EBV and human microRNAs co-target oncogenic and apoptotic viral and human genes during latency
2012; Springer Nature; Volume: 31; Issue: 9 Linguagem: Inglês
10.1038/emboj.2012.63
ISSN1460-2075
AutoresKasandra Riley, Gabrielle S. Rabinowitz, Therese A. Yario, Joseph M. Luna, Robert B. Darnell, Joan A. Steitz,
Tópico(s)Polyomavirus and related diseases
ResumoArticle30 March 2012free access EBV and human microRNAs co-target oncogenic and apoptotic viral and human genes during latency Kasandra J Riley Kasandra J Riley Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Gabrielle S Rabinowitz Gabrielle S Rabinowitz Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, Rockefeller University, New York, NY, USA Search for more papers by this author Therese A Yario Therese A Yario Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Joseph M Luna Joseph M Luna Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, Rockefeller University, New York, NY, USA Search for more papers by this author Robert B Darnell Robert B Darnell Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, Rockefeller University, New York, NY, USA Search for more papers by this author Joan A Steitz Corresponding Author Joan A Steitz Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Kasandra J Riley Kasandra J Riley Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Gabrielle S Rabinowitz Gabrielle S Rabinowitz Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, Rockefeller University, New York, NY, USA Search for more papers by this author Therese A Yario Therese A Yario Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Joseph M Luna Joseph M Luna Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, Rockefeller University, New York, NY, USA Search for more papers by this author Robert B Darnell Robert B Darnell Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, Rockefeller University, New York, NY, USA Search for more papers by this author Joan A Steitz Corresponding Author Joan A Steitz Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Author Information Kasandra J Riley1, Gabrielle S Rabinowitz1,2, Therese A Yario1, Joseph M Luna2, Robert B Darnell2 and Joan A Steitz 1 1Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT, USA 2Laboratory of Molecular Neuro-Oncology, Howard Hughes Medical Institute, Rockefeller University, New York, NY, USA *Corresponding author. Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, 295 Congress Avenue, BCMM136, New Haven, CT 06536, USA. Tel.:+203 737 4418; Fax:+203 624 8213; E-mail: [email protected] The EMBO Journal (2012)31:2207-2221https://doi.org/10.1038/emboj.2012.63 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Epstein–Barr virus (EBV) controls gene expression to transform human B cells and maintain viral latency. High-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) identified mRNA targets of 44 EBV and 310 human microRNAs (miRNAs) in Jijoye (Latency III) EBV-transformed B cells. While 25% of total cellular miRNAs are viral, only three viral mRNAs, all latent transcripts, are targeted. Thus, miRNAs do not control the latent/lytic switch by targeting EBV lytic genes. Unexpectedly, 90% of the 1664 human 3′-untranslated regions targeted by the 12 most abundant EBV miRNAs are also targeted by human miRNAs via distinct binding sites. Half of these are targets of the oncogenic miR-17∼92 miRNA cluster and associated families, including mRNAs that regulate transcription, apoptosis, Wnt signalling, and the cell cycle. Reporter assays confirmed the functionality of several EBV and miR-17 family miRNA-binding sites in EBV latent membrane protein 1 (LMP1), EBV BHRF1, and host CAPRIN2 mRNAs. Our extensive list of EBV and human miRNA targets implicates miRNAs in the control of EBV latency and illuminates viral miRNA function in general. Introduction MicroRNAs (miRNAs) are ∼22 nt noncoding RNAs that regulate gene expression in normal development (Ambros, 2011) and cancer (Di Leva and Croce, 2010). Through binding to an Argonaute (Ago) protein (human Ago1-4) and other RNA-induced silencing complex (RISC) components, miRNAs directly but imperfectly base-pair to mRNA targets and regulate translation and/or mRNA stability, resulting in modest declines in protein levels (Bartel, 2004) or in switch-like protein downregulation (Mukherji et al, 2011). Bioinformatic and biochemical studies of miRNA–mRNA base-pairing show that interactions typically occur via the 'seed' (minimally nts 2–7) of the miRNA (Lewis et al, 2005; Grimson et al, 2007) and that most human mRNAs are miRNA targets (Lim et al, 2005; Chi et al, 2009; Friedman et al, 2009). global identification of regulated mRNAs is key to understanding miRNA function but this has proven challenging, especially for viral miRNAs where mRNA target sites may not be evolutionarily conserved (Thomas et al, 2010). Epstein–Barr virus (EBV) infects >90% of the world population, commonly causing self-limiting infectious mononucleosis or rarely inciting a range of malignancies (Kutok and Wang, 2006), including nasopharyngeal carcinoma (NPC; Tao and Chan, 2007), gastric carcinoma (GC; Fukayama, 2010), lymphoproliferative disorders in immune compromised patients (Cesarman, 2011), and Burkitt's lymphoma (BL; Rowe et al, 2009). A gammaherpesvirus, EBV, may undergo lytic replication, releasing viral progeny, or instead initiate latency in one of three patterns (Latency I, II, III) involving limited gene expression that includes Epstein–Barr nuclear antigens (EBNA1–3), latent membrane proteins (LMP1 and 2), and the noncoding RNAs EBER1, EBER2, and EBV miRNAs (Delecluse et al, 2007). Viral miRNAs were discovered in EBV (Pfeffer et al, 2004), which expresses 44 mature miRNAs (Kozomara-Griffiths-Jones, 2011) from 25 precursor-miRNAs (pre-miRNAs), the most of any known human virus (Cai et al, 2006; Grundhoff et al, 2006; Lung et al, 2009; Zhu et al, 2009; Chen et al, 2010). EBV miRNAs are encoded in two genomic clusters (BHRF1 and BART) and are expressed at various levels in tumour samples and different cell lines during viral latency (Figure 1A) and lytic growth (Cai et al, 2006; Lung et al, 2009). Most (22/25) EBV pre-miRNAs have homologues in the closely related Rhesus lymphocryptovirus (rLCV; Cai et al, 2006; Riley et al, 2010; Walz et al, 2010), and three share sequences with human miRNAs: EBV miRNA BART1-3p exhibits seed identity (nts 1–7) with human miRNA miR-29a/b/c (Park et al, 2009), nts 2–7 are shared by BART5-5p and human miRNA miR-18a/b (Gottwein and Cullen, 2008), and nts 2–8 are identical in BART22-3p (Lung et al, 2009; Zhu et al, 2009), miR-520d-5p, and miR-524-5p (Bentwich et al, 2005; Landgraf et al, 2007). EBV can also alter host miRNA expression, notably enhancing the level of oncogenic miR-155 (Gatto et al, 2008; Lu et al, 2008; Rahadiani et al, 2008; Yin et al, 2008; Linnstaedt et al, 2010), a regulator of B-cell homeostasis (Rodriguez et al, 2007). Figure 1.HITS-CLIP analysis of EBV and human miRNAs in Jijoye cells. (A) Northern blotanalysis of EBV miRNA expression in B-cell lines. BJAB is an EBV−BL; BJAB-B1 is a P3HR1 EBV+ BJAB derivative; B95.8 is a marmoset LCLline infected with B95.8 EBV, which harbours a large deletion that eliminates expressionof most BART miRNAs; Daudi and Jijoye are patient-derived, EBV+ BLcell lines. All cell lines are EBV Latency III. U6 is a loading control. (B)Quality-filtered, raw sequencing reads of HITS-CLIP isolated, Ago-bound miRNAs (0 or 1mismatch) were aligned to human (blue) or EBV (red) miRNAs in miRBASE v.17 and groupedinto identical seed families. In all, 91% of total reads mapped to miRNAsequences, representing 295 unique seeds. (C) Individual miRNA sequencing readsfor the 44 EBV miRNAs in miRBASE v.17 are depicted in their relative genomic locationson the Type 2 EBV RefSeq map (below). The miRNAs deleted in the B95.8 strain are noted(red bar). Download figure Download PowerPoint Previously, specific EBV miRNAs were implicated in regulating host cell immunomodulatorytargets CXCL-11 (Xia et al, 2008) and MICB (Nachmani et al, 2009), pro-apoptotic targets BBC3/PUMA andBCL2L11/BIM (Choy et al, 2008; Marquitz et al, 2011), and transport targets TOMM22 and IPO7 (Dolken et al, 2010). EBV miRNAs have also been reported totarget three viral transcripts: the key transforming factor LMP1 (Lo et al, 2007), immunomodulatory protein LMP2A (Lung et al, 2009), and DNA polymerase BALF5 (Barth etal, 2008). Two phenotypic studies focusing on the BHRF1 miRNA cluster,which is highly expressed in B95.8-infected lymphoblastoid cells (LCLs), implicated EBVmiRNAs in cell cycle promotion (Seto et al, 2010;Feederle et al, 2011) and/or inhibition of apoptosisduring initial infection (Seto et al, 2010). Otherstudies characterized targets of BART miRNAs in non-B-cell cancers (NPC, GC), where they aremore highly expressed than in BL (Cai et al, 2006).Little is known about the roles of BART miRNAs in B cells, in part because they aredispensable for B-cell transformation (Robertson et al,1994); yet, they are expressed in all forms of EBV latency (Rowe et al, 2009). Crosslinking immunoprecipitation paired with high-throughput sequencing (HITS-CLIP)identifies genome-wide Ago–miRNA–mRNA ternary complexes from a cell line ortissue. Ultraviolet (UV) crosslinking induces covalent bonding between RNAs and associatedproteins inside cells. Ago is then stringently immunoprecipitated such that Ago-bound miRNAsand mRNA fragments can be separately purified and resulting cDNAs subjected tohigh-throughput sequencing (Chi et al, 2009). AgoHITS-CLIP can therefore interrogate host and viral miRNAs at their normal, unperturbedstoichiometries relative to each other and to mRNAs, providing a biologically relevant dataset (Khan et al, 2009). Here, we subjected unperturbed Jijoye (Latency III) BL cells to Ago HITS-CLIP. Subsequentbioinformatic analyses and luciferase reporter assays pinpointed three viral and >1500human mRNA targets of EBV miRNAs. The Jijoye strain was selected because, unlike the B95.8laboratory strain of EBV, it expresses all 44 EBV miRNAs reported in miRBASE (Kozomara-Griffiths-Jones, 2011), allowing the function of the fullcomplement of EBV miRNAs to be assessed. Indeed, our complete analysis identified viralmiRNAs regulating human and viral transcripts, as well as human miRNAs regulating human andviral transcripts. The 12 most highly expressed EBV miRNAs target mRNAs involved intranscription regulation, apoptosis, cell cycle control, and Wnt signalling. Only 10%of the transcripts targeted by EBV miRNAs are targeted by viral miRNAs alone. Rather, EBVmiRNAs co-target mRNAs with host miRNAs, in particular with members of the miR-17∼92miRNA cluster, a highly oncogenic group of miRNAs that induces lymphomas and inhibitsapoptosis (Matsubara et al, 2007; Ventura et al, 2008; Xiao et al, 2008), and with the highly abundant immunomodulatory miRNAs, miR-142-3p andmiR-155. Our study provides the first comprehensive picture of EBV miRNA function in Bcells. Results Global analysis of Ago HITS-CLIP in Latency III B cells To determine the role of EBV miRNAs in Latency III B cells, we first assessed EBV miRNAexpression in established B-cell lines (Figure 1A). While B95.8cells (resulting from infection with the B95.8 laboratory strain of EBV) express highlevels of some EBV miRNAs (Figure 1A, lane 3; Dolken et al, 2010; Feederle et al, 2011), a well-characterized ∼12 kb deletion eliminates expressionof the majority of the BART miRNAs (Pfeffer et al, 2004; Cai et al, 2006). Instead, weselected the BL cell line Jijoye, which detectably expresses all but 1 of the 44 reportedEBV mature miRNAs (Cai et al, 2006) (Figure 1A, lane 5) and therefore enables a complete analysis of EBV miRNAs.Jijoye cells were initially isolated from a patient tumour, and—like most culturedBL cell lines—exhibit Latency III gene expression (Pulvertaft,1964; Drexler and Minowada, 1998). We applied Ago HITS CLIP (developed by Chi et al (2009); reviewed by Darnell (2010)) to Jijoyecells in six biological replicates, three experiments each with anti-Ago monoclonalantibodies 2A8 (Nelson et al, 2007) or 11A9 (Rudel et al, 2008), which co-immunoprecipitate (co-IP)UV-crosslinked Ago-miRNA–mRNA complexes (Supplementary Figure 1A). After RNase trimming, the Ago–RNA complexes wereseparated by SDS–polyacrylamide gel electrophoresis, transferred to nitrocellulose,and the lower (LO∼110 kDa) and higher (HI∼118–130 kDa)molecular weight species separately isolated (Ago alone migrates at ∼100 kDa;Supplementary Figure 1B). After reversetranscription of extracted RNA and subsequent PCR, Ago-bound miRNAs (from one 11A9 co-IP)and mRNA samples (six total, three from each antibody) were subjected to Illuminahigh-throughput sequencing (read and alignment statistics in Supplementary Tables 1 and 2). The LO sample (Supplementary Figure 1B and C, lanes 3 and 4,Supplementary Table 1) yielded mainlymiRNAs (∼90% of 22.6 million raw sequencing reads), while the HI sample (Supplementary Figure 1B and C, lanes 6 and 7)contained both miRNAs and longer mRNA fragments. Of the total LO sequencing reads mapping with 0 or 1 mismatch to miRBASE v.17 (Kozomara-Griffiths-Jones, 2011) sequences, 25% were the 44known EBV miRNAs and 75% corresponded to 310 human miRNAs (Figure 1B). The most abundant miRNA was EBV miRNA BART10 (BART10-3p), representing ∼11% of miRNA reads (Supplementary Table 3). Because miRNA functionality is based on interactions between the miRNA seedand mRNA, we grouped the miRNA sequencing reads by seed family (identity in nts 1–8;Figure 1B). The miR-20a/106b family (UAAAGUGC) comprised∼11% of total Ago-bound miRNA reads, and the related miR-17/20b/93/519d family(CAAAGUGC) represented 9%. Together, the sum of sequencing reads for the members ofthe miR-17∼92 cluster (miR-17, 18a, 19a, 20a, 19b, 92a) and their associated seedfamilies (additionally including miR-18b, 19b, 20b, 32, 92b, 93, 106a/b, and 519d)represented ∼35% of all seeds. EBV miRNAs do not group into seed families, yetBART10-3p was so abundant that its seed alone (UACAUAAC) represented the second largestseed family (also ∼11%; Figure 1B). As in previous studies (Pfeffer et al, 2004; Cai et al, 2006), levels of different EBV miRNAs variedwidely, with the most abundant being BARTs originating from both clusters (Figure 1C). The 12 most highly expressed EBV miRNAs together comprise 90%of the EBV miRNA reads and 22% of total miRNA reads (Supplementary Table 3). Thus, this group was selected fordetailed analysis. While BHRF1-2 levels are equivalent in B95.8 and Jijoye cells, BHRF1-3levels are somewhat reduced, possibly due to a 1-nt mutation 159 nts downstream ofthe BHRF1-3 pre-miRNA, which was identified by HITS-CLIP and confirmed by cloning(unpublished observations). BHRF1-1 is not visible in northern blots (Figure 1A) because of a Jijoye-specific point mutation in pre-BHRF1-1 (Pfeffer et al, 2004; Cai et al, 2006), but is clearly present in our HITS-CLIP data (Supplementary Table 3). The Ago-bound mRNA sequencing reads (9.4–35.9 million raw reads in each of the sixreplicates) were collapsed and aligned to the human (hg18; Supplementary Tables 1 and 2A) and to the EBV genome(Dolan et al, 2006; NC_009334.1; Supplementary Table 2B). As expected for latentlyinfected cells, many more reads (99.4% of unique mappable reads) aligned to thehg18 than the EBV (0.6%) genome. Peak height (PH) and biologic complexity (BC) aremetrics assessing the quality of a given cluster (Chi et al, 2009); for our analysis we defined clusters as five overlapping readsreproduced in at least three of six experiments (PH ≥5, BC ≥3; see Supplementary Methods). Mapping these peaks to the humanRefSeq database yielded a distribution of Ago binding similar to that seen previously inHeLa cells and mouse brain (Chi et al, 2009), wherereproducible clusters primarily aligned to extended 3′-untranslated regions(3′UTRs; 40%) or to coding sequences (CDS; 31%); fewer 5′UTR,intron, and intergenic/noncoding RNA clusters were detected (Supplementary Figure 1D). Reproducible clusters weresearched for seed base-pairing potential (minimally nts 2–7) to Jijoye miRNAs. Asreported (Chi et al, 2009), the frequency of potentialmRNA-binding sites for a given seed sequence loosely correlated with miRNA abundance (Supplementary Figure 2). EBV and host miRNAs co-target three viral mRNAs in EBV Latency III The landscape of unique sequence reads (≥25 nts, 0–2 mismatches) on theEBV genome comprises four major peaks overlapping three mRNAs and the BART noncoding RNAs(Figure 2, bottom). EBNA2 is expressed in EBV Latency IIIto control transcription of several host and viral genes (Zimber-Strobl and Strobl, 2001). Our HITS-CLIP results show broad clusters ofAgo binding over the entire length of the EBNA2 transcript with few robust peaks (Figure 2A) or predicted miRNA-binding sites (Supplementary Figure 3). In contrast, distinct peaksreside within the 3′UTRs of BHRF1 (Figure 2B) and LMP1(Figure 2C) mRNAs. BHRF1 (discovered by Cleary et al (1986), reviewed by Cuconati-White (2002)) encodes a homologue of the host BCL2 anti-apoptotic protein and isexpressed at low levels in EBV Latency III (Kelly et al, 2009). Two large peaks on the BHRF1 3′UTR (BC=5 or 6) overlappredicted miRNA-binding sites for the most highly abundant human (miR-17 family,miR-142-3p) and EBV (BART10-3p) miRNAs (Figure 2B). Similarly, LMP1(reviewed by Middeldorp-Pegtel (2008)), which mimics the hostgene CD40 and activates multiple signalling pathways, has two distinctive3′UTR peaks with binding sites for EBV (BARTs 19-5p and 5-5p) and human (miR-17family) miRNAs (Figure 2C), as well as 'orphan' peakscontaining no predicted canonical miRNA seed-binding sites near the 3′ end (Chi et al, 2009). The peak on the EBV genome-labelled'BARTs' (Figure 2, below) represents Ago-boundpre-miRNAs of EBV BART miRNAs. Since the BART miRNAs are processed from noncodingtranscripts (Al-Mozaini et al, 2009) and thebase-pairing between the 5p and 3p miRNAs is highly bulged in most cases (Kozomara-Griffiths-Jones, 2011), it is unlikely that the BART miRNAsare self-targeting their own pre-miRNAs. Ago protein can both exist in the nucleus(Weinmann et al, 2009) and bind pre-miRNAs (Tan et al, 2009), though why these possibly nuclearpre-miRNAs bind Ago is not known. Figure 2.Viral and human miRNAs co-regulate EBV genes. RefSeq (NC_009334.1) map of theType 2 EBV genome (bottom, CDS grey, coordinates noted) aligned with HITS-CLIP deepsequencing reads (0 or 1 mismatch; ≥25 nts long) from six biological replicatesof Ago-bound RNAs in Jijoye BL cells (unique reads, one colour per biologicalreplicate). The EBV pre-miRNAs from the BART region are selectively and reproduciblyAgo-bound. Inset boxes are expanded, scaled views of the three major mRNA peaks(A, B, C). (A) The EBV nuclear antigen 2 (EBNA2) readsoverlap the entire EBNA2 CDS (grey). The precise coordinates of the 5′ and3′UTRs (black and white, respectively) of Jijoye EBV EBNA2 and EBNA-LP areunknown, as indicated by jagged boundaries. (B) The BHRF1 (Bam HI fragment Hrightward open reading frame 1) peak (coordinates are annotated ends of the UTRs)contains several robust, validated miRNA-binding sites designated below by verticalbars. (C) The latent membrane protein 1 (LMP1) reads map to the 3′UTR andindicate coordinate binding by at least two validated viral miRNAs and one human miRNAfamily. Download figure Download PowerPoint To validate our HITS-CLIP findings and confirm direct miRNA–mRNA interactions, wecloned the full-length Jijoye LMP1 3′UTR (Figure 3A) into adual-luciferase reporter vector. We first asked if inactivation of endogenous miR-17family miRNAs upregulates the level of luciferase. Since the miR-17 family of miRNAs ishighly expressed in all transfectable cell lines tested (unpublished observations), weemployed tiny locked nucleic acids (tiny LNAs): 8-mer, anti-seed sequence, modifiedoligonucleotides (Obad et al, 2011; Supplementary Table 4) to inactivate all miR-17 familymembers. Indeed, expression of the luciferase-LMP1 3′UTR increased with anti-miR-17,but not a control tiny LNA, in a dose-dependent manner (Figure 3B,left). Further, a control LMP1 3′UTR with point mutations in the expected miR-17family seed-binding site (Figure 3A) did not respond to anti-miR-17(Figure 3B, right). We then tested miRNAs with excellentbase-pairing potential to the LMP1 3′UTR: EBV miRNAs BART19-5p, BART5-5p, andBART11-5p, and human miRNAs miR-17-5p and miR-18a-5p. While miR-17-5p, BART19-5p, orBART5-5p synthetic miRNAs (Supplementary Table 4) repressed the WT (but not point mutant) LMP1 3′UTR, miR-18a-5p andBART11-5p did not (Figure 3C). Both miR-18a-5p and BART11-5pefficiently repressed artificial sensor reporters (unpublished observations). Thus,regulation of LMP1 is specific to BART5-5p and not miR-18a-5p, even though these miRNAsshare seed sequence identity (nts 2–7; Figure 3A). WhenBART19-5p and BART5-5p were co-transfected at the same total concentration of syntheticmiRNA, repression was further enhanced, indicating that these viral miRNAs cooperate torepress LMP1. Jijoye cell nucleofection of tiny LNAs (Figure 3D,lanes 1 and 2) or synthetic miRNAs (Figure 3D, lanes 3–6)resulted in up- or downregulation of endogenous LMP1 protein, respectively. We concludethat LMP1 mRNA is regulated by at least two EBV miRNAs and one host miRNA family. Figure 3.EBV LMP1 is co-regulated by EBV and human miRNAs. (A) The full-length LMP13′UTR (1215 nts; top) was cloned downstream of firefly luciferase in thepmiRGLO dual-luciferase vector, with either wild-type Jijoye sequence or point mutationsin putative miRNA-binding sequences (noted below, red nts). Relative locations ofproposed binding sites for BART19-5p (B19), hsa-miR-18 (18), BART5-5p (B5), andBART11-5p (B11), and the human miR-17 miRNA family (17; blue) are noted. Sequences of WTLMP1 and mutants disrupting the seed-binding site for BART19-5p (B19m), BART5-5p (B5m),BART11-5p (B11m), or miR-17 family (17m) are shown with potential base-pairing of themiRNAs to the WT mRNA sequences indicated. (B) HEK293T cells were co-transfectedwith a luciferase-LMP1 (WT or miR-17 family point mutant 17m) reporter and either water(mock), a negative control tiny LNA (Ctl), or the anti-miR-17 family tiny LNA (anti-17)at one of two concentrations. Firefly/Renilla luciferase ratios were normalized to the17m mock-transfected reporter. (C) HEK293T cells were co-transfected with afirefly luciferase-LMP1 (WT or point mutants from A) reporter and either40 nM total synthetic control miRNA duplex (CTL; scrambled sequence) or 1–2EBV miRNAs predicted to base-pair with the LMP1 3′UTR. Firefly/Renilla luciferaseratios were normalized to the same reporter transfected with the negative control miRNA(CTL). In all luciferase assays, mean values were from at least four independenttransfections. Error bars, s.d. P values from two-tailed Student'st-tests of noted sample relative to CTL, ***P<0.002.(D) Five million Jijoye cells were nucleofected with either a tiny LNA(55 pmol; lanes 1 and 2) or a synthetic miRNA duplex (10 pmol; lanes3–6). Endogenous LMP1 was detected at 48 h post-nucleofection by westernblot, and GAPDH was used as a loading control. Download figure Download PowerPoint We used a similar approach to validate BHRF1 repression by miR-17 family miRNAs,miR-142-3p, and BART10-3p (Figure 4A–D). Again, we observedsignificant upregulation of luciferase fused to the WT full-length BHRF1 3′UTR upontreatment with anti-miR-17 but not with the control tiny LNA (Figure 4B). Repression occurred upon addition of miR-17-5p, miR-142-3p, or BART10-3psynthetic miRNAs (Figure 4C). BART10-3p targets the BHRF13′UTR at two sites that both contribute to downregulation (Figure 4A and C). Co-transfection of equal parts miR-142-3p and BART10-3p yielded evengreater repression (Figure 4C). As expected, mutant reporters didnot respond to tiny LNAs or synthetic miRNAs (Figure 4B and C). Wealso tested the effects of miR-17-5p, miR-142-3p, and BART10-3p on BHRF1 protein levels.These synthetic miRNAs decreased co-transfected BHRF1 relative to the negative controlmiRNA (Figure 4D). Moreover, tiny LNA inhibition of miR-17-5p,miR-142-3p, and BART10-3p upregulated endogenous BHRF1 protein in Jijoye cells (Figure 4E), especially when used in combination (Figure 4E, lane 5). Together, these results confirm that EBV and host miRNAs co-repressthe BHRF1 mRNA, reducing protein output, and that host and viral miRNAs can have acumulative effect on targets. Figure 4.EBV BHRF1 is co-regulated by EBV and human miRNAs. (A) The full-length BHRF13′UTR (634 nts; top) with either the wild-type Jijoye sequence or mutationsin putative miRNA-binding sequences (below, red nts) was cloned downstream of fireflyluciferase in the pmiRGLO dual-luciferase vector. The relative locations of bindingsites for human miRNAs (17=hsa-miR-17 family, 142=hsa-miR-142-3p; blue)and two sites for EBV miRNA ebv-miR-BART10-3p (B10-1 and B10-2, red) are noted.Sequences are shown for the wild-type 3′UTR, for point mutants in the seed-bindingsite for miR-17-5p (17m), miR-142-3p (142m), or one of two sites for BART10-3p binding(denoted B10m1 and B10m2, assigned 5′ to 3′), and for the miRNAs. (B,C) HEK293T cells were co-transfected with a BHRF1-luciferase reporter and tinyLNAs or synthetic miRNAs as in Figure 3B and C. 'CTL' is synthetic BART5-5p, which does not have predicted binding sites in BHRF1. In allluciferase assays, mean values were from at least four independent transfections. Errorbars, s.d. P values from two-tailed Student's t-tests of notedsample relative to CTL, ***P<0.0001. (D) HEK293T cells wereco-transfected with pcDNA3-BHRF1 (full-length WT BHRF1, coordinates in Figure 2B) and the designated host or viral miRNA. BHRF1 protein levels weredetermined 24 h post-transfection by western blot. Neomycin phosphotransferase(Neo) from pcDNA3 and endogenous α-tubulin were the transfection/loading controls.(E) Five million Jijoye cells were nucleofected with 55 pmol total tinyLNA (lane 5 is a 1:1:1 mix). Endogenous full-length/cleaved PARP and BHRF1 levels weredetermined 48 h post-nucleofection by western blot, and endogenous GAPDH was aloading control. Download figure Download PowerPoint Lytic BHRF1 is anti-apoptotic, but little is known about its function in latency(Kelly et al, 2009); our HITS-CLIP data predict thatmany host apoptotic genes are also regulated by EBV miRNAs (Supplementary Table 5A). We therefore asked if miRNAsthat downregulate BHRF1 protein might enhance apoptosis in latent Jijoye cells. Eventhough nucleofection itself induces significant apoptosis in EBV+ Bcells (Hatton et al, 2011), nucleofection with tinyLNAs blocking miR-17 family miRNAs, miR-142-3p, and BART10-3p in latent Jijoye cells(Figure 4E) further increases both the low levels of endogenousBHRF1 and apoptosis, as indicated by increased PARP cleavage (Figure 4E). Thus, a phenotypic effect of BART10-3p is the inhibition of apoptosis, whichcould be explained by the involvement of 32 other apoptosis-associated genes predicted tobe regulated by BART10-3p (Supplementary Table 5B). EBV miRNAs target human mRNAs involved in transcription, apoptosis, Wntsignalling, and cell cycle control The limited number of Ago HITS-CLIP peaks aligning to the EBV genome predicts that thevast majority of EBV miRNA targets are human mRNAs. Our HITS-CLIP data indeed revealedhundreds of EBV miRNA-binding sites in human 3′UTRs and coding regions (Supplementary Figure 1D), including previouslyvalidated TOMM22, IPO7, BBC3/PUMA, and BCL2L11/BIM transcripts. However, we identifiedadditional and often more robust sites of miRNA binding in each of these 3′UTRs(Supplementary Figure 4). One advantage of the HITS-CLIP biochemical/bioinformatic approach is the ability toidentify all Ago–miRNA–mRNA ternary complexes simultaneously. For EBV, itseemed likely that the highly expressed, co-transcribed BART miRNAs might operatecoordinately. Thus, we examined genes targeted by the 12 most highly expressed EBV miRNAsin Jijoye cells (Supplementary Table 3) fortheir assignment to particular gene families or pathways. Specifically, we subjected the1664 human 3′UTRs with one or more canonical seed-binding sites (minimally nts2–7; clusters PH ≥5, BC ≥3) for the highly abundant EBV miRNAs to gene ontology (GO)analysis using DAVID v.6.7 (Huang da et al, 2009a;Huang da et al, 2009b). The most enriched categorieswere transcription regulation (20% of annotated genes;P<9×10−13, Fisher's exact tes
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