheNgenelevelincreased304-foldintheA549-Gicellsversusanincreaseof106-foldinthe
cells (Figure 1A, bottom panel). These findings are consistent with the temporal change in the
A549-Sccellsat8hexppreosssitoinn offe tchtei ovinral( Np .gie.)n.e.A Thte2 e4xphrespsi.oin., otfh theer eN wgeanes iancnreaosvede wri1th7 t,h0e0 t0im-feo olfd inifnecctiroen aseintheNgene
(Figure 1B), and was higher in the A549-Gi cells than in the A549-Sc cells. The N gene level increased
levelintheA549-Gicellsand5,000-foldincreaseintheA549-Sccells.Thesefindingsareconsistentwith
304-fold in the A549-Gi cells versus an increase of 106-fold in the A549-Sc cells at 8 h postinfection
ourpreviousfindi(np.gi.)s. A[1t 244] h. p.i., there was an over 17,000-fold increase in the N gene level in the A549-Gi cells and
5,000-fold increase in the A549-Sc cells. These findings are consistent with our previous findings [14].

Figure 1. Expressions of antiviral gene MX1 and TNF-α decrease upon HCoV 229E infection in A549-
Figure1.ExpressiGoi ncesllso. f(Aa) nAt5i4v9-iSrca alngd e-Gni eceMlls wXe1re ahnardvesTteNd fFo-r αdetderemcirneataiosne ouf Gp6oPDn eHxpCreossVion2 b2y 9wEestienrnf ectioninA549-Gi
blotting. -Actin was used as internal control. A549-Sc and -Gi cells were infected with HCoV-229E
cells.(A)A549-Scand-GicellswereharvestedfordeterminationofG6PDexpressionbywesternblotting.
β-Actinwasusedasinternalcontrol.A549-Scand-G5 icellswereinfectedwithHCoV-229E(0.1MOI)for
24hthenviralparticlewasharvestedandproductionwasdeterminedusingplaqueassay;(B)A549-Scand
-GicellswereinfectedwithHCoV-229E(0.1MOI)forindicatedtimepoints.ViralNgeneexpressionwas
determinedbyquantitative-PCR.DatawerenormalizedtothevalueofinfectedA549-Sccellsat2hp.i.;
(C)RNAwasharvestedfromHCoV-229E-infectedcellsatindicatedtimep.i..TNF-αgeneexpressionwas
determinedbyquantitative-PCR.DatawerenormalizedtothevalueofuninfectedA549-Sccells;(D)RNA
washarvestedfromHCoV229E-infectedcellsatindicatedtimepointsp.i..MX1geneexpressionwas
determinedbyquantitative-PCR.DatawerenormalizedtothevalueofuninfectedA549-Sccells.Values
representaverage˘SDofthreeexperiments.*p<0.05ascomparedtoA549-Sccells.
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ThediscrepancybetweentheviralreplicationinnormalcellsandG6PD-deficientcellscorrelates
with their antiviral gene expression. Numerous antiviral genes were found to be upregulated in
theA549andMRC-5cellsafterinfectionwithHCoV-229E(Table1). Theexpressionoftheantiviral
genesTNF-αandMX1wasstudiedintheinfectedA549-GiandA549-Sccells. TNF-αmRNAlevels
increased more than 400-fold in the A549-Sc cells at 2 h p.i., and returned to the original levels at
8hp.i. (Figure1C).TheinductionlevelwasconsiderablylowerintheA549-Gicells,withthemRNA
levelsshowingonlya250-foldincreaseat2hp.i. (Figure1C,whitebars). Likewise,theexpression
oftheMX1geneincreasedduringtheinfectioncourseofHCoV-229E,andwassignificantlyhigher
intheA549-SccellsthanintheA549-Gicells(Figure1D).ThelevelofMX1mRNAincreasedover
22.8-foldat2hp.i. and774.2-foldat8hp.iintheA549-Sccells(Figure1D).However,thelevelof
inductionwasalsoreducedby40%at2hp.i. andby28%at8hp.iintheA549-Gicells. Thedifference
ininducibilityofantiviralgenesincontrolversusG6PD-deficientcellswasobservedinthecaseof
EV71infection,whichincreasedTNF-αandMX1expressioninRDcells(TableS2andFigureS1). The
expressionofTNF-α(FigureS1B)andMX1(FigureS1C)genesinG6PD-knockdownRD-Gicellswas
significantlylowerthaninRD-Sccells. Furthermore,G6PD-knockdownincreasedthesusceptibilityto
EV71infection(FigureS1A,rightpanel;G6PDproteinexpressionwasshownintheleftpanel). These
findingssuggestthattheexpressionofTNF-αandMX1issuppressedinG6PD-deficientcells.
3.2. TNF-αKnockdownEnhancesViralReplicationinA549Cells
TNF-αisimplicatedinthemodulationofthevirallifecycleandtheregulationofantiviralgene
expression[17]. TotestwhetherTNF-αexpressionlimitsvirusreplication,weknockeddownTNF-α
expressioninA549cellsandexaminedtheirviralsusceptibility.PretreatmentoftheA549cellswithsiRNA
againstTNF-αsignificantlyreducedHCoV-229E-inducedTNF-αexpressionthroughoutthecourseof
infection(Figure2A).Moreover,therewasanincreaseinviralgeneexpression(Figure2B).Conversely,
TNF-αpretreatmentsignificantlyinhibitedviralreplicationinadosedependentmanner(Figure2C).These
findingssuggestthatTNF-αplaysadeterminantroleintheantiviralresponse.Thereducedinducibilityin
G6PD-deficientcellsmayaccountfortheincreaseintheviralreplicationofthesecells.
Viruses 2015, volume, page–page

Figure 2. TNF-α inhibits viral replication in A549 cells. (A,B) The A549 cells were treated with control
Figure2.TNF-αinhiobr iTtNsFv-αi sriRaNlAr efopr 2l4i cha atnido chnallienngeAd 5w4ith9 HcCeoVll s22.9E( Aof ,0B.1 )MTOIh. TeotaAl R5N4A9 wcase hllasrvewsteed raet treatedwithcontrol
indicated time and analyzed for TNF-α mRNA and viral N gene expression; (C) The A549 cells were
orTNF-αsiRNAforc2ha4llehngead nwdith cdihffearelnlte cnongceentdratwion iotfh TNHF-áC foor V24 h2 th2e9n Einfeoctfed0 w.1ith MHCOoVI 2.29TEo oft a0.1l MRONI Awasharvestedat
indicatedtimeandanfora 8l hy. zToetadl RfNoAr wTasN haFrv-eαstedm foRr aNnalAyzinag nvidral vN igrenael exNpregsseionn bey equxapntirtaetisvse-iPoCnR. ;V(aClue)s TheA549cellswere
represent average ± SD of three measurements. * p < 0.05, as compared to control siRNA-treated cells.
challengedwithdifferentconcentrationofTNF-áfor24htheninfectedwithHCoV229Eof0.1MOI
for8h.TotalRNAwasharvestedforanalyzingviralNgeneexpressionbyquantitative-PCR.Values
representaverage˘SDofthreemeasurements.*p<0.05,ascomparedtocontrolsiRNA-treatedcells.
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Table1.TimecoursepatternofantiviralgeneexpressionintheA549andMRC-5cellsuponHCoV229Einfection.
FoldIncrease
CellType Gene
0h 2h 4h 6h 8h 10h 24h
HCoV229ENgene N.D. 1 5.08˘1.30 20.92˘6.45 102.73˘15.24 946.55˘83.16 5566.24˘312.68
TNF-α 1 432.58˘23.35 71.32˘0.41 27.05˘0.89 8.39˘1.29 7.71˘0.75 236.52˘33.27
IFN-α 1 0.89˘0.11 1.76˘0.83 1.29˘0.43 2.30˘1.14 1.90˘0.87 5.27˘1.73
A549 IFN-β 1 1.31˘0.09 1.17˘0.15 1.43˘0.27 0.86˘0.29 1.81˘0.35 504.07˘42.68
OAS 1 1.77˘0.32 5.49˘0.22 9.73˘0.42 11.64˘2.53 15.16˘2.15 27.1˘1.25
PKR 1 8.99˘2.94 7.45˘1.48 7.96˘1.51 8.08˘1.46 7.47˘1.54 8.72˘1.97
MX1 1 123.69˘1.09 522.69˘50.95 736.82˘194.12 1390.95˘65.63 2381.91˘205.05 3709.11˘172.71
HCoV229ENgene N.D. 1 1.90˘0.12 6.96˘1.41 77.96˘14.32 311.13˘30.21 1145.40˘104.50
TNF-α 1 1441.36˘343.85 975.38˘63.78 303.71˘60.77 137.94˘24.09 35.49˘2.29 7955.68˘664.50
IFN-α 1 0.78˘0.20 0.93˘0.32 1.39˘0.54 0.79˘0.52 1.63˘0.24 0.88˘0.43
MRC-5 IFN-β 1 0.92˘0.02 0.89˘0.12 0.98˘0.29 0.84˘0.05 1.23˘0.92 1194.91˘36.47
OAS 1 11.27˘1.33 93.33˘0.41 289.97˘27.38 521.57˘170.79 997.31˘182.12 4775.85˘620.45
PKR 1 4.10˘1.84 4.18˘1.69 5.16˘2.13 4.33˘0.24 5.00˘1.45 5.07˘0.51
MX1 1 121.73˘44.20 512.70˘4.42 1345.64˘105.59 1774.51˘185.55 1966.26˘333.56 6117.88˘674.95
N.D.:NotDetected.
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3.3. TranscriptionoftheTNF-αandMX1GenesIsReducedinG6PD-KnockdownCellsUponVirusInfection
3.3. Transcription of the TNF-α and MX1 Genes Is Reduced in G6PD-Knockdown Cells Upon Virus Infection
The effect of the G6PD status on the expression of the TNF-α and MX1 genes may be due
The effect of the G6PD status on the expression of the TNF-α and MX1 genes may be due to
todifferencesbetweenthetranscriptionallevelsofG6PD-knockdownandcontrolcells. Totestthis
differences between the transcriptional levels of G6PD-knockdown and control cells. To test this
hypothesis,A549-ScandA549-Gicellsweretransfectedwithreporterconstructs,whichhavepromoters
hypothesis, A549-Sc and A549-Gi cells were transfected with reporter constructs, which have
oftheTNF-αandMX1genes(Figure3A).Thetransfectedcellswereassayedforluciferaseactivity
promoters of the TNF-α and MX1 genes (Figure 3A). The transfected cells were assayed for luciferase
afterinfectionwithHCoV-229E.TheTNF-αandMX1promoteractivityintheA549-Sccellsincreased
activity after infection with HCoV-229E. The TNF-α and MX1 promoter activity in the A549-Sc cells
significantlyat24hp.i. comparedwiththatofthemock-infectedcontrol(Figure3B).Thepromoter
increased significantly at 24 h p.i. compared with that of the mock-infected control (Figure 3B). The
activitiesoftheTNF-αandMX1geneswerereducedintheA549-Gicells. Moreover,lowerpromoter
promoter activities of the TNF-α and MX1 genes were reduced in the A549-Gi cells. Moreover, lower
activities, especially those of the TNF-α gene, were observed in the A549-Gi cells compared with
promoter activities, especially those of the TNF-α gene, were observed in the A549-Gi cells compared
theA549-Sccellsunderbasalconditions. Thesefindingssuggestthatthetranscriptionalactivitiesof
with the A549-Sc cells under basal conditions. These findings suggest that the transcriptional
antiviralgenesareaffectedbytheG6PDstatus.
activities of antiviral genes are affected by the G6PD status.

Figure 3. Promoter activities of antiviral genes, TNF-α and MX1, and NF-κB binding activity is highly
Figure3.Promoteractivitiesofantiviralgenes,TNF-αandMX1,andNF-κBbindingactivityishighly
correlated with G6PD status upon HCoV 229E infection. (A) Schematic presentation of the TNF-α (−1
correlatedwithG6PDstatusuponHCoV229Einfection. (A)SchematicpresentationoftheTNF-α
to −1101) and MX1 (−1 to −896) promoter region are shown. The transcriptional start site is marked
(´1to´1101)andMX1(´1to´896)promoterregionareshown. Thetranscriptionalstartsiteis
with an arrow (+1). NF-κB binding sites are indicated with labeled boxes; (B) A549-Sc and -Gi cells
markedwithanarrow(+1).NF-κBbindingsitesareindicatedwithlabeledboxes;(B)A549-Scand-Gi
were transfected with control, TNF-α and MX1 reporter plasmid for 24 h, and were untreated or
cellsweretransfectedwithcontrol,TNF-αandMX1reporterplasmidfor24h,andwereuntreatedor
infected with HCoV-229E at MOI of 0.1. Luciferase activity was analyzed at 24 h p. i., and expressed
infectedwithHCoV-229EatMOIof0.1.Luciferaseactivitywasanalyzedat24hp.i.,andexpressedas
as mean ± SD (n = 3) of fold change relative to vector control. * p < 0.05 as A549-Gi cells compared to
mean˘SD(n=3)offoldchangerelativetovectorcontrol. *p<0.05asA549-Gicellscomparedto
A549-Sc cells alone or infected with virus, respectively; (C) A549-Sc and -Gi cells were infected with
A549-Sccellsaloneorinfectedwithvirus,respectively;(C)A549-Scand-Gicellswereinfectedwith
HCoV-229E at MOI of 0.1 for indicated periods. Nuclear extracts were prepared, and DNA binding
HCoV-229EatMOIof0.1forindicatedperiods.Nuclearextractswereprepared,andDNAbinding
activity was assayed by EMSA. DNA-protein complexes were resolved by eletrophoresis and detected
activitywasassayedbyEMSA.DNA-proteincomplexeswereresolvedbyeletrophoresisanddetected
by chemiluminescence.
bychemiluminescence.
Journal 2015, volume, page–page; doi:10.3390/ www.mdpi.com/journal/journalName
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Journal 2015, volume, page–page
3.4. BindingActivityofNF-κBIsDiminishedinVirus-InfectedG6PD-KnockdownCells
3.4. Binding Activity of NF-κB Is Diminished in Virus-Infected G6PD-Knockdown Cells
BecausetheNF-κBbindingsiteswerefoundinTNF-α[45]andMX1[46]promotersequences
Because the NF-κB binding sites were found in TNF-α [45] and MX1 [46] promoter sequences
(Figure3A),G6PDdeficiencyprobablyinhibitedtheexpressionofthesegenesbyalteringtheNF-κB
(Figure 3A), G6PD deficiency probably inhibited the expression of these genes by altering the NF-κB
signaling. ThebindingactivityofNF-κBincreasedintheA549-SccellsuponHCoV-229Einfection,
signaling. The binding activity of NF-κB increased in the A549-Sc cells upon HCoV-229E infection,
reachingthemaximallevelat60minp.i. anddecreasingafterwards. ThebindingactivityofNF-κB
reaching the maximal level at 60 min p.i. and decreasing afterwards. The binding activity of NF-κB
washigherintheA549-SccellsthanintheA549-Gicells(Figure3C).ThesefindingssuggestthatG6PD
was higher in the A549-Sc cells than in the A549-Gi cells (Figure 3C). These findings suggest that
deficiencyleadstodecreasedNF-κBactivationinvirus-infectedhostcells.
G6PD deficiency leads to decreased NF-κB activation in virus-infected host cells.
ToTfou rftuhrethrecro cnofinrfmirmth teher orloeleo fofN NFF-κ-κBBi nint hthee vviirruuss--iinndduucceedd uupprreegguulalatitoionn oof fththe eTNTNF-Fα- αanadn dMMX1X 1
gengeesn,ews,e wtere tareteadtedA A545949c eclelsllsw witihthB BAAYY1 111--77008855,, aa ssppeecciiffiicc NNFF--κκBB iinnhhibibitiotor,r ,aanndd exeaxmaminiende dthteh teemtepmopraolr al
chacnhganegien itnh etheex epxrpersessiosinono fofa natnitviviriarallg geenneess aafftteerr iinnffeeccttiioonn.. BBAAYY 1111--77008855 sisgignnifiificacnatnlytl yrerdeudcuecde dtheth e
indinudctuicotnioonf otfh tehTe NTFN-Fα-αa nadndM MXX11g geenneess( F(Figiguurree SS22)).. TThheessee ddaattaa ssuuggggeests tththata tNNF-Fκ-Bκ Bacatcivtiavtiaotnio ins is
esseesnsteinatlitaol tvoi rvuirsu-isn-idnudcuecdeda natnitviivriarlalg geenneee exxpprreessssiioonn..
InIcno ncotrnatrsatstto toth teheT NTNF-Fα-αg geenneet hthaatt hhaass tthhee pprroommootteerr eennddoowweedd wwitihth NNF-Fκ-Bκ Bbibnidnidnign sgitseist e[s45[]4, 5],
littlliettlies kisn konwonwnab aobuotutth teher orloeleo fofN NFF-κ-κBBi ninM MXX11 pprroommootteerr rreegguullaattiioonn.. TThhe emmodoedsets itnicnrceraesae sien intheth e
propmroomteortaecr tiavcittiyviotyf thoef MthXe 1MgXen1 egreanisee sradioseusb tdcoounbcte rcnoinncgertnhiengin vthoelv ienmvoenlvteomfeNnFt -κofB NinFM-κXB1 ipn roMmXo1t er
regpurloamtioonte.rV raergiuoluastiorenp. oVratreirovues crteoprosrctoern vtaeicntionrgs caolnutcaiifneirnags ea gluecniefelrianskee gdentoe vlianrkieodu stof ovramrisouosf fMorXm1s goefn e
MX1 gene promoter were constructed, and their activities were examined in the infected cells. The
promoterwereconstructed,andtheiractivitieswereexaminedintheinfectedcells. Theconstruct
construct MX1P had the wild-type MX1 gene promoter, while the constructs MX1D and MX1M had
MX1Phadthewild-typeMX1genepromoter,whiletheconstructsMX1DandMX1Mhadnobinding
no binding sites or had mutant NF-κB binding sites (Figure 4A). HCoV-229E induced significant
sitesorhadmutantNF-κBbindingsites(Figure4A).HCoV-229Einducedsignificantinductionof
induction of the MX1P luciferase construct (Figure 4B). By contrast, the luciferase activity of MX1D
theMX1Pluciferaseconstruct(Figure4B).Bycontrast,theluciferaseactivityofMX1DandMX1M
and MX1M upon HCoV-229E infection was not significantly different from the uninfected control.
uponHCoV-229Einfectionwasnotsignificantlydifferentfromtheuninfectedcontrol. Thesefindings
These findings suggest that NF-κB plays a crucial role in the transcriptional regulation of the MX1
suggestthatNF-κBplaysacrucialroleinthetranscriptionalregulationoftheMX1gene.
gene.

Figure 4. The promoter activity of MX1 gene can be modulated by NF-κB in HCoV 229E-infected cells.
Figure4.ThepromoteractivityofMX1genecanbemodulatedbyNF-κBinHCoV229E-infectedcells.
(A) Schematic presentation of the MX1 promoter region (−1 to −896). The transcriptional start site is
(A)SchematicpresentationoftheMX1promoterregion(´1to´896).Thetranscriptionalstartsiteis
marked with an arrow (+1). Three ISREs and one NF-κB (−234 to −244) binding site are indicated with
markedwithanarrow(+1).ThreeISREsandoneNF-κB(´234to´244)bindingsiteareindicatedwith
labeled boxes. A MX1 promoter region devoid of NF-κB binding site (−244 to −235) was engineered
labeledboxes.AMX1promoterregiondevoidofNF-κBbindingsite(´244to´235)wasengineered
as described in Materials and Methods. The underlined letters indicates the nucleotides to be changed
asdescribedinMaterialsandMethods.Theunderlinedlettersindicatesthenucleotidestobechanged
in MX1M-mut, and the dotted line indicates the nucleotides deleted in MX1P-del; (B) A549 cells were
in MX1M-mut, and the dotted line indicates the nucleotides deleted in MX1P-del; (B) A549 cells
transfected with firefly and Renilla luciferase reporter plasmids containing MX1P, MX1D, or MX1M
weretransfectedwithfireflyandRenillaluciferasereporterplasmidscontainingMX1P,MX1D,or
sequence. Twenty-four hours after transfection, cells were infected with HCoV 229E of 0.1 MOI.
MX1Msequence. Twenty-fourhoursaftertransfection,cellswereinfectedwithHCoV229Eof0.1
Luciferase activities was assayed at 48 h after transfection, and firefly luciferase activity was
MOI.Luciferaseactivitieswasassayedat48haftertransfection,andfireflyluciferaseactivitywas
standardized by Renilla luciferase activity. The values are expressed relative to MX1P under basal
standardizedbyRenillaluciferaseactivity. ThevaluesareexpressedrelativetoMX1Punderbasal
condition. * p < 0.05 as compared to basal condition ones.
condition.*p<0.05ascomparedtobasalconditionones.
3.5. HSCARG Expression Is Enhanced in G6PD-Knockdown Cells
HSCARG was proposed as a cellular redox sensor of the NADPH/NADP+ ratio, and a regulator
of NF-κB signaling. HSCARG blocks IκB degradation and inhibits NF-κB activation [39]. The finding
2
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Viruses2015,7,6689–6706
3.5. HSCARGExpressionIsEnhancedinG6PD-KnockdownCells
HSCARGwasproposedasacellularredoxsensoroftheNADPH/NADP+ratio,andaregulator
ofNF-κBsignaling. HSCARGblocksIκBdegradationandinhibitsNF-κBactivation[39]. Thefinding
Journal 2015, volume, page–page
revealedasignificantlylowerNADPH/NADP+ratioinA549-GicellscomparedwithA549-Sccells;
thriesvienaclreeda sae ssitghneifpicoasnsitblyil iltoywthear tNHASDCPAHR/GNiAsDinPv+o lrvaetido iinn tAhe54re9g-Gulia ctieollns ocfoNmFp-aκrBeds iwgnitahli nAg5(4F9i-gSucr cee5llAs;) .
Tthheisl eivneclreoafsHesS tChAe RpGostsriabnilsitcyr itphtawt HasSeCleAvRaGte dis iinntvhoelvAe5d4 i9n- Gthiec erlelgsucloamtiopna roefd NwFi-tκhBt hsaigtnoafltihneg A(F5i4g9u-rSec
ce5lAls)(. FTighue rleev5eBl) .oIfn HaSdCdAitiRoGn, ttrhaenlsecvreiplto wfHasS CelAevRaGtepdr ointe tihnew Aa5s4h9i-gGhie creilnlst hcoemAp5a4r9e-Gd iwceitlhls tthhaatn oifn tthhee
AA54594-9S-cScc eclellsls( F(Figiguurere5 5CB).).T Ihne asdedreitsiuonlt,s tshueg lgevesetl tohfa HttShCeAGR6GP Dprsottaetiuns wisaisn hvioglhveerd inin ththee Are5g49u-lGatii ocnellosf
HtShCanA iRnG theex pAr5e4ss9i-oSnc ,ctehlelsr e(bFyigaufrfee c5tCin)g. TNhFe-sκeB ressigunltasl isnugggaensdt athnatitv tihrae lGre6sPpDo nsstaet.us is involved in the
regulation of HSCARG expression, thereby affecting NF-κB signaling and antiviral response.

FiFgiugurere5 .5.E Exxppreresssisoionno offH HSSCCAARRGGi innccrreeaasseessi inn GG66PPDD--kknnoocckkddoowwnn AA554499 cceellllss.. ((AA)) NNAADDPPHH aanndd NNAADDPP++
cocnotnetnetnwt wereered deteetremrminiendedb ybyU UPPLLCC,a, nanddN NAADDPPHH//NNAADDPP++ rraattiioo wwaass ccaallccuullaatteedd.. ** pp << 00..0055 aass ccoommppaarreedd
to A549-Sc cells; (B) The level of HSCARG mRNA was determined in A549-Sc and A549-Gi cells by
to A549-Sc cells; (B) The level of HSCARG mRNA was determined in A549-Sc and A549-Gi cells
quantitative-PCR. The level of HSCARG gene expression was normalized to that of A549-Sc cells. * p
byquantitative-PCR.ThelevelofHSCARGgeneexpressionwasnormalizedtothatofA549-Sccells.
< 0.05, as compared to A549-Sc cells; (C) HSCARG protein in A549-Sc and A549-Gi cells was
* p<0.05, as compared to A549-Sc cells; (C) HSCARG protein in A549-Sc and A549-Gi cells was
quantified by western blotting, and -actin serves as internal control. The relative change in HSCARG
quantifiedbywesternblotting,andβ-actinservesasinternalcontrol.TherelativechangeinHSCARG
protein level is shown as compared to A549-Sc cells. Numbers shown below the upper panel indicate
proteinlevelisshownascomparedtoA549-Sccells.Numbersshownbelowtheupperpanelindicate
the fold change in HSCARG expression relative to that of Sc cells.
thefoldchangeinHSCARGexpressionrelativetothatofSccells.
3.6. Enhancement of the Antiviral Response and Inhibition of Viral Gene Expression Are Observed in
3.6. EnhancementoftheAntiviralResponseandInhibitionofViralGeneExpressionAreObservedin
HSCARG-Knockdown Cells
HSCARG-KnockdownCells
To investigate the role of HSCARG in the antiviral response, A549-Gi cells were transfected with
ToinvestigatetheroleofHSCARGintheantiviralresponse,A549-Gicellsweretransfectedwith
HSCARG-specific siRNA and the nontargeting siRNA control. Treatment of the A549-Gi cells with
HSCARG-specificsiRNAandthenontargetingsiRNAcontrol. TreatmentoftheA549-Gicellswith
HSCARG-specific siRNA caused a significant reduction in the level of HSCARG mRNA (Figure 6A).
HSCARG-specificsiRNAcausedasignificantreductioninthelevelofHSCARGmRNA(Figure6A).
The HSCARG-specific and nontargeting-siRNA-treated cells were infected with HCoV-229E (at a
TheHSCARG-specificandnontargeting-siRNA-treatedcellswereinfectedwithHCoV-229E(ataMOI
MOI of 0.1), and assayed for NF-κB binding activity. The NF-κB binding activity was enhanced in the
of 0.1), and assayed for NF-κB binding activity. The NF-κB binding activity was enhanced in the
HSCARG-specific, siRNA-treated cells. The NF-κB binding activity in these cells was compared with
HSCARG-specific,siRNA-treatedcells. TheNF-κBbindingactivityinthesecellswascomparedwith
that of the nontargeting siRNA-treated cells (Figure 6B). The effect of the siRNA treatment on the
that of the nontargeting siRNA-treated cells (Figure 6B). The effect of the siRNA treatment on the
temporal change of the expression of the TNF-α and MX1 genes were studied in the infected cells.
temporalchangeoftheexpressionoftheTNF-αandMX1geneswerestudiedintheinfectedcells.
The levels of the transcripts of these genes were elevated in the HSCARG-specific, siRNA-treated
The levels of the transcripts of these genes were elevated in the HSCARG-specific, siRNA-treated
cells compared with the nontargeting siRNA-treated cells during the course of HCoV-229E infection
cellscomparedwiththenontargetingsiRNA-treatedcellsduringthecourseofHCoV-229Einfection
(Figure 6C,D). The elevation in the expression of the antiviral genes was accompanied by a converse
(Figure6C,D).Theelevationintheexpressionoftheantiviralgeneswasaccompaniedbyaconverse
reduction in the expression of the viral N gene (Figure 6E). The overexpression of HSCARG in the
reductionintheexpressionoftheviralN gene(Figure6E).TheoverexpressionofHSCARGinthe
A549 cells decreased NF-κB binding activity. Therefore, antiviral gene expression increased
A549cellsdecreasedNF-κBbindingactivity. Therefore,antiviralgeneexpressionincreasedcoronaviral
coronaviral replication (Figure S3). These findings suggest that HSCARG acts diminish NF-κB
replication(FigureS3). ThesefindingssuggestthatHSCARGactsdiminishNF-κBactivation,and
activation, and suppresses antiviral gene expression in G6PD-knockdown cells.
suppressesantiviralgeneexpressioninG6PD-knockdowncells.
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