Gene Expression Signature-Based Screening Identifies New Broadly Effective Influenza A Antivirals

18 Pages·2010·3.73 MB·French

Gene Expression Signature-Based Screening Identifies
New Broadly Effective Influenza A Antivirals
Laurence Josset1,2*, Julien Textoris3,4,5, Be´atrice Loriod3, Olivier Ferraris1, Vincent Moules1, Bruno
Lina1,2, Catherine N’Guyen3, Jean-Jacques Diaz4, Manuel Rosa-Calatrava1*
1CentreNationaldelaRechercheScientifique(CNRS)FRE3011VirologieetPathologieHumaine,Universite´Lyon1,Lyon,France,2LaboratoiredeVirologieCentrede
BiologieetdePathologieEst,HospicesCivilsdeLyon,Lyon,France,3InstitutNationaldelaSante´etdelaRechercheMe´dicale(INSERM)U928TechnologiesAvance´es
pourleGe´nomeetlaClinique,Universite´delaMe´diterrane´e,Marseille,France,4CentreNationaldelaRechercheScientifique(CNRS)UMR5534,CentreLe´onBe´rard,
CentredeGe´ne´tiqueMole´culaireetCellulaire,Universite´Lyon1,Lyon,France,5Serviced’anesthe´sieetdere´animationHoˆpitalNord,AssistancePublique-Hoˆpitauxde
Marseille,Marseille,France
Abstract
Classical antiviral therapies target viral proteins and are consequently subject to resistance. To counteract this limitation,
alternativestrategieshavebeendevelopedthattargetcellularfactors.Wehypothesizedthatsuchanapproachcouldalso
beusefultoidentifybroad-spectrumantivirals.TheinfluenzaAviruswasusedasamodelforitsviraldiversityandbecause
oftheneedtodeveloptherapiesagainstunpredictablevirusesasrecentlyunderlinedbytheH1N1pandemic.Weproposed
toidentifyagene-expressionsignatureassociatedwithinfectionbydifferentinfluenzaAvirussubtypeswhichwouldallow
theidentificationofpotentialantiviraldrugswithabroadanti-influenzaspectrumofactivity.Weanalyzedthecellulargene
expressionresponsetoinfectionwithfivedifferenthumanandavianinfluenzaAvirusstrainsandidentified300genesas
differentiallyexpressedbetweeninfectedandnon-infectedsamples.Themost20dysregulatedgeneswereusedtoscreen
theconnectivitymap,adatabaseofdrug-associatedgeneexpressionprofiles.Candidateantiviralswerethenidentifiedby
theirinversecorrelationtothequerysignature.Wehypothesizedthatsuchmoleculeswouldinduceanunfavorablecellular
environment for influenza virus replication. Eight potential antivirals including ribavirin were identified and their effects
were tested in vitro on five influenza A strains. Six of the molecules inhibited influenza viral growth. The new pandemic
H1N1virus,whichwasnotusedtodefinethegeneexpressionsignatureofinfection,wasinhibitedbyfiveoutoftheeight
identified molecules, demonstrating that this strategy could contribute to identifying new broad anti-influenza agents
acting on cellular gene expression. The identified infection signature genes, the expression of which are modified upon
infection,couldencodecellularproteinsinvolvedinthevirallifecycle.Thisisthefirststudyshowingthatgeneexpression-
basedscreeningcanbeusedtoidentifyantivirals.Suchanapproachcouldacceleratedrugdiscoveryandbeextendedto
otherpathogens.
Citation:JossetL,TextorisJ,LoriodB,FerrarisO,MoulesV,etal.(2010)GeneExpressionSignature-BasedScreeningIdentifiesNewBroadlyEffectiveInfluenzaA
Antivirals.PLoSONE5(10):e13169.doi:10.1371/journal.pone.0013169
Editor:Man-SeongPark,HallymUniversity,RepublicofKorea
ReceivedJune2,2010;AcceptedSeptember9,2010;PublishedOctober4,2010
Copyright: (cid:2) 2010 Josset et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkwassupportedbyCNRS(CentreNationaldelaRechercheScientifique),theHospicesCivilsdeLyon,theEuropeanVigilanceNetworkforthe
ManagementofAntiviralDrugResistance(VIRGIL),andbyagrantofthe"ProgrammedeRechercheA(H1N1)"co-ordinatedbytheInstitutdeMicrobiologieet
MaladiesInfectieuses(INSERM).Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected](LJ);[email protected](MRC)
Introduction vitro and in vivo inhibition of two different cellular pathways
without inducing resistance has been reported, and both are
Antiviral drug development is currently based on two currently undergoing preclinical trials(recently reviewedin [4]).
approaches:i)theconventionalapproachofinhibitingtheactivity Targeting cellular proteins may provide another crucial
of a viral enzyme which often leads to the emergence of drug advantage: if a cellular pathway is critical to the viral cycle,
resistant viruses due to viral genomic variability and ii) the more agents that target such a pathway should represent potential
recent approach of targeting cellular factors that are required for broad-spectrum antivirals. The influenza virus represents a
viralreplication.Indeed,codingforalimitednumberofproteins, constant threat to public health due to the emergence of new
viruses hijack the cellular machinery and rely on many host viral strains and is therefore an ideal model on which to test this
proteinsfortheirreplication.Themajorrecognizedadvantageof hypothesis.
targeting a host factor is therefore to limit the development of Belonging to the orthomyxoviridae family, influenza viruses
resistanceastheviruscannotreplaceamissingcellularprotein[1]. havegenomescomposedofsingle-strandedRNAandareclassified
Suchanapproachhasbeenusedinantiretroviraltherapywiththe into three types: A, B and C according to their internal protein
development of a CCR5 antagonist showing promise as an anti- sequences[5].TheinfluenzaAvirusesarefurthersubtypedbased
HIV drug [2]. We have also demonstrated that this strategy is on the antigenicity of the two envelope glycoproteins hemagglu-
efficient at inhibiting the replication of herpes viruses resistant to tinin(HA)[H1toH16]andneuraminidase(NA)[N1toN9].All
conventional antivirals [3]. In influenza research, the effective in influenza A subtypes are endemic in aquatic birds but only two,
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H1N1andH3N2,arepresentlycirculatingamonghumans.Since dose50%(TCID50)inMDCKcellsasdescribedinourprevious
the influenza genome is segmented, two different viral strains study [15].
infectingthesamecellareabletoreassorttheirgenomicsegments. Forthemicroarrayanalysis,A549cellswereinfectedfor24hat
Variability can also be due to the low fidelity of the viral RNA 37uCwithinfluenzavirusesatamultiplicityofinfection(moi)of1in
polymerase, whichcausesyearlyepidemicsowingtoan antigenic DMEMsupplementedwith2mML-glutamine,100Uofpenicil-
drift in glycoproteins. Novel pathogenic strains of the influenza lin/mL,100mgofstreptomycinsulfate/mLand0.5mgoftrypsin/
virus have also emerged with antigenically different HA and/or mL(infectionmedium).Thismoiwaschosentoensurethat100%of
NA and have caused three pandemics in the 20th century: the the cells were infected 24h postinfection. The microarray
Spanish influenza (H1N1) in1918, responsible forapproximately experimentswereperformedinfiveindependentreplicates.
50 million deaths; the Asian influenza (H2N2) in 1957 during For kinetics on A549 cells, confluent cells were infected with
which about 2–4 million people died; and the Hong Kong influenzavirusesatamoiof0.1or2foronehourunderaminimal
influenza (H3N2) in1968responsible for1–2million deaths [6]. volumeofinfectionmediumat37uC.Thecellswerethenoverlaid
Considering this pandemic potential and with up to 500,000 with fresh infection medium and incubated at 37uC. Samples of
annualdeathsworldwideduringusualwinteroutbreaks,influenza supernatants were collected at defined time points and stored at
Avirusesrepresentamajorpublichealthconcern[7].Prevention 280uCuntilendpointtitrationassays(TCID50)inMDCKcells.
reliesonvaccinationwhichhasseveralmajorlimitationsincluding
the lag time for vaccine preparation and the low vaccination 2 RNA preparation and hybridization to the gene chip
coverage rate. Once a patient becomes infected, the current Total RNA was extracted from cell pellets using an RNeasy
etiologic treatment of flu relies on M2 channel blockers or NA Mini Kit (Qiagen,Valencia,CA) for the BSL2 viruses. For H5N1
inhibitors[8].However,theseexistingtherapiesareinappropriate infections, total RNA was extracted with Trizol LS (Invitrogen).
foruseincasesofsevereinfectionandmaybelimitedduetothe mRNAs were labeled with 33P for the reverse transcription using
riskofrapidemergenceofdrugresistantviruses.Thusthereisan the Superscript III RT (Invitrogen), (a33P)dCTP and an
obvious need to complement existing therapies with new anti- oligodT25. Generated cDNAs were hybridized on home-made
influenza drugs. Nylon microarrays (HuSG9k) containing 9216 spotted IMAGE
To search for new antivirals, we hypothesized that common human cDNA clones, representing 8682 genes and 434 control
viral effects on cell metabolism should occur after infection with clones [16]. Further details on the HuSG9k microarray are
different avianandhuman influenzavirusesandthatthispattern available on the TAGC website (http://tagc.univ-mrs.fr/). All
shouldleadtotheidentificationofdrugseffectiveonallinfluenza membranes used in this study belonged to the same batch. After
A viruses potentially. We first sought to identify a common gene hybridizationandexposureonMicroImager,arrayswerescanned
expression signature following the infection with different human ina FujiBAS 5000machine andhybridization signalsquantified
and avian influenza A viruses. While several microarray analyses using the BZ Scan Software [17]. Primary data, in accordance
havealreadycomparedthepandemic1918H1N1virus[9,10]or with the proposed MIAME standards, are accessible through
some H5N1 strain [11,12] to other less pathogenic strains, our GEOSeriesaccessionnumberGSE22319(http://www.ncbi.nlm.
study is the first to demonstrate that a global influenza-induced nih.gov/geo/query/acc.cgi?acc=GSE22319).
gene-expression signature can be defined. This proof-of-concept
studywasconductedonahome-madenylonarrayusingahuman 3 Data normalization and analysis
pulmonary epithelial cell line infected by five influenza A virus Data files were loaded and analyzed with R (v2.9.2) and
subtypes (H1N1, H3N2, H5N1, H5N2 and H7N1). Using this Bioconductor(v2.4.1)[18],usingtheNylonArraylibrarydeveloped
signature, we determined if molecules disturbing this pattern of by the TAGC to support BZScan2 files (library available upon
infection would have a broad-influenza antiviral effect. By request). Raw data were normalized by quantile normalization.
consulting the Connectivity Map, a database of drug-associated Supervisedanalysis(supervisedmethodsaimatfindingasetofgenes
gene expression profiles [13,14], we identified molecules that whose expression profiles best correlate with a known phenotype)
induced gene expression changes after cell treatment that were betweengroupsInfectedandMocksampleswasconductedusingthe
mainly opposite to those induced by infection. These molecules SignificanceAnalysisofMicroarrayalgorithm(SAM)[19],usingthe
weretestedinvitrofortheireffectonthefivedifferentviruses.To siggenes library (v1.18.0) [20]. All statistical analyses involved
confirm our methodology, we took the opportunity of using the corrections for multiple comparisons (Benjamini and Hochberg)
newemergingpandemicH1N1virusasamodeltotesttheeffect [21]. Agglomerative hierarchical clustering was performed by the
of these moleculeson a newunknownvirus. pairwise average-linkage method using the Pearson correlation
distance(Cluster3.0,Eisen,StanfordUniversity).
Materials and Methods
4 Quantitative real-time RT-PCR validation
1 Cell lines and viruses To validate the microarray results with real-time RT-PCR
CellsofthehumanlungepithelialcelllineA549weregrownas assay,anothersetofA549cellswereinfectedwithinfluenzaviruses
monolayers in Dulbecco’s modified Eagle’s medium (DMEM) at a moi of 1 and total cell RNA was extracted at 24 hpi with
supplemented with 10% fetal bovine serum, 2 mM L-glutamine, TrizolLS(Invitrogen).FivehundredngoftotalRNAwerereverse
100U of penicillin/mL, and 100mg of streptomycin sulfate/mL transcribed using oligo(dT)18 andRevertAid M-MuLV (Fermen-
at 37uC. tas) according to the manufacturer’s instructions. OnemL of
Influenza viruses A/New Caledonia/20/99 (H1N1), A/Mos- cDNA was then amplified and analyzed in the 7500 Real Time
cow/10/99(H3N2),A/Lyon/969/09(H1N1SOIV),A/Turkey/ PCRSystem(AppliedBiosystems)usingthePlatinum(R)SYBR(R)
582/2006 (H5N1), A/Finch/England/2051/94 (H5N2), and A/ Green qPCR SuperMix-UDG kit (Invitrogen) according to the
Chicken/Italy/2076/99(H7N1)wereproducedinMDCKcellsin manufacturer’s instructions. Six genes were chosen according to
EMEM supplemented with 2 mM L-glutamine, 100U of penicil- theirlevelofexpression(Foldchangeinlog .2or,22)andthe
2
lin/mL, 100mg of streptomycin sulfate/mL and 1mg of trypsin/ availability of primers for the quantitative PCR (Table S1).
mL. Viruses were titrated to determine tissue culture infection Glyceraldehyde3-phosphatedehydrogenase(GAPDH)mRNAwas
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used as an internal control. The reaction mix contained a total 8 Neuraminidase assay
volume of 20mL and the thermal cycling consisted of UDG Standard fluorometric endpoint assays used to monitor NA
incubationat50uCfor2 min,40cyclesof95uCfor15sand60uC activity was recently shown to be suitable to quantify influenza
for33sforamplification.Alldatawerenormalizedtotheinternal virus in a high-throughput screening test [26]. Briefly, cell
standard GAPDH mRNA. For each single-well amplification supernatants (25ml) were transferred to a black 96-well plate
reaction, a threshold cycle (Ct) was observed in the exponential and 75ml of 29-(4-methylumbelliferyl)-alpha-N-acetylneuraminic
phase of amplification. Relative changes in gene expression were acid(MUNANA,SigmaChemicalCo.)toafinalconcentrationof
determined using the 2DDCt method as previously described [22] 50mMwereadded.Afterincubationoftheplateat37uCfor1hr,
and reported as the n-fold difference relative to a control cDNA 150mlstopsolution(0.05Mglycine,pH 10.4)wasaddedtoeach
(mock cells) prepared in parallel with the experimental cDNAs well and the fluorescence read on a FluoStar Opima (BMG
(infectedcells).StatisticalsignificancewascalculatedusingWelch’s Labtech) with excitation and emission filters of 355nm and
two sample t-test between mock and infected samples using R 460nm respectively. Relative fluorescence units (RFU) were
software. corrected by subtracting specific blanks, ie medium with or
without molecules.
5 In silico experiment: query the Connectivity Map with For the NA activity test on L3 viruses (H5N1), viruses were
the infection signature inactivated as previously described [27]. Cell supernatants were
To select potential antivirals, an unbiased in silico search for mixedwithfreshlypreparedTritonX-100toafinalconcentration
molecules that reverse the infection signature identified in the of 1% (vol/vol) Triton X-100 and incubated for 1 h at room
presentstudywasperformedusingthepubliclyavailableConnec- temperature. Theinactivated supernatants werethentransported
tivity Map database (build 02) [13]. The Connectivity Map (also outoftheBSL3totheBSL2laboratoryandusedforNAassaysas
known as CMAP) is a collection of genome-wide transcriptional described above.
data from cultured human cells treated with different kinds of Potential interference of test molecules on the NA enzymatic
molecules. The 20 most differentially expressed genes in the activity was tested by incubating the A/Moscow/10/99 (H3N2)
infectionstate(FoldChangeinlog2.2or,22)wereselectedfrom viral stock diluted in DMEM (107.8 TCID50/mL final) with
theinitial300genesetidentifiedbySAM.Thesewerethenmapped increasing concentrations of the test molecule (or DMEM for
to the U133Aprobesets in order toquery theConnectivity Map control) for 0.5h at room temperature. Specific blanks were
database. In total, 28 U133A probe sets mapped to the selected measured for each molecule. 25mL were used for the NA test as
genes from this study. The connectivity scores and p-values were describedaboveandresultswereexpressedasaratioofcorrected
obtainedusingtheCMAPalgorithm[13]. RFU of the sample to RFU of controls. Two independent
experiments were performed induplicate.
6 Molecules
2-aminobenzenesulfonamide (Sigma), calcium folinate (Sigma), 9 Viral growth assays in the presence of the molecules
harmol hydrochloride (MP Biomedical), merbromine (Sigma), For the viral growth assays in the presence of the molecules,
midodrine (Sigma) and ribavirin (Valeant Pharmaceuticals) were A549 cells were seeded into 96-well plates at 0.156105 cells per
dissolvedinsterilewatertoastockconcentrationof5g/L,5g/L, wellandculturedfor3daysto100%confluence.Cellswerethen
4 g/L, 3.4 g/L, 5 g/L and 10mM respectively. Rilmenidine washedwithDMEMandincubatedwithvariousconcentrationsof
(Sigma) was dissolved in dimethylsulfoxide (DMSO) to a stock the different molecules diluted in infection medium (DMEM
concentration of 13g/L and brinzolamide was in suspension at supplemented with 2 mM L-glutamine, 100U/mL penicillin,
10g/L inthecollyrium AZOPT. 100mg/mL streptomycin, 20mM HEPES and 0.5 mg/mL
Sulfameter (Sigma), pyrvinium (Sigma), moxalactam (Sigma) trypsin). Six hours after treatment, cells were infected with
andmethylbenzethoniumchloride(Sigma)weredissolvedinsterile influenza viruses at a moi of 2 or 0.2 by adding 25mL per well
water to a stock concentration of 50g/L. Alvespimycin (Sigma) of virus diluted in infection medium. Infection was allowed to
was dissolved in sterile water to a concentration of 0.03g/L. proceed for 65h at 37uC, 5% CO2 after which 25mL of
Sulodictil (Sigma) and DL-Thiorphan (Sigma) were dissolved in supernatant were collected for the NA activity test. Results are
DMSO toaconcentration of 50g/L. expressed as a ratio of corrected RFU of the sample to RFU of
control(incubationwithinfectionmediumwithouttestmolecule).
7 Viability assays To check for cytotoxicity, viability assays were performed in
parallel toeachviral growthassay.
Cell viability was measured by the neutral red assay, an
indicatorofcytotoxicityusedinculturesofdifferentcelllines[23]
with the same sensitivity as the MTT assay [24,25]. The neutral 10 Test of infection efficiency after cell or virus
redassayisbasedontheinitialprotocoldescribedbyBorenfreund pre-incubation with the molecules
and Puerner (1984) and determines the accumulation of the A549cellswereseededinto96-wellplatesat0.156105cellsper
neutral red dye in the lysosomes of viable, uninjured cells. Cells well and cultured for 3 days to 100% confluence. For the ’Cell
were seeded into 96-well plates and treated with molecules or Preincubation’test,cellswerewashedwithDMEMandincubated
solvent. 72h after treatment, cells were incubated for 3 h with with various concentrations of the different molecules diluted in
neutral red dye (100 mg/ml) dissolved in serum free medium 200mLperwellofinfectionmediumfor14h.Aftertwowashings
(DMEM).Cellswerethenwashedwithphosphatebufferedsaline withDMEM,cellswereinfectedwithinfluenzaA/Moscow/10/99
(PBS) and fixed in a formol/calcium mix (40%/10%) for 1min (H3N2)virusatamoiof7during15minandwashedtwicewith
beforebeinglysedwithEtOH/AcCOOH,(50%/1%)followedby infection medium. Infection was allowed to proceed for 5 h at
gentleshakingfor15minuntilcompletedissolutionwasachieved. 37uC. For the ‘Virus Preincubation’ assay, the molecules were
Absorbance at 550nm was measured using a microplate diluted in infectionmedium andA/Moscow/10/99 (H3N2)viral
spectrophotometer system (Microplate Reader 2001, BioWhit- stock (108.8 TCID /mL) was treated with increasing concentra-
50
taker) and results were presented as a ratioofcontrol values. tions of the molecules for 14h. Cells were then washed with
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DMEM and incubated for 15min with the virus and molecule A/Moscow/10/99 (H3N2) and avian A/Turkey/582/2006
mixdiluted12times.Infectionwasallowedtoproceedfor5 hat (H5N1), A/Finch/England/2051/94 (H5N2), and A/Chicken/
37uC. In both assays, the number of infected cells was estimated Italy/2076/99 (H7N1) influenza viral strains. These viruses are
withaNAtest.CellswerewashedwithPBSandlysedbyshaking hereinreferredtoasH1N1,H3N2,H5N1,H5N2andH7N1.A549
for 1h with 25mL per well of Triton 1X. The cell lysis extracts cells express both sialic acid a2,6- and a2,3-galactose receptors
were used for a neuraminidase test as described above. Results [30,31]andwereshowntobeinfectedbyhuman,avianandswine
were expressedasaratioofcorrected RFUof sampletoRFUof influenza viruses [32,33]. Infections were performed at 37uC, a
control (solvent treated infections). Statistical significance was temperature at which both human and avian influenza viruses
calculatedincomparisontoresultsforcontrolcellsusingtwotailed efficiently infect cell cultures [34] and at a moi of 0.1. In these
Welch ttest. conditions,therewasevidenceofproductiveviralreplicationofall
virusesbutwithsomekineticandyielddifferencesbetweenviruses,
11 EC and CC calculations as determined by infectious titers (TCID ) of supernatants of
50 50 50
Viability and antiviral data were analyzed using the following influenzavirusinfectedA549cells(Figure1).TheH5N1virustiters
three-parameter non linear logisticregression(l3) function [28] peakedhigherandearlier(24hpi)comparedtoothervirusestiters.
AvianH7N1andH5N2virusesreplicatedwithcorrectefficiencies,
similar to the human H3N2 virus. In contrast, the human H1N1
D
y~Dz virusstrainreplicatedslower(titerspeakedat65hpi)andgrewto
(cid:2)x(cid:3)B
1z lowertitersthanotherviruses(p-value,0.05at24hpi).
E
Todeterminethehostgene-responsetoinfection,totalcellular
RNA was extracted at 24 hpi and submitted to reverse
were y is the response, D is the upper limit (response when the transcriptioninthepresenceof33P.Eachconditionwasperformed
dose x is ‘infinite’), E is denoted EC or CC and is the dose in 5 independent replicates. All labeled cDNAs provided a good
50 50
producingaresponsehalf-waybetweentheupperlimitandlower radioactiveintensityandwerehybridizedontohome-madenylon
limit (0), and B is the relative slope around E. This model is the microarrays containing 8782 IMAGE cDNA clones. All hybrid-
shortened form of the four parameter logistic function where the izations were of good quality according to signals within
lower limit is fixed to 0. Results were obtained by fitting the l3 acceptable range, number of features present, and signals from
functionusingthepackagedrc[29]intheRStatisticalLanguage control spots.
(version2.7.1). Parameters of the l3 model were estimated and Supervised analysis of normalized gene expression data was
fitted curves were plotted only if the data set contained one conducted using theSAMalgorithm. Thisalgorithm was usedto
response ,D/2. identifygeneswhoseexpressionlevelsweresignificantlyalteredby
influenzainfection.WesetthedeltathresholdintheSAManalysis
Results to allow an acceptable false discovery rate (FDR) of 10%. We
found that the expression levels for a total of 300 genes
1GlobaltranscriptionalsignatureofinfluenzaAinfection (representing 3.4% of the genes considered present on the chip)
To characterize the global cellular gene-expression response to differed significantly between mock and infected samples (Table
influenza A infection, human pulmonary epithelium A549 cells S2). Using the DAVID Bioinformatics Resources database, we
wereinfected with human A/New Caledonia/20/99(H1N1) and annotatedthissignatureusingthegeneontology(GO)terms.This
Figure1.ComparisonofviralreplicationkineticsbetweeninfluenzavirusesinA549cells.A.A549cellswereinfectedatamoiof0.1with
influenza virus A/New Caledonia/20/99 (H1N1), A/Moscow/10/99 (H3N2), A/Turkey/582/2006 (H5N1), A/Finch/England/2051/94 (H5N2), and A/
Chicken/Italy/2076/99(H7N1)andsupernatantswerecollectedat24,41,48and65hpiforendpointtitrationassays.B.Eachbarrepresentsthemean
ofviraltitersat24hpifortwoindependentexperimentsandtiterswerestatisticallyanalyzedbytheonetailedWelcht-test;*:titersgreaterthan
H1N1titers(p-value,0.05),#:titerslowerthanH5N1titers(p-value,0.05).
doi:10.1371/journal.pone.0013169.g001
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revealed an enrichment of genes related to various cellular A subset of six genes with absolute fold changes in log (FCs)
2
processes such as protein complex biogenesis, membrane and above 2 was selected to validate the microarray analysis by
microtubule organization, DNA metabolic and catabolic process- quantitative RT-PCR (RT-qPCR) analysis: DNMT1, NTE and
es,cellproliferationregulation,cellcycleandcelldeath(Figure2). CAPN1thatwerefounddownregulatedininfectedcellsandG1P2,
Figure 2. Gene enrichment analysis of the 300 genes of the infection signature. Discriminatory genes were analyzed by DAVID for
associations with particular Gene Ontology terms. The negative Log (P-values) of enriched terms (plotted in bar) refer to how significant an
10
associationaparticularontologytermhaswiththegenelist.The30mostsignificantbiologicalprocessaregroupedaccordingtotheirbiological
meaning.
doi:10.1371/journal.pone.0013169.g002
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OAS1andICAM1thatwereupregulated.The6geneswerechosen 3020byH7N1and1455byH5N1.Themaindifferencebetween
atrandomamongthemost20dysregulatedgenesuponinfection. H1N1 and other viruses lay in the number of down-regulated
This quantification was performed on new samples equivalent to genesduringinfection.WhereasH3N2,H5N1,H5N2andH7N1
those used for the microarray analysis. Figure 3 shows the influenza viruses induced a down-regulation of most of the genes
confirmationbyRT-qPCRofthemicroarraydata.Foreachgene tested,asimilarnumberofgenesweredown-andup-regulatedby
andeachstrain,microarrayFCsarepresentedasablackboxplot H1N1 (highlighted by the blue vertical line in the heatmap
and RT-qPCR results are depicted as a gray histogram. Results Figure 4). As H1N1 viral titer was lower at 24 hpi than titers of
from RT-qPCR were in good agreement with the cDNA other viruses (Figure 1B), the scope of gene-expression changes
microarrayanalysesforfiveoutofsixgenestested.Indeed,except induced upon infection correlated, at least partially, to the viral
for CAPN1 (p-value=0.1), significant difference between infected replication efficiencyof thevirus-cell system usedinthisstudy.
andnoninfectedcellswasalsoobservedinquantitativeRT-PCR Interestingly, out of the 300 genes of the global infection
analysis(p-value,0.05,Welcht-test),similartoDNAmicroarray signature,only16wereupregulatedinallinfectedcells.These16
analysis. This result was acceptable considering that samples genes were associated to three GO biological process, including
analyzed by RT-qPCR were different from those used in the two related terms, ‘‘viral reproductive process’’ and ‘‘viral
microarray analysis. reproduction’’, that annotate genes encoding proteins involved
TovisuallycomparethechangesinmRNAabundanceforthe in the virus life cycle. Two genes were associated to these terms:
300 genes found to be influenced by influenza infection, ICAM1, which is the major receptor for human rhinovirus [35],
hierarchicalclusteringanalysisinbothdimensionswasperformed. and IRF7, which activates the expression of Epstein-Barr Virus
Results are depicted in the heatmap representation of Figure 4. Latent Membrane Protein 1 [36] (p-value=0.07 and 0.08,
Dendrogramsindicatethecorrelationbetweensamplesandgenes. respectively). While IRF7 has not been directly involved in
We verified that mock samples were sorted together vs infected influenza virus life cycle yet, ICAM1 was recently identified as a
ones. The H1N1 samples co-clustered with the mock samples proviralfactorthatmaybeco-optedbyinfluenzavirus[37].The
suggesting that infection with this strain induced few gene third associated biological process was the term ‘‘immune
expression changes. We verified this result by conducting a response’’ (p-value=0.04) annotating 4 genes (ICAM1, OASL,
virus-specificSAManalysisonthemockvsonevirussamples.For OAS1 and CFD). Therefore, the upregulated genes were mostly
aFDRof10%,only36geneswerefoundtoberegulatedbyH1N1 associatedwiththeimmunologicalresponse.Besides,sevenofthe
infectionincomparisonto2298genesbyH3N2,1510byH5N2, 16 genes were interferon stimulated genes (ISGs): IFITM1,
Figure3.Validationofmicroarrayresultsbyreal-timequantitativeRT-PCRanalysis.Expressionof6genesfromDNAmicroarrayanalysis
(blackcross)iscomparedwithreal-timequantitativePCRdata(greybar).Crossrepresentfoldchanges(mean6S.D.)givenbythemicroarrayanalysis =
Log (normalizedamountoftargetgeneininfectedsamples)-Log (normalizedamountoftargetgeneinmocksamples)(right).Theformulausedto
2 2
determinetheamountoftargetgeneininfectedcellsbyRT-qPCR,normalizedtoGAPDHandrelativetomockis:22DDCtwhereCtisthethresholdcycle
andDDCt = (Cttargetinfected–Ctgapdhinfected)–(Cttargetmock–Ctgapdhmock).BarrepresentedLog2expressionlevel(mean6S.D.)oftarget
genesintwoindependentquantitativeRT-PCRanalysis(left).
doi:10.1371/journal.pone.0013169.g003
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InfluenzaSignatureInversion
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InfluenzaSignatureInversion
Figure4.Hierarchicalclusteringandheatmapofthe300genesthatdiscriminatemockandinfectedsamples.Heatmaprepresenting
theexpressionlevelsofthe300genesdifferentiallyexpressedbetweeninfectedandmockcells.Redsquaresindicatetheexpressionlevelsabovethe
medianofthegeneabundanceacrossthesampleandgreensquarestheexpressionlevelsbelow.Themedianvalueswereclusteredhierarchicallyin
bothdimensions.Dendogramsindicatethecorrelationbetweengroupsofsamples(horizontal)andgenes(vertical).Verticallinesintherightportion
indicatethe2distinctgeneexpressionpatterns:upanddown-regulatedgenesduringinfection.Thebluelinestandsforthegenesdown-regulatedin
samplesinfectedwithH1N1influenzavirus.Geneexpressiondataforthe300genesarereproducedintableS2.
doi:10.1371/journal.pone.0013169.g004
ICAM1, IFIT3, OAS1, G1P2 (or ISG15), IRF7 and OASL. These bythemolecule,noneofthemoleculesavailableinthisdatabank
results were in accordance with previous studies showing the wereabletocompletelyreversetheinfectionsignature.
upregulation of immune response associated genes in samples
infected in vitro and in vivo with various influenza viruses 3 Evaluation of the antiviral potency of the selected
[9,11,12,37,38,39,40]. Gene expression levels in each group of drugs on H3N2 viral growth
samplesaredepicted inFigureS1.AllISGsweremarkedlymore
Weassessedtheeffectoftheeightselectedmoleculesoninfluenza
up-regulated in H5N1 infected cells than in other samples. This
replication in vitro. Cell viability, as assessed by the neutral red
hyperstimulation has been described in other transcriptional
assay, and viral growth, as quantified by a neuraminidase (NA)
studies [11,40,41] reinforcing the validity of the experimental
activitytest,wereconductedinparallel.BeforeusingtheNAactivity
cell-virus systemdeveloped inthepresent study.
test asanindirect measurementforviralimpairment,wechecked
firstly that the different influenza viruses used in this study had
2 In silico drug screening of the Connectivity Map sufficientneuraminidaseactivitiestobequantifiedusingthismethod
The Connectivity Map is a collection of genome-wide (FigureS2A).Foralltestedvirusesandforasignaltobackground
transcriptional expressiondatafromculturedhumancellstreated ratio between 2 and 70, the fluorescence was proportional to the
with bioactive small molecules [13]. The associated website amountofviruspresent(TCID /mL).Duringtheevaluationofthe
50
provides tools tofindmoleculesconnected tothequery signature drugpanel,allsignaltobackgroundratioswereincludedbetween2
i.e.anylistofgenesassociatedwithabiologicaltest.Thesimilarity and70.Secondly,wecontrolledthatthedifferentmoleculesdidnot
ofthequerysignaturetoeachofthereferenceexpressionprofilesis inhibittheenzymaticactivityofNAtobesurethatadropinRFU
assessed and quantified by a normalized score, from -1 for a would only reflect a drop of viral titer. While concentrations of
molecule that reverses the signature to +1 for a molecule which merbrominabove50mMandharmolabove500mMinhibitedNA
induces geneexpression changessimilar tothequery signature. activity,incubationoftheviruswithincreasingconcentrationsofthe
Our strategy was to query the Connectivity Map with a list of molecules otherwise resulted in no inhibition (Figure S2B).
genes differentially expressed in infected cells to find molecules Therefore, for these two molecules below these concentrations
that induced the opposite gene expression changes. We hypoth- and for other molecules of the drug panel, viral growth can be
esized that such molecules may influence host cell metabolism in assessedbyaneuraminidasetest.
such away that effectiveviral replication wouldbealtered. Evaluation of the drug panel was first conducted on influenza
A critical step in this screening was to define the query A/Moscow/10/99 (H3N2) virus. A549 cells were incubated with
signature. As the number of upregulated genes was very low increasingconcentrationsofthemoleculefor6hbeforeinfection.
(5.3%) in the list of 300 genes defined by the analysis, a lack of Thistimewaschosenbasedonthedurationoftreatmentindicated
specificity resulting from a loss of information for up-regulated intheConnectivityMaptoobtainsimilarcellularresponsebefore
genescouldbeintroducedindrugselectionifthesignaturewasnot infection [14]. Infection was allowed to proceed for 65h which
corrected for this bias. By selecting genes with the most drastic representsmultiplecyclesofinfection,howeversimilarresultswere
changes in level of expression (fold change in log . 2 or ,22), observed at 24and48hpi(Figure S3).
2
wewereabletodefineasignatureof20genesforinfluenzaAvirus Theviabilitydataoffiveindependentexperimentsaregivenin
infection with similar amounts of those up and down regulated Figure S4. The 50% cytotoxic concentrations (CC ) were
50
(Figure 5A,Table 1). determined by regression analysis. The CC of calcium folinate,
50
Byqueryingtheconnectivitymapwiththisconcisesignature,we 2-aminobenzenesulfonamide and midodrine could not be deter-
obtainedc-scoresfor6100instances,representingmorethan1000 mined since none of these molecules was cytotoxic at the highest
moleculesinvariousconditions[14].Weselectedthoseassociated tested dose.
with the most strongly anticorrelated signatures (negative enrich- The effect of each of the molecules on viral growth was tested
ment)andwhichhadap-valuelessthan0.5%(Figure5Band5C). usingtheH3N2virusatamoiof0.2and2.Dose-responsecurves
Applying this filtering step left us with eight candidate molecules: were fitted by regression analysis (Figure S5) and used to
harmol, rilmenidine, brinzolamide, ribavirin, calcium folinate, determine the 50% effective concentration (EC ) of each
50
2-aminobenzenesulfonamide, merbromin and midodrine (from molecule if at least one response was inferior to 50%. Selective
the most negatively correlated to the least negatively correlated indexes (SI) were calculated as CC /EC and used to classify
50 50
drug).Therelevanceofourselectionwassupportedbythefactthat selectedmoleculesasinactive(SI,2),weakinhibitors(2,SI,10),
ribavirin,analreadyknowninfluenzavirusinhibitor,wasidentified moderate inhibitors (10,SI,50) and strong inhibitors (SI.50)
withanegativeenrichmentof-0.83andapvalueof0.00157.Except (Figure5).Inagreementwithpreviousobservations[42],wenoted
for the topical antiseptic merbromin, theother selected molecules that SI were dependent on the moi, since molecules are more
havevarioustherapeuticindications(depictedinTableS3)butare effective at lower moi. In our conditions, at a moi of 0.2, two
notreferencedasantivirals. molecules (calcium folinate and 2-aminobenzenesulfonamide)
Graphs in Figure 5C report how the different genes of the were ineffective, two (harmol and merbromin) were weak
infectionsignaturebehaveintheexpressionprofileoftheselected inhibitors, two (brinzolamide and midodrine) were moderate
molecules.Althoughthegenesdown-regulatedduringinfectionare inhibitorsandone(ribavirin)wasastronginhibitor.Atamoiof2,
generallyup-regulatedinresponsetothemoleculeandconversely whereas brinzolamide was reclassified as a weak inhibitor, the
theup-regulatedgenesofthesignaturearegloballydown-regulated othermoleculesremainedinthesameclassdespitetheirSIbeing
PLoSONE | www.plosone.org 8 October2010 | Volume 5 | Issue 10 | e13169
InfluenzaSignatureInversion
PLoSONE | www.plosone.org 9 October2010 | Volume 5 | Issue 10 | e13169
InfluenzaSignatureInversion
Figure5.Geneexpression-basedscreeningidentifieseightpotentialantiviralmolecules.A.Listofcellulargeneschosentoquerythe
ConnectivityMap.Acircumscribedsignatureofinfectionwasderivedfromthe300genesdiscriminatingmockandinfectedsamplesbyselecting
geneswithafoldchange.2or,22.Thefoldchangeisdefinedastheratioofthemeangeneexpressionininfectedsamplestothemeanforthe
correspondingmockinfectioninlog .Thisselectionresultedinalistof20genes,12beingup-regulatedduringinfectionand8down-regulated(see
2
Table 1). These genes constituted the signature used to query the online database Connectivity Map. B. Drugs with significant enrichment to
influenzavirusinfectionsintheConnectivityMap.Significancecut-offwassetatp-value,0.005.Thepermutationp-valueestimatesthelikelihood
thattheresultswouldbeobservedbyrandomchance.Mean:thearithmeticmeanoftheconnectivityscoresforthepost-dosechanges(orinstances)
bythegivenmolecule;n:thenumberofinstancesofagivenmoleculeintheCMAPdatabase;Enrichment:ameasureoftheenrichmentofthose
instancesintheorderlistofallinstances,Positiveenrichmentscoresareofinterestifperturbagensinducingthebiologicalstaterepresentedbythe
signatureusedtoproducetheresultaresought.Likewise,ifreversalorrepressionofthebiologicalstateencodedinthequerysignatureisrequired,
perturbagenswithnegativeenrichmentscoresareofinterest.Thespecificityvalueisdefinedasthefrequencyatwhichtheenrichmentofasetof
instancesequalsorexceedsthatofthesamesetofinstancesinqueriesexecutedon312published,experimentallyderivedsignaturesusingthe
MolecularSignaturesDatabase.Lowervaluesareassociatedwithagreaterspecificity;thenonnullpercentagerepresentsameasureofthesupport
fortheconnectionbetweenasetofinstancesandasignatureofinterestbasedonthebehavioroftheindividualinstancesinthatset.C.8molecules
arenegativelyconnectedtoinfluenzavirusinfection(p-value,0.005).Agraphicalrepresentationofthelocationofthesignatureofinfectionis
depictedforeachmolecule,takingtheinstancewiththemostnegativeconnectivityscoreofeachmolecule.Thex-axisrepresentsthegenesofthe
expressionprofileofthemolecule,rankorderedaccordingtotheirdifferentialexpressionrelativetothecontrol.Thelocationofeachgeneofthe
infectionsignatureisappreciatedalongthex-axis.
doi:10.1371/journal.pone.0013169.g005
weaker. As an example, the CC for midodrine was superior to used to obtain 1550.7mM of rilmenidine- and the EC was
50 50
4250mMandEC wascomprisedbetween322mM(moiof0.2) comprised between 1.0% (moi of 0.2) and 1.8% (moi of 2). The
50
and 532mM (moi of 2). Concerning rilmenidine, which was EC ofrilmenidinewassignificantlydifferentfromthatofDMSO
50
dissolved in DMSO, it was not possible to conclude on an effect. at a moiof 2(p=2e24)but not at a moiof0.2 (p=0.85).
DMSO has previously been shown to be cytotoxic and to inhibit However,evenifthismoleculeisconsideredineffectiveagainst
influenzainfectionabove4%(vol/vol)[43]howeveritisstillused theH3N2influenzavirus,wedidobtainaveryhighconfirmation
asamajorsolventformoleculesinhigh-throughputscreening.In rate (62.5%) in comparison with the hit rate of classical high-
thisstudy,theCC forDMSOwas2.9%(v/v)-theconcentration throughput screening (3.2% in [43]). This clearly indicates that
50
Table1. Genesof thecircumscribed signature ofinfection.
Gene Genesymbol I.M.A.G.E.CloneID FoldChange
TranscriptionRegulation
Etsvariantgene3 ETV3 1473929 7.24
Myocyteenhancerfactor2D MEF2D 4209 5.13
FBJmurineosteosarcomaviraloncogenehomologB FOSB 79022 22.69
DNA(cytosine-5-)-methyltransferase1 DNMT1 768241 22.06
AnkyrinrepeatandBTB(POZ)domaincontaining2 ABTB2 204456 2.28
Immuneresponse
ISG15ubiquitin-likemodifier,G1P2 ISG15 742132 3.48
29,59-oligoadenylatesynthetase1,40/46kDa OAS1 666703 2.07
ComplementfactorD(adipsin),DF CFD 666128 2.99
Cellsurfaceprotein
Patatin-likephospholipasedomaincontaining6,NTE PNPLA6 431990 22.01
Heparansulfateproteoglycan2 HSPG2 3339 22.23
Solutecarrierfamily2(facilitatedglucosetransporter) SLC2A2 207963 2.23
PTPRFinteractingprotein,bindingprotein1(liprinbeta1) PPFIBP1 263094 2.04
Intercellularadhesionmolecule1(CD54) ICAM1 3383 3.36
Lysophosphatidicacidreceptor1 LPAR1 505524 3.63
Enzyme
Hyaluronoglucosaminidase4 HYAL4 668088 2.90
Asparagine-linkedglycosylation2homolog ALG2 150443 2.35
Calpain1,(mu/I)largesubunit CAPN1 70555 22.20
Proteinphosphatase1,PP1 PPP1R14D 140525 23.13
Nuclearprotein
Karyopherin(importin)alpha6 KPNA6 16650 22.21
HypotheticalproteinHSPC111 NOP16 505242 22.08
FoldChange = log2(Inf/Mock).
doi:10.1371/journal.pone.0013169.t001
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