Research Article RNA-Seq Based Transcriptome Analysis of the...

Research ArticleRNA-Seq Based Transcriptome Analysis of the Type I InterferonHost Response upon Vaccinia Virus Infection of Mouse Cells

Bruno Hernaacuteez1 Graciela Alonso1 Juan Manuel Alonso-Lobo1 Alberto Rastrojo1

Cornelius Fischer23 Sascha Sauer23 Begontildea Aguado1 and Antonio Alcamiacute1

1Centro de Biologıa Molecular Severo Ochoa Consejo Superior de Investigaciones Cientıficas-Universidad Autonoma deMadrid (CSIC-UAM) 28049 Madrid Spain2Max Planck Institute for Molecular Genetics 14195 Berlin Germany3Max Delbruck Center for Molecular Medicine Robert-Rossle-Str 10 13092 Berlin Germany

Correspondence should be addressed to Antonio Alcamı aalcamicbmcsices

Received 14 October 2016 Accepted 9 January 2017 Published 9 February 2017

Academic Editor Frank A Schildberg

Copyright copy 2017 Bruno Hernaez et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Vaccinia virus (VACV) encodes the soluble type I interferon (IFN) binding protein B18 that is secreted from infected cells andalso attaches to the cell surface as an immunomodulatory strategy to inhibit the host IFN response By using next generationsequencing technologies we performed a detailed RNA-seq study to dissect at the transcriptional level the modulation of the IFNbased host response byVACV and B18 Transcriptome profiling of L929 cells after incubationwith purified recombinant B18 proteinshowed that attachment of B18 to the cell surface does not trigger cell signalling leading to transcriptional activation Consistentwith its ability to bind type I IFN B18 completely inhibited the IFN-mediated modulation of host gene expression Addition ofUV-inactivated virus particles to cell cultures altered the expression of a set of 53 cellular genes including genes involved in innateimmunity Differential gene expression analyses of cells infected with replication competent VACV identified the activation of abroad range of host genes involved in multiple cellular pathways Interestingly we did not detect an IFN-mediated response amongthe transcriptional changes induced by VACV even after the addition of IFN to cells infected with a mutant VACV lacking B18This is consistent with additional viral mechanisms acting at different levels to block IFN responses during VACV infection

1 Introduction

Type I interferons (IFNs) constitute a family of relatedcytokines (IFN-120572 subtypes IFN-120573 and other IFN familymembers) that bind a commonandheterodimeric cell surfacereceptor (IFNAR) and play an important role in the firstline of defence against virus infections [1ndash3] After initialmolecular recognition of the invading virus by host cellpattern recognition receptors (PRRs) these IFNs are secretedand bind cognate cellular receptors to exert their functioneither locally or distally This binding initiates the Januskinase (JAK)signal transducers and activators of transcrip-tion (STAT) signalling cascade to trigger the activation ofdiverse host genes depending on cell type with potentantiviral activity that contributes to the establishment of anantiviral state in the adjacent healthy cells and the activationof the apoptotic program to eliminate infected cellsThus the

main purpose of the IFN response is to limit virus replicationand infection spreading [4]

Vaccinia virus (VACV) is the most studied member ofthe Poxviridae family of large DNA viruses with cytoplasmicreplication VACV is the vaccine used to eradicate smallpoxmore than 30 years ago and constitutes an excellent model toanalyze the evasion of the IFN based host response to viralinfection Viruses have to neutralize the antiviral activity ofIFNs and in this sense VACV and other poxviruses seemto be unique encoding a plethora of genes to this effect(reviewed in [2 3 5 6]) Among others VACV encodesthe A46 and A52 protein to inhibit toll-like receptor (TLR)signalling that leads to IFN production [7] and VH1 todephosphorylate STAT1 and STAT2 [8 9] but also diverseproteins to specifically inhibit the antiviral activity of someIFN-induced genes This is the case of the E3 and K3

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2017 Article ID 5157626 12 pageshttpsdoiorg10115520175157626

2 Journal of Immunology Research

proteins that employ two different mechanisms to counter-act double-stranded RNA-dependent protein kinase (PKR)effector functions [10 11] Additionally E3 binds the productof the IFN-stimulated gene 15 (ISG15) to prevent its antiviralaction [12] But one the most efficient strategies employedby poxviruses to avoid IFN effects is to encode IFN bindingproteins that are secreted from infected cells to prevent theinteraction of IFNs with their cellular receptors In the case ofVACV strain Western Reserve (WR) the type I IFN bindingprotein is encoded by theB18R gene (B19R in theCopenhagenstrain) A relevant role of this protein in VACV pathogenesiswas soon assigned since the lack of B18R expression afterintranasal infection of mice resulted in an attenuated virusindicating that blocking the IFN host response is crucial forthe development of VACV infection [13]The B18 protein hasno amino acid sequence similarity to cellular IFN receptorsand in contrast to the cellular counterparts binds IFN120572120573from a broad range of host species [13] The protein issynthesized early after VACV infection is secreted into themedium and is found as a soluble form or anchored to thecell surface [14 15] This binding to the cell surface has beenshown to occur via interaction of the B18 amino terminuswith glycosaminoglycans (GAGs) [16] and allows B18 toprevent the establishment of an IFN-induced antiviral statein cells surrounding the infection site

In the present study by using RNA sequencing with theIllumina technology (RNA-seq) and differential gene expres-sion analyses we have further analyzed the ability of B18 toblock the IFN based response in a mouse fibroblast cell lineWe also extend the study to VACV-infected cells to identifychanges in host gene expression profile induced by VACV ora VACV mutant lacking the B18R gene (VACVΔB18) withspecial emphasis on the inhibition of the type I IFN-inducedhost cell response

2 Materials and Methods

21 Cell Culture and Reagents Mouse L929 cells were used toobtain RNA samples for high-throughput sequencing whileBSC-1 cells (African green monkey kidney origin) were usedto prepare virus stocks Recombinant His-tagged VACV B18protein was expressed in the baculovirus system and purifiedas previously described [17] Protein purity was checked onCoomassie blue-stained SDS-PAGE and quantified by geldensitometry Murine recombinant IFN-120572 subtype A waspurchased from PBL Assay Science (gt95 pure) diluted inphosphate-buffered saline and maintained at minus70∘C untiluse

22 Viruses and Infections Virulent VACV strain WR andthe correspondent VACV mutant lacking B18R expression(VACVΔB18 [14]) were grown in BSC-1 cells and stocks ofsemipurified virus were prepared by sedimentation througha 36 sucrose cushion L929 cells were infected with VACVor VACVΔB18 with a multiplicity of infection of 5 plaqueforming units (pfu)cell in order to ensure the infectionof all cells to obtain a representative RNA-seq profile ofeach condition After adsorption of virus for 1 h at 37∘Cthe virus-containing medium was removed and cells were

washed twice with phosphate-buffered saline and replacedwith fresh culture medium supplemented with 2 fetalbovine serum Infected cells were then incubated at 37∘Cand harvested at 4 or 8 h postinfection (hpi) by scrappingWhere indicated IFN (50 unitsml) was added to the infectedcultures at 4 hpi and the incubation extended at 37∘C to 9 hpiInactivation of viruseswas performed as previously described[18] by incubation with 2120583gml psoralen (4-9-aminomethyl-trioxsalen Sigma) for 10min and then UV-irradiated for10min with 225 Jcm2 in a Stratalinker 1800 Completeinactivation (gt108-fold reduction in pfu) was confirmed byplaque assay in BSC-1 cells

23 RNA Extraction and Illumina RNA-Seq Library Prepa-ration Immediately after harvesting the samples total cel-lular RNA was isolated from 12 times 106 L929 cells usingSV Total RNA Isolation System (Promega) RNA sampleswere quantified on a spectrophotometer (NanoDrop ND-1000 Thermo Scientific) and quality-analyzed in an Agi-lent 2100 Bioanalyzer (Agilent Technologies Santa ClaraCA US) All samples exhibited a RNA integrity number(RIN) over 9 The sequencing libraries were generated withTruSeq RNA Sample Prep Kit v2 Set A (Illumina) Brieflypoly(A) containing mRNA molecules were purified in tworounds using oligo(dT) attached magnetic beads from 1120583gof total RNA After chemical fragmentation mRNA frag-ments were reverse-transcribed and converted into double-stranded cDNA molecules Following end-repair and dA-tailing paired-end sequencing adaptors were ligated to theends of the cDNA fragments using TruSeq PE Cluster Kit v3-cBot-HS (Illumina)

24 Deep Sequencing and Sequence Analysis Libraries weresequenced using TruSeq SBS Kit v3-HS (Illumina) on anIllumina Hiseq 2000 machine at the Max Planck Insti-tute for Molecular Genetics Berlin More than 108 100 ntpaired-end reads were obtained from each sample andafter quality assessment with package FastQC (httpwwwbioinformaticsbabrahamacukprojectsfastqc) the fastqfiles containing these reads were mapped to the mousegenome (build GRCm38 from Mus musculus C57BL6Jstrain) together with the VACV WR genome (GenebankAY2433121) using Tophat v204 with default parameters [19]Only those reads aligned against mouse genomewere consid-ered in a differential gene expression analysis with Cuffdiff(Cufflinks v210 software [19]) Since biological duplicatesof samples from untreated cells were available all compar-isons were performed against this sample using the defaultmode of Cuffdiff which is the most suitable for our kindof data Pathway analysis of the significantly differentiallyexpressed genes detected was performed using IngenuityPathway Analysis (IPA) software Creation of proportionalVenndiagrams and gene expression heatmapswere generatedwith the R ldquoVennDiagram v169rdquo and ldquoGplotsrdquo packagesrespectively The raw RNA-seq data has been deposited atthe European Nucleotide Archive (ENA) under the projectnumber PRJEB15047

Journal of Immunology Research 3

25 mRNA Expression by Real-Time-PCR (RT-PCR) Toevaluate the expression levels of selected genes by RT-PCR1 120583g of DNA-free total RNA isolated from L929 cells (threebiological replicates per condition) was used for first strandcDNA synthesis with iScript cDNA Synthesis (BioRad) usingoligo(dT) and random primers Quantitative polymerasechain reaction (qPCR) analysis was performed using FastSYBR Green PCR Master Mix (Applied Biosystem) withthree technical replicates for each biological replicateaccording to the manufacturerrsquos recommendation in an ABI7900 HT system (Applied Biosystem) Gene-specific qPCRprimers were designed using primer3Plus (httpwwwbi-oinformaticsnlcgi-binprimer3plusprimer3pluscgi) anddescribed in Table S1 in Supplementary Material availableonline at httpsdoiorg10115520175157626 Amplificationwas real-time-monitored and allowed to proceed in theexponential phase until fluorescent signal reached asignificant value (Ct) The fold change was determined usingthe 2minusΔΔC(t) method [20]

3 Results

31 The Type I IFN Cellular Response Is Inhibited in thePresence of the VACV B18 Protein To characterize theinhibitory role of the VACV type I IFN binding proteinB18 on IFN signalling we analyzed the RNA-seq profile ofmouse L929 cells incubated with recombinant B18 beforeand after IFN treatment We first determined the effect oftype I IFN on global cellular gene expression and performedhigh-throughput RNA sequencing on total RNA obtainedfrom cells mock-treated or treated with 50 unitsml ofIFN-120572 for 4 h Under these conditions we identified a setof 46 significantly differentially expressed genes (SDEGs)after IFN treatment when compared to mock-treated cells(Table S2) Most of them (42 genes) were found to beupregulated in response to IFN while only 4 genes weredownregulated This set of IFN-stimulated genes (ISGs) con-tained several genes with previously known direct antiviralactivity such as APOL9 BST2 (Tetherin) DDX58 (RIG-1)EIF2AK2 (PKR) IFITM3 ISG15 MX2 OAS-1 PARP12 orTRIM We also identified some ISGs involved in the positiveregulation of IFN production such as IRF9 STAT1 STAT2TRIM21 or TRIM30 and others encoding immunomodula-tory molecules such as IL15 H2-Q1 (HLA-B) or UBC Weconsidered this our high-confidence gene set and performeda pathway enrichment analysis that mainly identified IFNrelated canonical pathways as statistically significant enriched(Figure 1(c)) indicating that L929 cells used in this studyexhibited an appropriate biological response to IFN

However when cells were incubated with 045 120583gml ofrecombinant protein B18 2 h before IFN addition we couldnot detect any significant change in cellular gene expressionindicating that the IFN-induced cell response was efficientlyprevented by the addition of B18 (Figure 1) At the same timeit was confirmed that this concentration of B18 effectivelyprotected against the antiviral effects of IFN (50Uml) usingVesicular stomatitis virus infection in HeLa cells (data notshown)

B18 is secreted from VACV-infected cells and has beenpreviously shown to interact with GAGs at the surface ofuninfected neighbouring cells to exert its inhibitory function[16] This ability of B18 opens up the possibility of triggeringadditional signalling cascades after binding to GAGs on thecell surface To test this possibility cells were incubatedfor 4 h with the same amount of recombinant B18 usedpreviously but in the absence of IFN Importantly under theseconditions no significant changes in the gene expressionprofile could be observed when compared to mock-treatedcells indicating no activation of host gene expression istriggered after the addition of B18 to cells (Figure 1)

To confirm these results we selected three of thegenes upregulated after IFN addition from the RNA-seqdata (APOL9 IRF9 and OAS-1) together with other threegenes whose expression was unaffected (DBF4 GAPDHand MPRL2) and determined by RT-qPCR their expressionlevels As expected we found significant increased geneexpression for APOL9 IRF9 and OAS-1 after IFN inductionand concordant with the results from RNA-seq the additionof B18 prior to IFN prevented this upregulation keepingtheir expression values similar to those found in untreatedcells (Figure 2) Moreover DBF4 GAPDH and MPRL2expression determined by RT-qPCR remained unaffectedafter IFN induction or B18 incubation as seen in the RNA-seq data (Figure 2)

32 VACV-Induced Changes on Cellular Gene ExpressionProfile Searching for the initial response to VACV infectionwe first explored by RNA-seq the transcriptomes of cellsinfected with UV-inactivated VACV and compared it withmock-treated cells After alignment only 017 of total readsmatched the viral genome (Table S3) mostly correspondingto early VACV genes according to the temporal expression ofVACVORFs previously defined [21] Under these conditionswe could identify changes in the expression of a modest set of53 cellular genes (Table S4) Among these the upregulationof some genes controlled by the NF-120581B complex such asCCL5 (RANTES) H2-Q1 (HLA-B) the protein phosphataseDUSP5 involved in negative regulation of MAP kinasesthe transcription factor FOSB or SERPINE1 and also thedownregulation of the macrophage migration inhibitoryfactor (MIF) CXCL1 ABCG1 SOD3 and negative regulatorof NF-120581B TRIB3 could represent the initial response to virusinfection in the absence of viral genome replication Howeverthe absence of type I IFN or IFN effectors should be noted

By contrast at 4 hpi with replication competent VACVaround 30 of total reads matched the virus genome anda total of 2228 cellular genes were significantly differentiallyexpressed compared to uninfected cells 887 upregulated and1341 downregulated (Table 1) In order to gain a comprehen-sive understanding of the transcriptomic changes induced byVACV infection we evaluated pathway enrichment by theseSDEGs using Ingenuity Pathway Analysis (IPA) softwareAt this time postinfection the analysis identified severealterations in cellular energy metabolism since tricarboxylicacid (TCA) cycle mitochondrial dysfunction glycolysis oroxidative phosphorylation represented the most significantly


q lt 005 = 0 not shown 0)q gt 005 = 13718 not shown 53)


Sig (q lt 005 = 0 not shown 0)Nosig (

Sig (Nosig (

Sig (Nosig (

q gt 005 = 13645 not shown 73)

B18 + IFN

minus6

6

4

2

0

minus2

minus4

minus8

minus10

0 5 100 5 10

15

minus10

minus 5

minus5

minus5

0

5

10

10

minus5

5

5

0

0

IFNB18

(a)

MX2RTP4DDX58XAF1IGTPOASL1OAS1BISG15RHODPARP14TRIM21APOL9BOAS1GSLFN2OAS1AAPOL9ATRIM30ASAMD9LSLFN5IRGM1AY036118PARP12IRF9RNF213STAT2TOR3AH2-Q1IL15STAT1IFI35GM6548EIF2AK2KCNE4LGALS3BPBST2EGR1PNPLA6TRIM25IFITM3MRGPRFIER3AKR1B3WDR43

UBCCD3EAP

RPS19-ps2minus4 0 4

IFN B18 +IFNB18

(b)

Canonical pathways

Interferon signaling

Activation of IRF by cytosolic patternrecognition receptorsRole of pattern recognition receptors inRecognition of bacteria and virusesRole of RIG1-like receptors in antiviral innateimmunityCommunication between innate and adaptiveimmune cells

787

648

415

233

173

Ratio

0139

0067

0037

0041

0018

Molecules

OAS1 IFI35 STAT2 IRF9 STAT1

DDX58 STAT2 IRF9 STAT1 ISG15

OAS1 DDX58 Oas1b EIF2AK2

DDX58 TRIM25

HLA-B IL15

(1)

(2)

(3)

(4)

(5)

minuslog(p value)

(c)

Figure 1 Effect of B18 on type I IFN response L929 cells were incubated with recombinant B18 protein (B18) with mouse IFN120572 (IFN) orwith recombinant B18 and then mouse IFN120572 (B18 + IFN) and analyzed by RNA-seq (a) Corresponding119872119860 plots representing expressionof all cellular genes 119872 value (log 2 fold change) from each transcript between untreated and indicated sample cells is plotted against 119860(overall average log expression level) of each untreated and indicated pair Red dots indicate SDEGs when compared to untreated cells afterdifferential expression analysis (b) Heatmap of SDEGs identified after IFN treatmentThe heatmap displays the fold change expression (log 2)in the indicated samples relative to results for untreated cells The colour scale is shown at the bottom of the heatmap (c) Enriched canonicalpathways after IPA analysis with SDEGs identified after IFN induction


Table 1 Alignment of Illumina reads and number of differentially expressed genes from infected cells with the indicated viruses

PLWUV VACV VACV 4hpi VACV 9 hpi VACVΔB18 4 hpi VACVΔB18 9 hpiTotal reads aligneda 150810958 117443318 181794658 139017860 155220273Viral readsb 260091 (017) 37708314 (321) 144727059 (796) 25497885 (184) 90326955 (582)Cellular SDEGs Up Down Up Down Up Down Up Down Up Down

18 24 887 1341 2398 3753 660 1013 1238 2309aAligned to either mouse or VACVWestern Reserve genomesbAligned exclusively to VACVWestern Reserve genome

Fold

incr

ease

ove

r unt

reat

ed ce

lls

B18IFN

IFN + B18

IRF9 APOL9GAPDH OAS1aMRPL2 DBF4

lowast

lowast

lowast

0

2

4

6

8

lt 001lowastp

Figure 2 Confirmation of RNA-seq data by RT-qPCR after IFNinduction Gene expression for the indicated genes was assessed byRT-qPCR from L929 cells after incubation with B18 50 unitsmlIFN120572 or IFN togetherwith B18120573-Actin genewas used as a referenceto normalize the data Expression values are shown as fold changecompared to untreated cells (meanplusmn SEMand significant differencesare displayed)

enriched pathways (Figure 3(a)) Some of these pathwayswere predicted to be inhibited indicating that infection wassuppressing levels of a broad variety of proteins involved inenergy metabolism early in infection Signalling related tocell proliferation and differentiation was also found to beclearly affected during VACV infection and examples wereERKMAPK or PI3KAKT signalling pathways that weremodified Differential expression of pathways specificallyassociated with cell-cycle arrest such as G1S and G2MDNAdamage checkpoints p53 signalling or ATM signallingwere also enriched following infection Other significantlyenriched pathways such as actin signalling Rac signallingand integrin signalling were related to cell migration and areconsistent with the previously described VACV-induced cellmotility during infection [22]

Most of these enriched pathways altered at 4 hpi werealso found to be modified later in infection (at 9 hpi Fig-ure 3(b)) showing higher 119901 values However at 9 hpi theanalysis detected a striking overrepresentation of cellulargenes involved in the modulation of protein translationWe observed the downregulation of 165 genes encodingribosomal proteins and 45 encoding translation initiationfactors As a consequence the most significantly enrichedpathways identified at this time postinfection included EIF2signalling and regulation of EIF4 and p7056K and mTORsignalling (Figure 3)

33 The Absence of B18 during VACV Infection Does NotMarkedly Alter the Cellular Gene Expression Profile Previousanalysis revealed the absence of IFN related pathways amongenriched pathways altered after VACV infection suggestingthe viral downmodulation of type I IFN based host responsesConsistent with this some of the ISGs with previously knownantiviral activity such as APOL9A APOL9B OAS1a orOAS1g exhibited lower expression levels in VACV-infectedcells at 4 hpi as compared to mock-infected cells We nextevaluated the impact of B18 absence on cell host responseduring infection by using a VACV deletion mutant lack-ing expression (VACVΔB18) L929 cells were infected withVACVΔB18 and the gene expression profile was determinedat 4 and 9 hpi and then compared to mock-infected cells Asshown in Table 1 20 and 60 of the total sequencing readscorresponded to viral genes at 4 and 9 hpi respectively At 4 hafterVACVΔB18 infection a total of 1973 cellular SDEGswereidentified when compared to mock-infected cells and thecorresponding pathway enrichment analysis with these genesrevealed that although 188 SDEGs were found exclusivelydifferentially expressed during VACVΔB18 infection mostof these changes in gene expression were similar to thoseinduced at the same times during wild type VACV infectionand no additional pathways were found among the 200 mostsignificant enriched pathways (Figure 4)

34 Inhibition of the ISG Signature during VACV InfectionIs Not Exclusively Dependent on B18 We also analyzed theIFN-mediated innate immune response after IFN treatmentof infected cells To this end the expression levels of the ISGspreviously identified after IFN treatment were determinedby RNA-seq under various conditions L929 cells were either(i) infected with wild type VACV and treated or not withIFN at 5 hpi once the IFN inhibitor B18 had been producedand secreted (ii) infected with VACVΔB18 and then treatedor not with IFN (in the absence of B18) or (iii) infectedwith VACVΔB18 and supplemented with recombinant B18before IFN addition In all cases total RNA was isolatedat 9 hpi (4 h after IFN addition) and processed as indicatedbefore The results showed that addition of IFN to VACV-infected cells did not result in a clear activation pattern ofthe ISGs analyzed We did not observe any difference in theISG profile in cells infected with wild type VACV in theabsence or presence of IFN most likely due to the blockingaction of secreted B18 to prevent IFN engagement with IFNcellular receptors Surprisingly even in cells infected withVACVΔB18 not producing B18 the addition of IFN did not


Autophagy

Rac signaling

ERKMAPK signalingOxidized GTP and dGTP

detoxificationGlycerol-3-phosphate shuttle

Ascorbate recycling (cytosolic)

Glycolysis I

Superoxide radicals degradation

PI3KAKT signaling

ATM signaling

Fatty acid 120573-oxidation I

TNFR2 signaling

IL-17A signaling in fibroblasts

ERK5 signaling

p38 MAPK signalingFc120574 receptor-mediated

phagocytosis in macrophages andIntegrin signaling

Folate transformations I

Tight junction signaling

GADD45 signaling

Actin cytoskeleton signaling

RAR activationCaveolar-mediated endocytosis

signalingVDRRXR activation

Epithelial adherens junctionsignaling

p53 signaling

Phenylethylamine degradation I

Ephrin receptor signaling

Oxidative phosphorylationCell cycle G1S checkpoint

regulationCell cycle G2M DNA checkpoint

regulationGlucocorticoid receptor signaling

Hypoxia signaling in thecardiovascular system

NRF2-mediated oxidative stressresponse

Clathrin-mediated endocytosissignaling

Mitochondrial dysfunctionAryl hydrocarbon receptor

signalingtRNA charging

TCA cycle II (eukaryotic)

7 (19)16 (15)26 (14)2 (67)2 (67)2 (67)2 (67)6 (24)3 (43)19 (15)11 (18)11 (18)7 (24)8 (23)12 (19)19 (16)16 (17)30 (15)4 (44)26 (16)6 (32)32 (15)29 (15)14 (20)15 (19)24 (16)18 (18)3 (75)28 (16)20 (18)14 (22)12 (24)42 (15)15 (23)31 (17)32 (17)

34 (20)31 (22)15 (38)12 (52)

Inosine-5998400-phosphatebiosynthesis II

2 310 4 5 876

minuslog(p value)

(a)

Insulin receptor signaling

PPAR signaling

SAPKJNK signaling

GADD45 signaling

Apoptosis signaling

phagosome maturation

Isoleucine degradation I

Cell cycle G1S checkpoint regulation

Superpathway of cholesterol biosynthesis

FAK signaling

RhoA signaling

Protein kinase A signaling

HGF signaling

Cysteine biosynthesis III (mammalia)

p70S6K signaling

IL-8 signaling

Aryl hydrocarbon receptor signaling

ILK signaling

Rac signaling

RAR activation

IGF-1 signalingCell cycle G 2M DNA damage

checkpoint regulationActin cytoskeleton signaling

Glucocorticoid receptor signaling

ERKMAPK signaling

Integrin signaling

Superpathway of methionine degradation


PI3KAKT signaling

Ephrin receptor signalingRemodeling of epithelial adherens

NRF2-mediated oxidative stress response

Protein ubiquitination pathway

mTOR signaling

Regulation of eIF4 and p70S6K signaling

Oxidative phosphorylation

Mitochondrial dysfunction

EIF2 signaling

50 (37)38 (40)38 (40)12 (63)37 (42)47 (39)10 (71)29 (45)16 (57)37 (43)13 (65)43 (40)128 (33)44 (42)12 (71)13 (68)49 (41)70 (38)57 (41)72 (39)46 (44)74 (39)44 (45)27 (55)84 (39)104 (38)79 (42)83 (41)22 (69)18 (78)59 (48)78 (45)42 (62)

85 (47)116 (46)95 (51)80 (55)69 (63)

104 (61)118 (64)

10 1550 20 25 30

minuslog(p value)

DNA methylation and transcriptionalrepression signaling

junctions

Methionine degradation I(to homocysteine)

(b)

Figure 3 IPA analysis of differentially expressed host genes in VACV-infected cells The list of SDEGs identified after VACV infection wasused in a pathway enrichment analysis with IPA software The 40 most significant pathways identified at 4 hpi (a) and 9 hpi (b) after VACVinfection are shown The 119909-axis represents the 119901 value indicating the significance of enrichment for the corresponding gene set The valuesare plotted in a negative log10 scale

result in an evident IFN based response and the expressionlevels of the ISGs analyzed were similar to those found inVACVΔB18-infected cells in the presence of recombinant B18protein and wild type VACV-infected cells (Figure 5)

In an independent assay with additional RNA sampleswe could confirm these results by RT-qPCR in cells infectedwith wild type VACV or VACVΔB18 in the same condi-tions described above We first verified the expression of


745 1481 198

VACVΔB18

VACV

(a)

2958 3184 359

VACVΔB18

VACV

(b)

Figure 4 Effect of B18 absence on host gene expression duringVACV infection Venn diagrams showing the number of overlapped transcriptscorresponding to cellular genes differentially expressed between VACV- and VACVΔB18-infected cells at 4 hpi (a) and 9 hpi (b) are displayed

the viral genes WR092 and WR127 in all infected culturesand observed increasing expression values from 4 to 9 hpiindependently of the addition of IFN On the contrary butin concordance with results from the RNA-seq the expres-sion values of the ISGs determined by RT-qPCR (APOL9IRF9 and OAS1a) during wild type VACV or VACVΔB18infections and independently of the addition of IFN weresimilar in all cases to those detected in nontreated cells(Figure 6) Finally no significant modification of cellularGAPDH expression levels determined by RT-qPCR wasobserved at 4 h after infection while a slight decrease wasobserved at 9 h during wild type VACV or VACVΔB18infection in either the absence or presence of IFN

4 Discussion

The secreted type I IFN binding protein B18 from VACVrepresents a unique strategy employed by poxviruses to evadethe host IFN response Its important contribution to thevirulence of VACV and ectromelia virus a related mouse-specific virus that also encodes a B18 orthologue has beendemonstrated inmousemodels of infection [13 23]This anti-IFN activity has also been identified in the highly virulentvariola virus and monkeypox virus [17] In this report wehave addressed the ability of the secreted type I IFN bindingprotein to modulate the expression of host genes regulatedby IFN using an RNA-seq approach to monitor the globalexpression of host and viral genes

First we evaluated the impact of type I IFN on thegene expression profile required to induce an antiviral stateand protect cells from infection In the case of L929 mousefibroblasts we found the expression of 46 genes affectedby the addition of IFN-120572 Consistent with previous resultsdemonstrating the ability of B18 to block IFN effects [13ndash16] the modulation of host gene expression by IFN could beefficiently prevented by the action of B18

Using the same RNA sequencing approach the incuba-tion of cells with purified B18 protein did not cause anysignificant change in gene expression suggesting that nocell signalling is triggered by B18 This result is of particu-lar relevance since after secretion from infected cells B18

interacts with GAGs at the surface of infected and adjacentuninfected cells [14 16] and GAGs have been shown toregulatemultiple signalling pathwaysThis is the case of somegrowth factors such as fibroblast hepatocyte or vascularendothelial growth factors [24ndash26] where the participationof GAGs is essential for receptor-ligand engagement and theresulting signalling In contrast our results clearly indicatethat the interaction of B18 with GAGs at the surface of L929cells does not trigger any detectable cell signalling leading tochanges in host gene expression Consistent with our resultsit has been reported that addition of purified recombinant B18to primary mouse plasmacytoid cells does not induce type IIFNproduction whereas these cells were able to produce typeI IFN in response to TLR ligands [27]

In this report we have determined the cellular transcrip-tome profile to investigate the changes in host expressionduring VACV infection The host reaction to VACV seemsto start immediately after infection as deduced from theset of genes differentially expressed 4 h after infection withUV-inactivated VACV Among these we found some NF-120581B regulated genes such as the proinflammatory chemokineRANTESCCL5 gene genes involved in the regulation ofMAPK activity or the downregulation of antigen presentationrelated gene H2Q1 among others It was somehow surprisingthat we did not detect more transcriptional activation in cellsincubated with UV-inactivated virus which should be ableto attach and enter the cell This may suggest that PRRs thatactivate cells in response to VACV infection detect mainlythe viral genome that is being transcribed or replicatedrather than the small amount of virus particles that enterinitially the cell (with viral proteins and DNA) Also theincoming virus particle contains VH1 a phosphatase knownto inhibit STAT1 and STAT2 activation and may also preventIFN responses in the absence of virus replication as it wasinitially described [8] Previous reports described the abilityof inactivated VACV WR and monkeypox virus to inducethe synthesis of IFN or IFN-inducible genes in plasmacytoiddendritic cells and macrophages respectively [27 28] but wecould not detect the activation of these genes in L929 cellsTwo factors could contribute to explaining these differencesthe earlier timepoints analyzed compared with the previous


VACVVACVΔB18

adsorption

+minus IFN

RNAextraction

RNAextraction

9876543210 (hpi)

(+minus rB18)

(a)

VACVΔB18

+ + + +

+minus

minus

minus

minus

minusminus

minus

minus

minus

minusminus

minus

9 9 9 994 4

minus4 minus2 0 2 4

MX2RTP4DDX58XAF1IGTPOASL1OAS1BISG15RHODPARP14TRIM21APOL9BOAS1GSLFN2OAS1AAPOL9ATRIM30ASAMD9LSLFN5IRGM1AY036118PARP12IRF9RNF213STAT2TOR3AH2-Q1IL15STAT1IFI35GM6548EIF2AK2KCNE4LGALS3BPBST2EGR1PNPLA6TRIM25IFITM3MRGPRFIER3AKR1B3WDR43CD3EAPUBCGM6863GM10275RPS19-PS2

Virushpi na

IFNrB18

VACV

(b)

Figure 5 ISGs expression during VACV infection (a) Diagram showing the experimental conditions to obtain RNA samples from infectedcells (b) Heatmap shows the expression levels of the ISGs previously identified relative to untreated cells where indicated recombinant B18protein (rB18) andor mouse IFN120572 werewas added to cells at 2 and 5 hpi respectively

studies or the fact that the levels of IFN production are celltype dependent Plasmacytoid dendritic cells are consideredto be the professional type I IFN producing cells after viralinfections [29 30] and secrete much more IFN-120572 than othercell types

By contrast a drastic change in the host gene expressionprofile occurred after 4 h of infection with replication com-petent VACV mainly affecting biological functions relatedto metabolism cell death and survival cell development andproliferation and cell movement Previous studies with HeLacells using microarrays or deep RNA sequencing showed ageneral decrease in the cellularmRNAs uponVACV infection[21 31] In our study as previously reported by Yang et al[21] we found that the proportion of VACV mRNA wasapproximately 30 of the total mRNA andmore than 50 ofmodified cellular genes were downregulated at 4 hpi which isindicative of the virus-induced degradation of cellularmRNAthat precedes host translation shutoff [32] This effect waseven more pronounced at 9 hpi Since the aim of our studywas to analyze themodulation of type I IFN responses by B18we selected the times postinfection that allowed the synthesis

and secretion into the supernatant of an effective amount ofthe B18 protein which is produced at early times of infectionUnder these conditions we focused on type I IFN responsesupon VACV infection

Our RNA-seq data from VACV-infected samples is inconcordance with previous reports showing that unlike thehighly attenuated modified VACV Ankara (MVA) strainvirulent VACV WR infection of mice or dendritic cellcultures did not raise IFN-120572 responses It is also known thatthe lack of a functional B18R gene and other IFN inhibitors inMVA allows the development of an IFN based host responseduring infection [33] However our data indicated that theinfection with VACVΔB18 lacking expression of the secretedtype I IFN inhibitor equally failed to raise an effective IFNhost response and VACVΔB18 infection proceeded similarlyto VACV infection in L929 cells This result corroboratesthe existence of additional viral mechanisms to inhibit theinduction of type I IFN responses as previously indicatedby Waibler and cols during VACV infection of pDCs [27]While B18 remains as the only identified secreted type I IFNinhibitor during the last years diverse VACV genes have been


WR092

APOL9

OAS1

WR127

IRF9

GAPDH

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

10

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8 Fo

ld in

crea

se re

l to

VAC

V

0

2

4

6

8

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

4hp

i

Fold

incr

ease

rel

to V

ACV4

hpi

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

Figure 6 Confirmation of RNA-seq data by RT-qPCR during VACV infection Gene expression for the indicated genes was assessed by RT-qPCR from VACV- or VACVΔB18-infected L929 cells at 4 hpi and 9 hpi IFN was added to infected cells at 5 hpi where indicated Expressionlevels ofWR092 andWR127 VACV genes were also determined tomonitor the progress of infection and relativized to VACV 4hpi expressionvalues Mean from 3 biological replicates plusmn SEM and significant differences are displayed


shown to have a direct role in the inhibition of the IFNproduction or the inhibition of the IFN signal transductionthat takes place after type I IFN binding to cellular IFNAR[2] Our results showed the lack of a functional IFN responseduring VACV infection in the absence of B18 and even afterthe addition of exogenous IFN-120572 indicating that the IFNsignalling downstream of IFNAR is impaired after VACVinfection We speculate that the virion associated phos-phatase VH1 which dephosphorylates STAT1 and STAT2 toblock downstream IFN-120572 signalling [8 9] may contributetogether with other VACV genes to explaining this lackof IFN responses during VACV infection in the absence ofB18 function In the cellular experimental system used hereall cells were initially infected with VACV and hence theinhibition of the IFN response by B18 cannot be appreciatedThe effect of B18 on virus replication in cell cultures treatedwith IFN is evident under other circumstances such aswhen IFN is added a few hours after infection as wasillustrated in a previous report [14] Deletion of the typeI IFN binding protein in the VACV strain NYVAC hasbeen reported to trigger the activation of IRF3 IRF7 andSTAT1 and to increase the production of ISGs in humanmonocytes in a transcriptomic analysis using microarrays[34] The reasons for the different results reported in theprevious report may be due to a different response in humanmonocytes or to the use of a highly attenuated VACV strainlacking many immunomodulatory genes such as C4L N1Lor N2L which have been implicated in the modulation ofintracellular signalling events [35ndash37] Also the recombinantviruses used in the NYVAC transcriptional studies have notbeen controlled for the potential inadvertent selection ofmutations during the generation of the recombinant virusesthrough the construction of revertant viruses or sequencingof the complete viral genomes and thus the presence ofadditional mutations in other genes that may influence thereported results cannot be formally ruled out [38 39]

The contribution of the secreted type I IFN binding pro-tein to virus virulence and immune evasion becomes evidentin mouse models of VACV and ectromelia virus infectionwhere mutant viruses show an attenuated phenotype thatis dramatic in the mousepox model [13 23] In the animalhost the expression of a secreted IFN inhibitor is relevantto efficiently block the protective effects of IFN which isproduced in response to infection and is able to trigger IFN-mediated antiviral activities in neighbouring cells and restrictvirus spread [23]

5 Conclusion

We have used RNA-seq to study by the modulation of thetype I IFN response by VACV and the secreted type IIFN binding protein B18 This analysis identified cellularpathways modulated during VACV infection or inducedby UV-inactivated virus particles VACV B18 was a potentinhibitor of the type I IFN response consistent with its abilityto bind with high affinity IFN and to prevent its interactionwith cellular IFNAR VACVΔB18 inhibits the IFN responseto an extent similar to that of wild type VACV indicatingthat VACV encodes numerous mechanisms to block the IFN

response and that the contribution of B18 to immune evasionis more evident in infected mice than in tissue culture Wealso show that the interaction of B18 with cell surface GAGsdoes not trigger a specific host response leading to changes inhost gene expression The RNA-seq methodology allows theevaluation of the global gene expression in infected cells andthe modulation of IFN responses by the VACV type I IFNbinding protein Future RNA-seq studies in VACV-infectedmicemay dissect better the ability of B18 tomodulate the typeI IFN-mediated response in different tissues

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contributions

Bruno Hernaez and Graciela Alonso equally contributed tothis work

Acknowledgments

This work was supported by the European Sequencingand Genotyping Infrastructure (Seventh Framework Pro-grammeunderGrantAgreement no 262055) and the SpanishMinistry of Economy and Competitiveness and EuropeanUnion (European Regional Developmentrsquos Funds FEDER)(Grant SAF2015-67485-R) Bruno Hernaez was funded by aJAE postdoctoral contract (Spanish Research Council) andGraciela Alonso was supported by a FPI studentship fromthe Spanish Ministry of Economy and Competitiveness Theauthors also thankDAguirre deCarcer for helpful commentsand the Genomics and Next Generation Sequencing Serviceat Centro de Biologıa Molecular Severo Ochoa for theirsupport

References

[1] O Haller G Kochs and F Weber ldquoThe interferon responsecircuit induction and suppression by pathogenic virusesrdquoVirology vol 344 no 1 pp 119ndash130 2006

[2] G L Smith C T O Benfield C Maluquer de Motes et alldquoVaccinia virus immune evasion mechanisms virulence andimmunogenicityrdquo Journal of General Virology vol 94 no 11 pp2367ndash2392 2013

[3] G A Versteeg and A Garcıa-Sastre ldquoViral tricks to grid-lockthe type I interferon systemrdquo Current Opinion in Microbiologyvol 13 no 4 pp 508ndash516 2010

[4] WM SchneiderMD Chevillotte andCM Rice ldquoInterferon-stimulated genes a complex web of host defensesrdquo AnnualReview of Immunology vol 32 pp 513ndash545 2014

[5] A Alcami ldquoViral mimicry of cytokines chemokines and theirreceptorsrdquo Nature Reviews Immunology vol 3 no 1 pp 36ndash502003

[6] B Perdiguero and M Esteban ldquoThe interferon system andvaccinia virus evasion mechanismsrdquo Journal of Interferon andCytokine Research vol 29 no 9 pp 581ndash598 2009


[7] A Bowie E Kiss-Toth J A Symons G L Smith S K Dowerand L A J OrsquoNeill ldquoA46R and A52R from vaccinia virusare antagonists of host IL-1 and toll-like receptor signalingrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 97 no 18 pp 10162ndash10167 2000

[8] P Najarro P Traktman and J A Lewis ldquoVaccinia virus blocksgamma interferon signal transduction viral VH1 phosphatasereverses Stat1 activationrdquo Journal of Virology vol 75 no 7 pp3185ndash3196 2001

[9] B AMann J H Huang P Li et al ldquoVaccinia virus blocks Stat1-dependent and Stat1-independent gene expression induced bytype I and type II interferonsrdquo Journal of Interferon andCytokineResearch vol 28 no 6 pp 367ndash380 2008

[10] K Carroll O Elroy-Stein B Moss and R Jagus ldquoRecombinantvaccinia virus K3L gene product prevents activation of double-stranded RNA-dependent initiation factor 2120572-specific proteinkinaserdquo Journal of Biological Chemistry vol 268 no 17 pp12837ndash12842 1993

[11] H-W Chang and B L Jacobs ldquoIdentification of a conservedmotif that is necessary for binding of the vaccinia virus E3L geneproducts to double-stranded RNArdquo Virology vol 194 no 2 pp537ndash547 1993

[12] S Guerra A Caceres K-P Knobeloch I Horak and MEsteban ldquoVaccinia virus E3 protein prevents the antiviral actionof ISG15rdquo PLoS Pathogens vol 4 no 7 Article ID e10000962008

[13] J A Symons A Alcamı and G L Smith ldquoVaccinia virusencodes a soluble type I interferon receptor of novel structureand broad species soecificityrdquo Cell vol 81 no 4 pp 551ndash5601995

[14] A Alcamı J A Symons and G L Smith ldquoThe vaccinia virussoluble alphabeta interferon (IFN) receptor binds to the cellsurface and protects cells from the antiviral effects of IFNrdquoJournal of Virology vol 74 no 23 pp 11230ndash11239 2000

[15] O R Colamonici P Domanski S M Sweitzer A Larnerand R M L Buller ldquoVaccinia virus B18R gene encodes atype I interferon-binding protein that blocks interferon 120572transmembrane signalingrdquo Journal of Biological Chemistry vol270 no 27 pp 15974ndash15978 1995

[16] I Montanuy A Alejo and A Alcami ldquoGlycosaminoglycansmediate retention of the poxvirus type I interferon bindingprotein at the cell surface to locally block interferon antiviralresponsesrdquo FASEB Journal vol 25 no 6 pp 1960ndash1971 2011

[17] M Fernandez deMarcoMdel A Alejo PHudson I KDamonand A Alcami ldquoThe highly virulent variola and monkeypoxviruses express secreted inhibitors of type I interferonrdquo TheFASEB Journal vol 24 no 5 pp 1479ndash1488 2010

[18] K Tsung J H Yim W Marti R M L Buller and J ANorton ldquoGene expression and cytopathic effect of vaccinia virusinactivated by psoralen and long-wave UV lightrdquo Journal ofVirology vol 70 no 1 pp 165ndash171 1996

[19] C Trapnell A Roberts L Goff et al ldquoDifferential gene andtranscript expression analysis of RNA-seq experiments withTopHat and Cufflinksrdquo Nature Protocols vol 7 no 3 pp 562ndash578 2012

[20] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[21] Z Yang D P Bruno C A Martens S F Porcella and B MossldquoSimultaneous high-resolution analysis of vaccinia virus andhost cell transcriptomes by deep RNA sequencingrdquo Proceedings

of the National Academy of Sciences of the United States ofAmerica vol 107 no 25 pp 11513ndash11518 2010

[22] C M Sanderson M Hollinshead and G L Smith ldquoThevaccinia virus A27L protein is needed for the microtubule-dependent transport of intracellular mature virus particlesrdquoJournal of General Virology vol 81 no 1 pp 47ndash58 2000

[23] R-H Xu M Cohen Y Tang et al ldquoThe orthopoxvirus type IIFN binding protein is essential for virulence and an effectivetarget for vaccinationrdquo Journal of Experimental Medicine vol205 no 4 pp 981ndash992 2008

[24] S Ashikari-Hada H Habuchi Y Kariya and K Kimata ldquoHep-arin regulates vascular endothelial growth factor165-dependentmitogenic activity tube formation and its receptor phospho-rylation of human endothelial cells Comparison of the effectsof heparin and modified heparinsrdquo The Journal of BiologicalChemistry vol 280 no 36 pp 31508ndash31515 2005

[25] L M McDowell B A Frazier D R Studelska et al ldquoInhibitionor activation of apert syndrome FGFR2 (S252W) signaling byspecific glycosaminoglycansrdquo Journal of Biological Chemistryvol 281 no 11 pp 6924ndash6930 2006

[26] T F Zioncheck L Richardson J Liu et al ldquoSulfated oligosac-charides promote hepatocyte growth factor association andgovern its mitogenic activityrdquo Journal of Biological Chemistryvol 270 no 28 pp 16871ndash16878 1995

[27] Z Waibler M Anzaghe T Frenz et al ldquoVaccinia virus-mediated inhibition of type I interferon responses is a multi-factorial process involving the soluble type I interferon receptorB18 and intracellular componentsrdquo Journal of Virology vol 83no 4 pp 1563ndash1571 2009

[28] K H Rubins L E Hensley D A Relman and P O BrownldquoStunned silence gene expression programs in human cellsinfected with monkeypox or vaccinia virusrdquo PLoS ONE vol 6no 1 Article ID e15615 2011

[29] M Colonna A Krug and M Cella ldquoInterferon-producingcells on the front line in immune responses against pathogensrdquoCurrent Opinion in Immunology vol 14 no 3 pp 373ndash3792002

[30] Y-J Liu ldquoIPC professional type 1 interferon-producing cellsand plasmacytoid dendritic cell precursorsrdquo Annual Review ofImmunology vol 23 pp 275ndash306 2005

[31] S Guerra L A Lopez-Fernandez A Pascual-Montano MMunoz K Harshman and M Esteban ldquoCellular gene expres-sion survey of vaccinia virus infection of human HeLa cellsrdquoJournal of Virology vol 77 no 11 pp 6493ndash6506 2003

[32] A P Rice and B E Roberts ldquoVaccinia virus induces cellularmRNA degradationrdquo Journal of Virology vol 47 no 3 pp 529ndash539 1983

[33] Z Waibler M Anzaghe H Ludwig et al ldquoModified vacciniavirus Ankara induces toll-like receptor-independent type Iinterferon responsesrdquo Journal of Virology vol 81 no 22 pp12102ndash12110 2007

[34] J Delaloye A Filali-Mouhim M J Cameron et alldquoInterleukin-1- and type I interferon-dependent enhancedimmunogenicity of an NYVAC-HIV-1 Env-Gag-Pol-Nefvaccine vector with dual deletions of type I and Type IIinterferon-binding proteinsrdquo Journal of Virology vol 89 no 7pp 3819ndash3832 2015

[35] G DiPerna J Stack A G Bowie et al ldquoPoxvirus protein N1Ltargets the I-120581B kinase complex inhibits signaling to NF-120581B bythe tumor necrosis factor superfamily of receptors and inhibitsNF-120581B and IRF3 signaling by toll-like receptorsrdquo Journal ofBiological Chemistry vol 279 no 35 pp 36570ndash36578 2004


[36] SW J Ember H Ren B J Ferguson andG L Smith ldquoVacciniavirus protein C4 inhibits NF-120581B activation and promotes virusvirulencerdquo Journal of General Virology vol 93 no 10 pp 2098ndash2108 2012

[37] B J Ferguson C T O Benfield H Ren et al ldquoVaccinia virusprotein N2 is a nuclear IRF3 inhibitor that promotes virulencerdquoJournal of General Virology vol 94 no 9 pp 2070ndash2081 2013

[38] C E Gomez B Perdiguero J L Najera et al ldquoRemoval ofvaccinia virus genes that block interferon type I and II pathwaysimproves adaptive and memory responses of the HIVAIDSvaccine candidate NYVAC-C in micerdquo Journal of Virology vol86 no 9 pp 5026ndash5038 2012

[39] K V Kibler C E Gomez B Perdiguero et al ldquoImprovedNYVAC-based vaccine vectorsrdquo PLoSONE vol 6 no 11 ArticleID e25674 2011

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


proteins that employ two different mechanisms to counter-act double-stranded RNA-dependent protein kinase (PKR)effector functions [10 11] Additionally E3 binds the productof the IFN-stimulated gene 15 (ISG15) to prevent its antiviralaction [12] But one the most efficient strategies employedby poxviruses to avoid IFN effects is to encode IFN bindingproteins that are secreted from infected cells to prevent theinteraction of IFNs with their cellular receptors In the case ofVACV strain Western Reserve (WR) the type I IFN bindingprotein is encoded by theB18R gene (B19R in theCopenhagenstrain) A relevant role of this protein in VACV pathogenesiswas soon assigned since the lack of B18R expression afterintranasal infection of mice resulted in an attenuated virusindicating that blocking the IFN host response is crucial forthe development of VACV infection [13]The B18 protein hasno amino acid sequence similarity to cellular IFN receptorsand in contrast to the cellular counterparts binds IFN120572120573from a broad range of host species [13] The protein issynthesized early after VACV infection is secreted into themedium and is found as a soluble form or anchored to thecell surface [14 15] This binding to the cell surface has beenshown to occur via interaction of the B18 amino terminuswith glycosaminoglycans (GAGs) [16] and allows B18 toprevent the establishment of an IFN-induced antiviral statein cells surrounding the infection site

In the present study by using RNA sequencing with theIllumina technology (RNA-seq) and differential gene expres-sion analyses we have further analyzed the ability of B18 toblock the IFN based response in a mouse fibroblast cell lineWe also extend the study to VACV-infected cells to identifychanges in host gene expression profile induced by VACV ora VACV mutant lacking the B18R gene (VACVΔB18) withspecial emphasis on the inhibition of the type I IFN-inducedhost cell response

2 Materials and Methods

21 Cell Culture and Reagents Mouse L929 cells were used toobtain RNA samples for high-throughput sequencing whileBSC-1 cells (African green monkey kidney origin) were usedto prepare virus stocks Recombinant His-tagged VACV B18protein was expressed in the baculovirus system and purifiedas previously described [17] Protein purity was checked onCoomassie blue-stained SDS-PAGE and quantified by geldensitometry Murine recombinant IFN-120572 subtype A waspurchased from PBL Assay Science (gt95 pure) diluted inphosphate-buffered saline and maintained at minus70∘C untiluse

22 Viruses and Infections Virulent VACV strain WR andthe correspondent VACV mutant lacking B18R expression(VACVΔB18 [14]) were grown in BSC-1 cells and stocks ofsemipurified virus were prepared by sedimentation througha 36 sucrose cushion L929 cells were infected with VACVor VACVΔB18 with a multiplicity of infection of 5 plaqueforming units (pfu)cell in order to ensure the infectionof all cells to obtain a representative RNA-seq profile ofeach condition After adsorption of virus for 1 h at 37∘Cthe virus-containing medium was removed and cells were

washed twice with phosphate-buffered saline and replacedwith fresh culture medium supplemented with 2 fetalbovine serum Infected cells were then incubated at 37∘Cand harvested at 4 or 8 h postinfection (hpi) by scrappingWhere indicated IFN (50 unitsml) was added to the infectedcultures at 4 hpi and the incubation extended at 37∘C to 9 hpiInactivation of viruseswas performed as previously described[18] by incubation with 2120583gml psoralen (4-9-aminomethyl-trioxsalen Sigma) for 10min and then UV-irradiated for10min with 225 Jcm2 in a Stratalinker 1800 Completeinactivation (gt108-fold reduction in pfu) was confirmed byplaque assay in BSC-1 cells

23 RNA Extraction and Illumina RNA-Seq Library Prepa-ration Immediately after harvesting the samples total cel-lular RNA was isolated from 12 times 106 L929 cells usingSV Total RNA Isolation System (Promega) RNA sampleswere quantified on a spectrophotometer (NanoDrop ND-1000 Thermo Scientific) and quality-analyzed in an Agi-lent 2100 Bioanalyzer (Agilent Technologies Santa ClaraCA US) All samples exhibited a RNA integrity number(RIN) over 9 The sequencing libraries were generated withTruSeq RNA Sample Prep Kit v2 Set A (Illumina) Brieflypoly(A) containing mRNA molecules were purified in tworounds using oligo(dT) attached magnetic beads from 1120583gof total RNA After chemical fragmentation mRNA frag-ments were reverse-transcribed and converted into double-stranded cDNA molecules Following end-repair and dA-tailing paired-end sequencing adaptors were ligated to theends of the cDNA fragments using TruSeq PE Cluster Kit v3-cBot-HS (Illumina)

24 Deep Sequencing and Sequence Analysis Libraries weresequenced using TruSeq SBS Kit v3-HS (Illumina) on anIllumina Hiseq 2000 machine at the Max Planck Insti-tute for Molecular Genetics Berlin More than 108 100 ntpaired-end reads were obtained from each sample andafter quality assessment with package FastQC (httpwwwbioinformaticsbabrahamacukprojectsfastqc) the fastqfiles containing these reads were mapped to the mousegenome (build GRCm38 from Mus musculus C57BL6Jstrain) together with the VACV WR genome (GenebankAY2433121) using Tophat v204 with default parameters [19]Only those reads aligned against mouse genomewere consid-ered in a differential gene expression analysis with Cuffdiff(Cufflinks v210 software [19]) Since biological duplicatesof samples from untreated cells were available all compar-isons were performed against this sample using the defaultmode of Cuffdiff which is the most suitable for our kindof data Pathway analysis of the significantly differentiallyexpressed genes detected was performed using IngenuityPathway Analysis (IPA) software Creation of proportionalVenndiagrams and gene expression heatmapswere generatedwith the R ldquoVennDiagram v169rdquo and ldquoGplotsrdquo packagesrespectively The raw RNA-seq data has been deposited atthe European Nucleotide Archive (ENA) under the projectnumber PRJEB15047



3 Results











Sig (Nosig (

Sig (Nosig (

q gt 005 = 13645 not shown 73)

B18 + IFN

minus6

6

4

2

0

minus2

minus4

minus8

minus10

0 5 100 5 10

15

minus10

minus 5

minus5

minus5

0

5

10

10

minus5

5

5

0

0

IFNB18

(a)


UBCCD3EAP

RPS19-ps2minus4 0 4

IFN B18 +IFNB18

(b)

Canonical pathways



787

648

415

233

173

Ratio

0139

0067

0037

0041

0018

Molecules




DDX58 TRIM25

HLA-B IL15

(1)

(2)

(3)

(4)

(5)

minuslog(p value)

(c)






Fold

incr

ease

ove

r unt

reat

ed ce

lls

B18IFN

IFN + B18


lowast

lowast

lowast

0

2

4

6

8

lt 001lowastp







Autophagy

Rac signaling




Glycolysis I


PI3KAKT signaling

ATM signaling


TNFR2 signaling


ERK5 signaling





GADD45 signaling





p53 signaling












7 (19)16 (15)26 (14)2 (67)2 (67)2 (67)2 (67)6 (24)3 (43)19 (15)11 (18)11 (18)7 (24)8 (23)12 (19)19 (16)16 (17)30 (15)4 (44)26 (16)6 (32)32 (15)29 (15)14 (20)15 (19)24 (16)18 (18)3 (75)28 (16)20 (18)14 (22)12 (24)42 (15)15 (23)31 (17)32 (17)

34 (20)31 (22)15 (38)12 (52)


2 310 4 5 876

minuslog(p value)

(a)


PPAR signaling

SAPKJNK signaling

GADD45 signaling

Apoptosis signaling





FAK signaling

RhoA signaling


HGF signaling


p70S6K signaling

IL-8 signaling


ILK signaling

Rac signaling

RAR activation




ERKMAPK signaling

Integrin signaling



PI3KAKT signaling




mTOR signaling




EIF2 signaling

50 (37)38 (40)38 (40)12 (63)37 (42)47 (39)10 (71)29 (45)16 (57)37 (43)13 (65)43 (40)128 (33)44 (42)12 (71)13 (68)49 (41)70 (38)57 (41)72 (39)46 (44)74 (39)44 (45)27 (55)84 (39)104 (38)79 (42)83 (41)22 (69)18 (78)59 (48)78 (45)42 (62)

85 (47)116 (46)95 (51)80 (55)69 (63)

104 (61)118 (64)

10 1550 20 25 30

minuslog(p value)


junctions


(b)





745 1481 198

VACVΔB18

VACV

(a)

2958 3184 359

VACVΔB18

VACV

(b)



4 Discussion







VACVVACVΔB18

adsorption

+minus IFN

RNAextraction

RNAextraction

9876543210 (hpi)

(+minus rB18)

(a)

VACVΔB18

+ + + +

+minus

minus

minus

minus

minusminus

minus

minus

minus

minusminus

minus

9 9 9 994 4

minus4 minus2 0 2 4


Virushpi na

IFNrB18

VACV

(b)







WR092

APOL9

OAS1

WR127

IRF9

GAPDH

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

10

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8 Fo

ld in

crea

se re

l to

VAC

V

0

2

4

6

8

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

4hp

i

Fold

incr

ease

rel

to V

ACV4

hpi

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i





5 Conclusion



Competing Interests




Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















3 Results











Sig (Nosig (

Sig (Nosig (

q gt 005 = 13645 not shown 73)

B18 + IFN

minus6

6

4

2

0

minus2

minus4

minus8

minus10

0 5 100 5 10

15

minus10

minus 5

minus5

minus5

0

5

10

10

minus5

5

5

0

0

IFNB18

(a)


UBCCD3EAP

RPS19-ps2minus4 0 4

IFN B18 +IFNB18

(b)

Canonical pathways



787

648

415

233

173

Ratio

0139

0067

0037

0041

0018

Molecules




DDX58 TRIM25

HLA-B IL15

(1)

(2)

(3)

(4)

(5)

minuslog(p value)

(c)






Fold

incr

ease

ove

r unt

reat

ed ce

lls

B18IFN

IFN + B18


lowast

lowast

lowast

0

2

4

6

8

lt 001lowastp







Autophagy

Rac signaling




Glycolysis I


PI3KAKT signaling

ATM signaling


TNFR2 signaling


ERK5 signaling





GADD45 signaling





p53 signaling












7 (19)16 (15)26 (14)2 (67)2 (67)2 (67)2 (67)6 (24)3 (43)19 (15)11 (18)11 (18)7 (24)8 (23)12 (19)19 (16)16 (17)30 (15)4 (44)26 (16)6 (32)32 (15)29 (15)14 (20)15 (19)24 (16)18 (18)3 (75)28 (16)20 (18)14 (22)12 (24)42 (15)15 (23)31 (17)32 (17)

34 (20)31 (22)15 (38)12 (52)


2 310 4 5 876

minuslog(p value)

(a)


PPAR signaling

SAPKJNK signaling

GADD45 signaling

Apoptosis signaling





FAK signaling

RhoA signaling


HGF signaling


p70S6K signaling

IL-8 signaling


ILK signaling

Rac signaling

RAR activation




ERKMAPK signaling

Integrin signaling



PI3KAKT signaling




mTOR signaling




EIF2 signaling

50 (37)38 (40)38 (40)12 (63)37 (42)47 (39)10 (71)29 (45)16 (57)37 (43)13 (65)43 (40)128 (33)44 (42)12 (71)13 (68)49 (41)70 (38)57 (41)72 (39)46 (44)74 (39)44 (45)27 (55)84 (39)104 (38)79 (42)83 (41)22 (69)18 (78)59 (48)78 (45)42 (62)

85 (47)116 (46)95 (51)80 (55)69 (63)

104 (61)118 (64)

10 1550 20 25 30

minuslog(p value)


junctions


(b)





745 1481 198

VACVΔB18

VACV

(a)

2958 3184 359

VACVΔB18

VACV

(b)



4 Discussion







VACVVACVΔB18

adsorption

+minus IFN

RNAextraction

RNAextraction

9876543210 (hpi)

(+minus rB18)

(a)

VACVΔB18

+ + + +

+minus

minus

minus

minus

minusminus

minus

minus

minus

minusminus

minus

9 9 9 994 4

minus4 minus2 0 2 4


Virushpi na

IFNrB18

VACV

(b)







WR092

APOL9

OAS1

WR127

IRF9

GAPDH

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

10

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8 Fo

ld in

crea

se re

l to

VAC

V

0

2

4

6

8

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

4hp

i

Fold

incr

ease

rel

to V

ACV4

hpi

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i





5 Conclusion



Competing Interests




Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Sig (Nosig (

Sig (Nosig (

q gt 005 = 13645 not shown 73)

B18 + IFN

minus6

6

4

2

0

minus2

minus4

minus8

minus10

0 5 100 5 10

15

minus10

minus 5

minus5

minus5

0

5

10

10

minus5

5

5

0

0

IFNB18

(a)


UBCCD3EAP

RPS19-ps2minus4 0 4

IFN B18 +IFNB18

(b)

Canonical pathways



787

648

415

233

173

Ratio

0139

0067

0037

0041

0018

Molecules




DDX58 TRIM25

HLA-B IL15

(1)

(2)

(3)

(4)

(5)

minuslog(p value)

(c)






Fold

incr

ease

ove

r unt

reat

ed ce

lls

B18IFN

IFN + B18


lowast

lowast

lowast

0

2

4

6

8

lt 001lowastp







Autophagy

Rac signaling




Glycolysis I


PI3KAKT signaling

ATM signaling


TNFR2 signaling


ERK5 signaling





GADD45 signaling





p53 signaling












7 (19)16 (15)26 (14)2 (67)2 (67)2 (67)2 (67)6 (24)3 (43)19 (15)11 (18)11 (18)7 (24)8 (23)12 (19)19 (16)16 (17)30 (15)4 (44)26 (16)6 (32)32 (15)29 (15)14 (20)15 (19)24 (16)18 (18)3 (75)28 (16)20 (18)14 (22)12 (24)42 (15)15 (23)31 (17)32 (17)

34 (20)31 (22)15 (38)12 (52)


2 310 4 5 876

minuslog(p value)

(a)


PPAR signaling

SAPKJNK signaling

GADD45 signaling

Apoptosis signaling





FAK signaling

RhoA signaling


HGF signaling


p70S6K signaling

IL-8 signaling


ILK signaling

Rac signaling

RAR activation




ERKMAPK signaling

Integrin signaling



PI3KAKT signaling




mTOR signaling




EIF2 signaling

50 (37)38 (40)38 (40)12 (63)37 (42)47 (39)10 (71)29 (45)16 (57)37 (43)13 (65)43 (40)128 (33)44 (42)12 (71)13 (68)49 (41)70 (38)57 (41)72 (39)46 (44)74 (39)44 (45)27 (55)84 (39)104 (38)79 (42)83 (41)22 (69)18 (78)59 (48)78 (45)42 (62)

85 (47)116 (46)95 (51)80 (55)69 (63)

104 (61)118 (64)

10 1550 20 25 30

minuslog(p value)


junctions


(b)





745 1481 198

VACVΔB18

VACV

(a)

2958 3184 359

VACVΔB18

VACV

(b)



4 Discussion







VACVVACVΔB18

adsorption

+minus IFN

RNAextraction

RNAextraction

9876543210 (hpi)

(+minus rB18)

(a)

VACVΔB18

+ + + +

+minus

minus

minus

minus

minusminus

minus

minus

minus

minusminus

minus

9 9 9 994 4

minus4 minus2 0 2 4


Virushpi na

IFNrB18

VACV

(b)







WR092

APOL9

OAS1

WR127

IRF9

GAPDH

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

10

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8 Fo

ld in

crea

se re

l to

VAC

V

0

2

4

6

8

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

4hp

i

Fold

incr

ease

rel

to V

ACV4

hpi

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i





5 Conclusion



Competing Interests




Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Fold

incr

ease

ove

r unt

reat

ed ce

lls

B18IFN

IFN + B18


lowast

lowast

lowast

0

2

4

6

8

lt 001lowastp







Autophagy

Rac signaling




Glycolysis I


PI3KAKT signaling

ATM signaling


TNFR2 signaling


ERK5 signaling





GADD45 signaling





p53 signaling












7 (19)16 (15)26 (14)2 (67)2 (67)2 (67)2 (67)6 (24)3 (43)19 (15)11 (18)11 (18)7 (24)8 (23)12 (19)19 (16)16 (17)30 (15)4 (44)26 (16)6 (32)32 (15)29 (15)14 (20)15 (19)24 (16)18 (18)3 (75)28 (16)20 (18)14 (22)12 (24)42 (15)15 (23)31 (17)32 (17)

34 (20)31 (22)15 (38)12 (52)


2 310 4 5 876

minuslog(p value)

(a)


PPAR signaling

SAPKJNK signaling

GADD45 signaling

Apoptosis signaling





FAK signaling

RhoA signaling


HGF signaling


p70S6K signaling

IL-8 signaling


ILK signaling

Rac signaling

RAR activation




ERKMAPK signaling

Integrin signaling



PI3KAKT signaling




mTOR signaling




EIF2 signaling

50 (37)38 (40)38 (40)12 (63)37 (42)47 (39)10 (71)29 (45)16 (57)37 (43)13 (65)43 (40)128 (33)44 (42)12 (71)13 (68)49 (41)70 (38)57 (41)72 (39)46 (44)74 (39)44 (45)27 (55)84 (39)104 (38)79 (42)83 (41)22 (69)18 (78)59 (48)78 (45)42 (62)

85 (47)116 (46)95 (51)80 (55)69 (63)

104 (61)118 (64)

10 1550 20 25 30

minuslog(p value)


junctions


(b)





745 1481 198

VACVΔB18

VACV

(a)

2958 3184 359

VACVΔB18

VACV

(b)



4 Discussion







VACVVACVΔB18

adsorption

+minus IFN

RNAextraction

RNAextraction

9876543210 (hpi)

(+minus rB18)

(a)

VACVΔB18

+ + + +

+minus

minus

minus

minus

minusminus

minus

minus

minus

minusminus

minus

9 9 9 994 4

minus4 minus2 0 2 4


Virushpi na

IFNrB18

VACV

(b)







WR092

APOL9

OAS1

WR127

IRF9

GAPDH

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

10

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8 Fo

ld in

crea

se re

l to

VAC

V

0

2

4

6

8

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

4hp

i

Fold

incr

ease

rel

to V

ACV4

hpi

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i





5 Conclusion



Competing Interests




Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Autophagy

Rac signaling




Glycolysis I


PI3KAKT signaling

ATM signaling


TNFR2 signaling


ERK5 signaling





GADD45 signaling





p53 signaling












7 (19)16 (15)26 (14)2 (67)2 (67)2 (67)2 (67)6 (24)3 (43)19 (15)11 (18)11 (18)7 (24)8 (23)12 (19)19 (16)16 (17)30 (15)4 (44)26 (16)6 (32)32 (15)29 (15)14 (20)15 (19)24 (16)18 (18)3 (75)28 (16)20 (18)14 (22)12 (24)42 (15)15 (23)31 (17)32 (17)

34 (20)31 (22)15 (38)12 (52)


2 310 4 5 876

minuslog(p value)

(a)


PPAR signaling

SAPKJNK signaling

GADD45 signaling

Apoptosis signaling





FAK signaling

RhoA signaling


HGF signaling


p70S6K signaling

IL-8 signaling


ILK signaling

Rac signaling

RAR activation




ERKMAPK signaling

Integrin signaling



PI3KAKT signaling




mTOR signaling




EIF2 signaling

50 (37)38 (40)38 (40)12 (63)37 (42)47 (39)10 (71)29 (45)16 (57)37 (43)13 (65)43 (40)128 (33)44 (42)12 (71)13 (68)49 (41)70 (38)57 (41)72 (39)46 (44)74 (39)44 (45)27 (55)84 (39)104 (38)79 (42)83 (41)22 (69)18 (78)59 (48)78 (45)42 (62)

85 (47)116 (46)95 (51)80 (55)69 (63)

104 (61)118 (64)

10 1550 20 25 30

minuslog(p value)


junctions


(b)





745 1481 198

VACVΔB18

VACV

(a)

2958 3184 359

VACVΔB18

VACV

(b)



4 Discussion







VACVVACVΔB18

adsorption

+minus IFN

RNAextraction

RNAextraction

9876543210 (hpi)

(+minus rB18)

(a)

VACVΔB18

+ + + +

+minus

minus

minus

minus

minusminus

minus

minus

minus

minusminus

minus

9 9 9 994 4

minus4 minus2 0 2 4


Virushpi na

IFNrB18

VACV

(b)







WR092

APOL9

OAS1

WR127

IRF9

GAPDH

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

10

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8 Fo

ld in

crea

se re

l to

VAC

V

0

2

4

6

8

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

4hp

i

Fold

incr

ease

rel

to V

ACV4

hpi

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i





5 Conclusion



Competing Interests




Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















745 1481 198

VACVΔB18

VACV

(a)

2958 3184 359

VACVΔB18

VACV

(b)



4 Discussion







VACVVACVΔB18

adsorption

+minus IFN

RNAextraction

RNAextraction

9876543210 (hpi)

(+minus rB18)

(a)

VACVΔB18

+ + + +

+minus

minus

minus

minus

minusminus

minus

minus

minus

minusminus

minus

9 9 9 994 4

minus4 minus2 0 2 4


Virushpi na

IFNrB18

VACV

(b)







WR092

APOL9

OAS1

WR127

IRF9

GAPDH

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

10

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8 Fo

ld in

crea

se re

l to

VAC

V

0

2

4

6

8

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

4hp

i

Fold

incr

ease

rel

to V

ACV4

hpi

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i





5 Conclusion



Competing Interests




Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















VACVVACVΔB18

adsorption

+minus IFN

RNAextraction

RNAextraction

9876543210 (hpi)

(+minus rB18)

(a)

VACVΔB18

+ + + +

+minus

minus

minus

minus

minusminus

minus

minus

minus

minusminus

minus

9 9 9 994 4

minus4 minus2 0 2 4


Virushpi na

IFNrB18

VACV

(b)







WR092

APOL9

OAS1

WR127

IRF9

GAPDH

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

10

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8 Fo

ld in

crea

se re

l to

VAC

V

0

2

4

6

8

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

4hp

i

Fold

incr

ease

rel

to V

ACV4

hpi

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i





5 Conclusion



Competing Interests




Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















WR092

APOL9

OAS1

WR127

IRF9

GAPDH

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

1

2

3

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8

10

Fold

incr

ease

ove

r unt

reat

ed ce

lls

0

2

4

6

8 Fo

ld in

crea

se re

l to

VAC

V

0

2

4

6

8

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

4hp

i

Fold

incr

ease

rel

to V

ACV4

hpi

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i

IFN

VACV

+ IF

N

VACV

ΔB18

+IF

N

VACV

ΔB18

+IF

N +

B18

VACV

ΔB184

hpi

VACV

4hp

i

VACV

ΔB189

hpi

VACV

9hp

i





5 Conclusion



Competing Interests




Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















5 Conclusion



Competing Interests




Acknowledgments


References
















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article RNA-Seq Based Transcriptome Analysis of the...

Documents

Transcript of Research Article RNA-Seq Based Transcriptome Analysis of the...