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The Human Cytomegalovirus Strain DB Activates Oncogenic Pathways in Mammary Epithelial Cells

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Background: Human cytomegalovirus (HCMV) establishes a persistent lifelong infection and increasing evidence indicates HCMV infection can modulate signaling pathways associated with oncogenesis. Breast milk is an important route of HCMV transmission
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  Research Paper The Human Cytomegalovirus Strain DB Activates Oncogenic PathwaysinMammary Epithelial Cells Amit Kumar a,1 , Manoj Kumar Tripathy a,1 , Sébastien Pasquereau a,1 , Fatima Al Moussawi a,b , Wasim Abbas c ,Laurie Coquard a , Kashif Aziz Khan a , Laetitia Russo d , Marie-Paule Algros d , Séverine Valmary-Degano d ,Olivier Adotevi e,f  , Stéphanie Morot-Bizot c , Georges Herbein a,g, ⁎ a Department Pathogens & In  󿬂 ammation-EPILAB, UPRES EA4266, University of Franche-Comté (UFC), University of Bourgogne France-Comté (UBFC), F-25030 Besançon, France b Lebanese University, Beyrouth, Lebanon c  Apex Biosolutions, F-25000 Besançon, France d Department of Pathology, CHRU Besançon, F-25030 Besançon, France e INSERM UMR1098, University of Bourgogne Franche-Comté, Besançon, France f  Department of Medical Oncology, CHRU Besancon, F-25030 Besancon, France g Department of Virology, CHRU Besancon, F-25030 Besancon, France a b s t r a c ta r t i c l e i n f o  Article history: Received 29 September 2017Received in revised form 9 March 2018Accepted 13 March 2018Available online 28 March 2018 Background: Humancytomegalovirus(HCMV)establishesapersistentlife-longinfectionandincreasingevidenceindicatesHCMVinfectioncanmodulatesignalingpathwaysassociatedwithoncogenesis.Breastmilkisanimpor-tantrouteofHCMVtransmissioninhumansandwehypothesizedthatmammaryepithelialcellscouldbeoneof the main cellular targets of HCMV infection. Methods:  The infectivity of primary human mammary epithelial cells(HMECs) was assessed following infectionwith the HCMV-DB strain, a clinical isolate with a marked macrophage-tropism. The impact of HCMV-DB infec-tiononexpressionofp53andretinoblastomaproteins,telomeraseactivityandoncogenicpathways(c-Myc,Akt,Ras, STAT3) was studied. Finally the transformation of HCMV-DB infected HMECs was evaluated using soft agarassay. CTHcells (CMVTransformedHMECs)were detectedinprolonged cultures of infectedHMECs.Tumorfor-mation was observed in NOD/SCID Gamma (NSG) mice injected with CTH cells. Detection of long non codingRNA4.9 (lncRNA4.9) gene was assessed in CTH cells, tumors isolated from xenografted NSG mice and biopsiesof patients with breast cancer using qualitative and quantitative PCR. Results:  We found that HCMV, especially a clinical strain named HCMV-DB, infects HMECs  in vitro . The clinicalstrain HCMV-DB replicates productively in HMECs as evidenced by detection of early and late viral transcriptsand proteins. Following infection of HMECs with HCMV-DB, we observed the inactivation of retinoblastomaand p53 proteins, the activation of telomerase activity, the activation of the proto-oncogenes c-Myc and Ras,the activation of Akt and STAT3, and the upregulation of cyclin D1 and Ki67 antigen. Colony formation was ob-served in soft agar seeded with HCMV-DB-infected HMECs. Prolonged culture of infected HMECs resulted inthe development of clusters of spheroid cells that we called CTH cells (CMV Transformed HMECs). CTH cellswhen injected in NOD/SCID Gamma (NSG) mice resulted in the development of tumors. We detected in CTHcells thepresence of a HCMVsignature corresponding to a sequence of the longnoncoding RNA4.9 (lncRNA4.9)gene. We also found the presence of the HCMV lncRNA4.9 sequence in tumors isolated from xenografted NSGmiceinjectedwithCTHcellsandinbiopsiesofpatientswithbreastcancerusingqualitativeandquantitativePCR. Conclusions:  Our data indicate thatkey molecular pathways involvedinoncogenesis areactivated inHCMV-DB-infected HMECs that ultimately results in the transformation of HMECs  in vitro  with the appearance of CMV-transformed HMECs (CTH cells) in culture. CTH cells display a HCMV signature corresponding to a lncRNA4.9 Keywords: CytomegalovirusHCMV HCMV-DBHMECsOncogenesisTransformationCTH cellslncRNA4.9EBioMedicine 30 (2018) 167 – 183  Abbreviations: ChIP, chromatin immunoprecipitation; HMECs, human mammary epithelial cells; HCMV,humancytomegalovirus; MOI, multiplicity of infection; UV, ultraviolet rays;HI, heat inactivated; Rb, retinoblastoma; hTERT, human telomerase reverse transcriptase; IE, immediate early; LA, late antigen. ⁎  Corresponding author at: Department Pathogens & In 󿬂 ammation-EPILAB, EA4266, University of Bourgogne Franche-Comte, 16 route de Gray, F-25030 Besançon, Cedex, France. E-mail addresses:  sebastien.pasquereau@univ-fcomte.fr, (S. Pasquereau), mp1algros@chu-besancon.fr, (M.-P. Algros), sdeganovalmary@chu-besancon.fr, (S. Valmary-Degano), Olivier.adotevi@univ-fcomte.fr, (O. Adotevi), smorot@apexlabo.com, (S. Morot-Bizot), georges.herbein@univ-fcomte.fr. (G. Herbein). 1 AK, MKT, SP contributed equally to the work.https://doi.org/10.1016/j.ebiom.2018.03.0152352-3964/© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Contents lists available at ScienceDirect EBioMedicine  journal homepage: www.ebiomedicine.com  genomicsequenceandgiverisetofastgrowingtriple-negativetumorsinNSGmice.AsimilarlncRNA4.9genomicsequence was detected in tumor biopsies of patients with breast cancer.©2018TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction Worldwide breast cancer is the most common cancer diagnosedamongwomen (Warner,2011). Notably, majorityof the breast cancersclassi 󿬁 ed as carcinomas have been found to be srcinated from themammary epithelial cells lining the duct responsible for convertingmost precursors into milk constituents and transporting them to themammary lumen (Dimri et al., 2005). Breast cancer exhibits heteroge-neous molecular characteristics and utilizing gene expression patternsseveral types of breast cancer have been identi 󿬁 ed including a normalbreast epithelial-like group, a luminal epithelial cell type A, a luminalepithelial cell type B, an ErB2-overexpressing group, a basal-like groupand a claudin low group (Lehmann et al., 2011). Etiological factors in-volved in breast cancer include genetic and environmental risk factors(Hüsing et al., 2012), and among these latter viruses could be involvedwith close to one- 󿬁 fth of all cancers in the world caused by infectiousagents (Zur Hausen, 2009).The human cytomegalovirus (HCMV), a highly host speci 󿬁 c patho-gen, is a member of   Betaherpesviridae  family. HCMV generally causesasymptomatic to mild infection in immunocompetent host. However,itsinfectioninimmunocompromisedhostmayresultinseriouscompli-cations (Coaquette et al., 2004). HCMV infects a broad range of cells in-cluding monocytes, macrophages,  󿬁 broblasts, endothelial cells,epithelial cells, stromal cells, hepatocytes, smooth muscle cells, andneural stem/progenitor cells (Belzile et al., 2014; Khan et al., 2009; Lepiller et al., 2013; Wang and Shenk, 2005). Although HCMV clinical isolates display a broad cellular tropism infecting among others  󿬁 bro-blasts and epithelial cells, the growth of laboratory HCMV strains is re-stricted to  󿬁 broblasts (Wang and Shenk, 2005). In infected patients,thebloodmonocytesandtissuemacrophagesareregardedasanimpor-tant HCMV cellular reservoir responsible for the dissemination of virusand may also act as a site for the establishment of latency (Hargettand Shenk, 2010; Khan et al., 2009; Smith et al., 2004). Noteworthy, HCMV has the ability to induce a distinct in 󿬂 ammatory (M1) and im-munosuppressive (M2) macrophages polarization (Chan et al., 2009).In addition, macrophage polarization into M1/M2 phenotype is associ-ated with the secretion of cytokines that could play a pivotal role inviral replication and  󿬁 tness, and favor breast cancer promotion(Grivennikov et al., 2010; McKinney et al., 2014; Teng et al., 2012). Role of HCMV in in 󿬂 ammatory diseases and cancer has been wellspeculated (Cobbs et al., 2002; Lepiller et al., 2011; Söderberg-Nauclér, 2006). Earlier studies demonstrated that HCMV was able to induce the in vitro  transformation of human embryo lung  󿬁 broblasts (Clantonet al., 1983; Geder et al., 1976). More recently, HCMV DNA or antigen hasbeenfoundintumortissuesfrombrain(glioblastoma,medulloblas-toma), colon, prostate, liver and breast cancer (Banerjee et al., 2015;Baryawno et al., 2011; Bhattacharjee et al., 2012; Harkins et al., 2010; Samanta et al., 2003; Taher et al., 2013). Besides a direct role of HCMV  in cellular transformation, HCMV could infect the tumor tissue andactsasacofactorinamplifyingmechanismsofoncogenesis,aparadigmcalled as oncomodulation (Michaelis et al., 2009).Previously, we isolated a clinical HCMV strain from a 30-year-oldpregnant woman named as HCMV-DB (KT959235), which is highlymacrophage-tropic, triggers a M2 phenotype and upregulates theproto-oncogene Bcl-3 (Khan et al., 2009). HCMV-DB strain is closefrom other primary clinical isolates which also infect macrophagessuch as PH and TR strains (Suppl. Fig. S1) (Michaelis et al., 2009).There is scarcity of direct evidence suggesting the involvement of HCMV in transformation of human mammary epithelial cells (HMECs)(Herbein and Kumar, 2014). Here, we assessed the potential directoncogenic role of HCMV in primary human mammary epithelial cells(HMECs)  in vitro  and  in vivo . 2. Materials and Methods  2.1. Reagents Anti-p53, anti-Rb,anti-Ras, anti-pAktThr308, anti-pAktSer473,anti-Akt and anti-cyclinD1 antibodies were purchased from Cell signaling(Danvers, MA, USA). Anti-pSTAT3, anti-STAT3 and anti-Myc antibodieswere purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti- β -actin antibody was purchased from Sigma-Aldrich (St. Louis, MO,USA). Anti-pp65, anti-pp85 (pUL25), anti-pp71 (pUL82), anti-IE1 andanti-IE2antibodieswerepurchasedfromSantaCruzBiotechnology.Re-combinantRaf1-GSTwaspurchasedfromMillipore(Molsheim,France).  2.2. Cell Cultures Human primary mammary epithelial cells (HMECs) were obtainedfrom Life Technologies (Carlsbad, CA, USA). MDA-MB-231 and MCF-7cells were provided by Institut Hiscia (Arlesheim, Switzerland).HMECs were cultivated in HMEC medium (Life Technologies, Carlsbad,CA, USA) supplemented with HMEC supplement and bovine pituitaryextract (Life Technologies, USA). Cell viability assay was performed aspreviously described (Khan et al., 2009). Cultures were free of mycoplasma.  2.3. HCMV Infection of HMECs AD169isahighlypassagedlaboratorystrainofHCMVoriginallyiso-latedfromtheadenoidsofachild(Murphyetal.,2003).Theclinicaliso-late HCMV-DB was isolated in our laboratory from a cervical swabspecimen from a 30-year-old pregnant woman (Khan et al., 2009).Cell-free virus stocks were prepared by propagating AD169 in MRC5human 󿬁 broblasts, meanwhile HCMV-DB were grown in macrophages,as described previously (Khan et al., 2009). The TB40/E strain was iso-lated from a throat wash of a bone marrow transplant recipient(Tomasec et al., 2005). MRC5 human  󿬁 broblasts were cultured as de-scribed previously (Coaquette et al., 2004; Khan et al., 2009). HMECs (1×10 6 )andMRC5cells(1×10 6 )wereinfectedatamultiplicityofin-fection(MOI)of1or10for2hat37°C,washedthoroughly(threetimeswith 1× PBS),and covered withfreshmedium.Wherespeci 󿬁 ed,HCMV heat-inactivatedat95°Cfor10minortreatedwithUV(1200 μ   J/cm 2 for15 min)were used ascontrols.Supernatants were clari 󿬁 ed by centrifu-gation and stored at − 80 °C until use. Virus titers were determined byplaque-forming assay in MRC5 human  󿬁 broblasts as described previ-ously(Khanetal.,2009).ThepurityofourHCMVstockswascon 󿬁 rmedby the absence of detection of other viruses (HSV-1, HSV-2, varicella-zostervirus,Epstein-Barrvirus,adenovirus,BKvirus)usingPCRscreen-ing (data not shown). Following HCMV infection of HMECs, viral repli-cation was assessed by the appearance of a cytopathic effect (CPE) inthe cultures and by detection of IE1, IE2, pp65 and pp85 by westernblotting and IE1 antigen (clone E13, Argene-Biosoft, Varihes, France)usingimmuno 󿬂 uorescencemicroscopy(NikonEclipseE400,Kanagawa, Japan).ForthedetectionofHCMVmRNAusingRT-PCRassay,totalRNAwas extracted from uninfected, UV-treated and HCMV-DB infectedHMECs with RNeasy mini kit (Qiagen). Total 2  μ  g of RNA was reversetranscribed into cDNA with Superscript III RT (Life Technologies) usingoligo (dT) primers. The 5  μ  l of reverse transcription reaction productwas ampli 󿬁 ed using primers against IE1, US28 and UL82 primers as 168  A. Kumar et al. / EBioMedicine 30 (2018) 167  – 183  described previously (Lepiller et al., 2013). The beta-globin gene wasampli 󿬁 ed as an internal control (sense, 5 ′ -TCCCCTCCTACCCCTACTTTCTA-3 ′ ; antisense, 5 ′ -TGCCTGGACTAATCTGCAAGAG-3 ′ ). The PCR prod-uct was electrophoresed on a 2% agarose gel containing ethidium bro-mide. Quanti 󿬁 cation of viral titer in cell culture supernatants wasperformed by qPCR as previously described (Khan et al., 2009). TheUL128,UL131,UL133andUL138geneswereampli 󿬁 edusingthefollow-ing primers: UL128 (sense, 5 ′ -GATTCGCGGGATCGTCACCA-3 ′ ; anti-sense, 5 ′ -TCACTGGAGCATATAGCCCA-3 ′ ); UL131 (sense,5 ′ -ATGTGTATGATGTCTCATAATAAAGC-3 ′ ; antisense, 5 ′ -TCAACGTGACGTCCACGAGC-3 ′ ); UL133 (sense, 5 ′ -AGACTCCGTAATCGACCTCC-3 ′ ; antisense,5 ′ -GATGACCGTATTGGACCATGTC-3 ′ ) and UL138 (sense, 5 ′ -ACGATCTGCCGCTGAATGTC-3 ′ ; antisense, 5 ′ -ACAGCTCGCAACAGCGGATC-3 ′ ).Quanti 󿬁 cation of c-Myc (MYC) and cyclin-D1 (CCND1) transcripts inHMECsinfectedwithHCMV-DB(moi=1,day1postinfection)andun-infectedHMECswasperformedusingthehumanbreastcancerRT 2 pro- 󿬁 ler PCR assay (PAHS-131ZA) (Qiagen). HCMV entry assays wereperformed as previously described (Khan et al., 2009).  2.4. Western Blotting  Cellularextracts of HMECs either uninfected or infected withHCMV or UV-treated HCMV strains were used to examine the expression of p53, Rb, pRb, c-Myc, Ras, pAkt(Thr308), pAkt(Ser473), Akt, pSTAT3,STAT3, cyclin D1, IE1, IE2, pp65, pp85 and  β -actin protein by westernblotting as described previously (Khan et al., 2009). Where speci 󿬁 ed,protein levels were quanti 󿬁 ed by densitometry using ImageJ 1.40 soft-ware (National Institutes of Health, Bethesda, MA, USA).  2.5. Pull-Down Assay ForGST-pulldownassay,20 μ  gofGST-Raf(Millipore)proteinswereimmobilized on glutathione agarose beads (Thermo Fisher Scienti 󿬁 c,Rochester, NY), washed  󿬁 ve times with 1× PBS, and were incubatedovernight at 4 °C with 500  μ  g of lysates. The suspension was thenwashed three times with 1× PBS, denaturized and the expression of active-Ras was analyzed by SDS-PAGE and autoradiography.  2.6. Immunoprecipitation HMECs were mock-treated or were infected with HCMV strains(MOI = 1). At day 3 post infection, the cell lysates were precleared byadding 50  μ  l of protein A magnetic beads (Millipore) for 1 h at 4 °C.The clear supernatants were removed, combined with anti-p53 anti-body (Cell Signaling) or isotype control antibody (Millipore) and incu-bated overnight at 4 °C. The lysates were further incubated with 50  μ  lprotein A magnetic beads (Millipore) at 4 °C for 2 h. Immune complexwere washed in the presence of protease inhibitors (Roche, Meylan,France) and bound protein was eluted with sample buffer and run on10% SDS-PAGE gels. Expression of IE2 was determined by westernblotting.  2.7. Chromatin Immunoprecipitations Assay p53 and H3K9me3 chromatin immunoprecipitations (ChIPs) wereperformed with EZ-Magna ChIP ™ A - Chromatin ImmunoprecipitationKit (Millipore). Brie 󿬂 y, 10 7 HMECs were infected with HCMV-DB andatday1postinfectioncellswere 󿬁 xedandsonicatedaspermanufactur-er's instructions. Sonication conditions were as follows: 9 pulses, 40%amplitude, pulse duration 9 s and gap between each cycle 30 s. p53and H3K9me3 ChIP assays were performed as described previously(Soria et al., 2010). DNA was puri 󿬁 ed and analyzed using PCR followedby agarose gel electrophoresis.  2.8. Flow Cytometry Analysis For proliferation assays, HMECs were left uninfected or were in-fected with HCMV. Proliferationwasmeasured usingthequanti 󿬁 cationof Ki67Ag expression by intracellular 󿬂 ow cytometry asdescribed pre-viously(Lepilleretal.,2013).Todiscriminatebetweeninfectedandun-infected HMECs, the pp71 HCMV expression was detected by  󿬂 owcytometry using an anti-pp71 antibody.  2.9. Assessment of Telomerase Activity Telomerase activity was assayed by using TRAPEZE Telomerase de-tectionkitasrecommendedbythemanufacturer(Chemicon,Temecula,CA). Control and HCMV-infected HMECs were suspended in 3-[(3-cholamidopropyl) dimethyl ammonio] propanesulfonic acid (CHAPS)lysis buffer (10 mMTris  –  HCl [pH 7.5], 1 mM MgCl 2 ,1 mM EGTA,0.1 mM phenylmethylsulfonyl  󿬂 uoride, 5 mM P3-mercaptoethanol,0.5% CHAPS, 10% glycerol) for 20 min on ice and centrifuged at20,000  g   for 20 min at 4 °C. The amount of protein in the supernatantswas determined using a DC Protein Assay kit (Bio-Rad Laboratories,Hercules, CA). For each assay of telomerase activity, 1  μ  g of proteinwasused, and 30 PCR cycles were performed after the elongation reac-tionusingtelomeraseprimers.ThePCRproductswereresolvedbypoly-acrylamide gel electrophoresis and visualized by staining with SYBR Green I (Roche). For the detection of hTERT transcripts, total cellularRNA was isolated from uninfected or HCMV-infected HMECs with anRNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was synthesizedwith OligodT20 primers using a SuperScript III First-Strand SynthesisSystem (RT-PCR; Invitrogen Life Technologies, Carlsbad, CA). hTERTmRNA expression was analyzed by RT-PCR with primers speci 󿬁 c forhTERT mRNA (accession no. AF015950): 5 ′ -CGGAAGAGTGTCTGGAGCAA-3 ′  and 5 ′ -GGATGAAGCGGAGTCTGGA-3 ′ .  2.10. Soft Agar Colony Formation Assay Colony formation in soft agar seeded with uninfected HMECs,HMECs infected with wild-type or heat-inactivated HCMV, MCF-7 cellsand MDA-MB-231 cells was assayed using Cell Biolabs Cytosolic CellTransformation Assay kit (Colorimetric assay, CB135; Cell Biolabs Inc.,San Diego, CA) as per the manufacturer's protocol. Starting 1 day postinfection, cells were incubated for 14 days (15 days post-infection) inthe semisolid agar medium before solubilization and detection usingthe provided MTT solution for quanti 󿬁 cation of the formation of colo-nies in soft agar (Cayman Chemical, Ann Arbor, MI) and a microplatereader(  A 570nm ).ColonieswereobservedunderanOlympusmicroscope(Center Valley, PA). Following DNA extraction from soft agar colonies,theHCMVmajorimmediateearlypromoter(MIEP)andbeta-globinse-quences were ampli 󿬁 ed using the following primers: MIEP (sense, 5 ′ -TGGGACTTTCCTACTTGG-3 ′ ; antisense, 5 ′ -CCAGGCGATCTGACGGTT-3 ′ )and beta-globin (sense, 5 ′ -TCCCCTCCTACCCCTACTTTCTA-3 ′ ; antisense,5 ′ -TGCCTGGACTAATCTGCAAGAG-3 ′ ).  2.11. Isolation and Growth of CTH Cells HMECs cultures were infectedwith HCMV-DBata MOI of 1.Severalclustersofspheroid-cellswereobservedinHMECsinfectedwithHCMV-DBaroundday20postinfectioninsomeofthecultures.Thesespheroid-cell clusters detected in HMEC cultures infected with HCMV-DB weregently detached and the  󿬂 oating detached cells named CTH cells werecultured in HMEC Ready medium (Cat#12752010, Gibco, Grand Island,NY) for numerous passages, currently  N 150 passages.  2.12. Animals Six-week-oldfemaleNOD/SCIDGamma(NSG)micepurchasedfromCharles River Laboratories (L'Arbresle, France) were kept under strict 169  A. Kumar et al. / EBioMedicine 30 (2018) 167  – 183  pathogen-freeconditionsattheCentralAnimalfacilitiesofUniversityof Franche-Comté (noti 󿬁 cation d'autorisation n° 05085.02). Cell viabilitywas determined by trypan blue staining and cells were counted usinghaemocytometer. After harvesting and during injection, cells weremaintained at 4 °C. Two million of CTH cells,  󿬁 ve million cells of unin-fected HMECs, two million of MCF-7 cells and MDA-MB-231 cellssuspended in100 μ  lof serum-freeDulbecco'smodi 󿬁 edEagle's mediumtogether with 100  μ  l of Matrigel at the day 0, were injected into themammary fat pad of NSG mice. Mice were checked twice in a weekfor tumor growth. Once tumor development was detectable by eye,tumordimensionsweremeasuredusingverniercaliper.Tumorvolumewascalculatedusingtheformula:volume(cm 3 )=(d×d×D)/2wheredistheshortestdiameterandDisthelongestdiameter.Onday37postinjection,micewerekilledaccordingtotheguidelinesoftheanimaleth-ical committee. Tumors were retrieved from the mice and analyzed forthe presence of MIEP and lncRNA4.9 sequences from HCMV-DB usingPCR.  2.13. Immunohistochemistry of Mice Tumors Formalin- 󿬁 xed tumors retrieved from the mice were embedded inwax and sections (5  μ  m) were prepared using standard methods andstained with eosin and hematoxylin. Sections were processed andstained individually for hematoxylin and eosin staining, ER (1:1,Roche), PR (1:1, Roche), HER2 (1:1, Roche), vimentin (1:400, Leica),E-cadherin (1:50, Dako), CK5/6 (1:50, Dako), GATA3 (1:100, CellMarque),CK20(1:5000,Biocare),andGCDFP(1:1000,CellMarque)be-fore observation by microscopy.  2.14. Detection of HCMV lncRNA 4.9 Genomic Sequence in CTH Cells, Xenografted Mice Tumor and Human Breast Cancer Tissue Total DNA from uninfected HMEC, HCMV-DB infected HMEC, CTHcells and MRC5 cultures was isolated. The retrieved mice tumors weregrinded in liquid nitrogen and DNA was isolated using QIAamp DNAmini kit (Qiagen, Valencia, CA) as per manufacture's guidelines. Geno-mic DNA isolated from patient breast tumor biopsies and from healthyhuman breast tissue was provided by the regional tumor bank (BB-0033-00024 Tumorothèque Régionale de Franche-Comté).Presence of HCMV was determined by qualitative and quantitativePCR where speci 󿬁 ed using a set of HCMV-DB lncRNA 4.9 gene primers(sense, 5 ′ -GTGAACCGATACGGGTGCAG-3 ′ ; antisense, 5 ′ -CATTTGAACAGAGAAAGGTGG-3 ′ ). An amplicon of 126 bp corresponding to HCMV-DBlncRNA4.9genewasampli 󿬁 ed ascon 󿬁 rmed bySanger's sequencing(Genoscreen,Lille,FranceandGATC,Köln,Germany).WealsoscreenedCTH cells, mice tumors and human breast biopsies (tumor and healthytissue) for the presence of MIEP sequence using PCR assay (sense, 5 ′ -TGGGACTTTCCTACTTGG-3 ′ ; antisense, 5 ′ -CCAGGCGATCTGACGGTT-3 ′ ).As a positive control, DNA isolated from HCMV-DB infected HMECsandfromHCMV-DBviralstockwasincludedinthestudy.Equalamountof DNA was analyzed by PCR using HCMV-DB MIEP and lncRNA 4.9primers. As equal loading control  β -globin gene was ampli 󿬁 ed (sense,5 ′ -TCCCCTCCTACCCCTACTTTCTA-3 ′ ; antisense, 5 ′ -TGCCTGGACTAATCTGCAAGAG-3 ′ ). Ampli 󿬁 ed product was electrophoresed in 2% agarosegel stained with Sybr green I nucleic acid stain.  2.15. Phylogenetic Analysis Phylogenetic analysis was determined amongseveral HCMV strains(described in Table 1) with respect to the  UL144  gene as previously re-ported (Waters et al., 2010). Multi-sequence alignments (MSA) wereperformedusingCLUSTALWwithfollowingparameters:agapopeningpenalty of 15 and gap extension penalty of 6.66. Phylogenetic tree wasconstructed using the neighbor-joining method. Each clustering wascon 󿬁 rmed by the bootstrap method with 1000 replicates. The analysiswas conducted using MEGA7 (http://www.megasoftware.net/).  2.16. Statistical Analysis The reported values are the means and SD of independent experi-ments. Statistical analysis was performed using Mann Whitney  U   test,and differences were considered signi 󿬁 cant at a value of P  b  0.05.Microsoft Excel was used to construct the plots. 3. Results  3.1. HCMV Permissively Infects and Completes its Life Cycle in HMECs AlthoughHCMVproteinsandDNAhavebeenreportedinbreastcan-cer tissue (Harkins et al., 2010; Taher et al., 2013), so far only few at- tempts have been made to investigate HCMV replication in HMECs in vitro  (Twite et al., 2014). We infected HMECs with two strains of HCMV, HCMV-DB a clinical isolate which is macrophage-tropic, closeto PR and TH strains and belongs to UL144 genotype C (Suppl Fig. S1)(Khan et al., 2009), and the extensively passaged laboratory strainAD169 that has lost the ability to ef  󿬁 ciently replicate in endothelialand epithelial cells (Sinzger et al., 2008; Wang and Shenk, 2005). We observed productive replication of HCMV-DB in infected HMECs withpeak viral titer at day 12 post infection (Fig. 1a, left panel). AD169 didnotreplicateinHMECs(Fig.1a,leftpanel).Thelevelofproductiveinfec-tion of the HCMV-DB strain in HMECs was much lower than what was  Table 1 Phylogeneticanalysisbasedon UL144 genewasdeterminedamongseveralHCMVstrains.Strain name Abbreviation Accessionnumber1 HHV5 strain DB, complete genome DB KT9592352 HHV5 transgenic strain Towne, complete genome Towne GQ1210413 HHV5 strain AD169, complete genome AD169 FJ5275634 HHV5 strain TB40/E clone Lisa, complete genome TB40/E KF2973395 HHV5 transgenic strain Merlin, complete genome Merlin GU1790016 HHV5 strain Toldeo, complete genome Toledo GU9377427 HHV5 strain VR1814, complete genome VR1814 GU1792898 HHV5 strain Davis, complete genome Davis JX5121989 HHV5 strain JP, complete genome JP GQ22197510 HHV5 strain TR, complete genome TR KF02160511 HHV5 PH-BAC isolate, complete genome PH AC146904 Fig.1. HCMV-DBinfectsandreplicatesinHMECs.a.GrowthkineticsofHCMVinHMECsandMRC5cells.Leftpanel.HMECswereinfectedindividuallywithtwoHCMVstrains(HCMV-DBandAD169)(MOI=1).At2h postinfectioninoculumwas removedandcellswerewashedthreetimeswith1×PBSfollowed by threewashes withHMECmedia.Atseveraltimepoints(up to day 25) supernatants were collected and viral growth kinetics were determined by realiversity of Bourgogne France-Comté (UBFC), F-25030 time qPCR. Right panel. MRC5 cellswereinfectedwithHCMV-DBandAD169(MOI=1)andsupernatantwascollectedatseveraltimepoints(uptoday5).ViralgrowthkineticswasdeterminedusingrealtimeqPCR.Resultsrepresent means (±SD)of three independent experiments. b. Replicative virus is detected insupernatants harvested from HMECsinfected with HCMV-DB. Supernatants collected fromHMECsinfectedwithHCMV-DBatseveraltimepoints(uptoday21)wereusedtoinfectMRC5cellsandatday5postinfectioncellswere 󿬁 xed.ExpressionofIE1antigenwasdeterminedbyimmuno 󿬂 uorescenceasdescribedinMaterialsandMethods.DirectinfectionofMRC5cellsbyHCMV-DB(MOI=1)wasusedasapositivecontrol.SupernatantsharvestedfromHMECsinfected with UV-inactivated HCMV-DB (MOI = 1) were used as a negative control.Right panel. The curves represent the number of IE1 positive foci detected in MRC5 cultures treatedwith supernatants harvestedfromHMECs infected with HCMV-DBand UV-inactivatedHCMV-DBrespectively.Resultsrepresentmeans (±SD) of threeindependent experiments. c.De-tection of a typical cytopathic effect at day 15 post-infection in HMECs infected with HCMV-DB (MOI = 1) (Upper right panel). MRC5-infected with HCMV-DB were used as a positivecontrol (Lower right panel). Mock-infected HMECs and MRC5 cells were included as negative controls. Magni 󿬁 cation 100×. d. Detection of IE1 antigen in HMECs infected with HCMV-DB at day 12 post infection. Presence of IE1 antigen was assessed in HMECs cells infected with HCMV-DB (two positive cultures are shown) and AD169 strains at day 12 post infectionbyimmuno 󿬂 uorescenceassayasdescribedinMaterialsandMethods.Ascontrols,uninfectedHMECsandMRC5cells,HMECsinfectedwithUV-treatedHCMV-DBandMRC5cellsinfectedwith HCMV-DB are shown. Magni 󿬁 cation 20×.170  A. Kumar et al. / EBioMedicine 30 (2018) 167  – 183  observedinMRC5 󿬁 broblasts,usingaPCRassay(Fig.1a,rightpanel).TodeterminewhetherHCMVDNAdetectionbyPCRassayrepresentsgen-uine replicative virus, we harvested supernatants of HMECs infectedwith HCMV-DB strain up to day 21 post infection. Harvested superna-tants were used to infect MRC5 cells and the number of infected cellswas determined by using IE1 immuno 󿬂 uorescence staining (Fig. 1b).Using harvested supernatants to infected MRC5 cells, we observed lowlevels of HCMV-DB replication in HMECs (Fig. 1b). In contrastsupernatants of HMECs infected with UV-inactivated HCMV-DB failedto replicate in MRC5 cells (Fig. 1b). MRC5 cells directly infected withHCMV-DB were used as a positive control (Fig. 1b).Although limited, a typical cytopathic effect was observed in HMECcultures directly infected with HCMV-DB (Fig. 1c). In contrast toAD169, HCMV-DB was able to start its life cycle in HMECs asevidencedby expression of IE1 genes determined by immuno 󿬂 uorescence assay(Fig. 1d). We detected both viral transcripts and proteins of various     d  e  v   i  r  e   d    C   E   M   H   t  n  a   t  a  n  r  e  p  u  s      H     C     M     V   -     D     B     U     V   -     H     C     M     V   -     D     B D7 D12 D 15 D 21    P  o  s   i   t   i  v  e   C  o  n   t  r  o   l D5 MRC5 cells 0510152025D5 D7 D12 D15 D21    I  m  m  u  n  o   f   l  u  o  r  e  s  c  e  n   t  c  e   l   l  s  p  e  r  w  e   l   l HCMV-DBUV-HCMV-DB bc  Uninfected HCMV-DB MRC-5HMEC   s   C   E   M   H   1   2   d  p   i UV-HCMV-DBMOCK HCMV-DB AD169    M   R   C   5   5   d  p   i Positive control d MOCK HCMV-DB a 024681012D7 D9 D12 D15 D17 D19 D21 D23 D25    )   l  m   /  s  e   i  p  o  c   d  n  a  s  u  o   h   t   (    d  a  o   l    l  a  r   i   V Time post infection HCMV-DB AD169   s   C   E   M   H Time post infection    M   R   C   5   V   i  r  a   l   l  o  a   d   (   l  o  g  c  o  p   i  e  s   /  m   l   ) 1,00E+001,00E+011,00E+021,00E+031,00E+041,00E+050H 2H 5H D1 D3 D5 171  A. Kumar et al. / EBioMedicine 30 (2018) 167  – 183
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