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. 2012 May 2;31(9):2207-21.
doi: 10.1038/emboj.2012.63. Epub 2012 Mar 30.

EBV and human microRNAs co-target oncogenic and apoptotic viral and human genes during latency

Kasandra J Riley  1 , Gabrielle S Rabinowitz, Therese A Yario, Joseph M Luna, Robert B Darnell, Joan A Steitz
Affiliations

EBV and human microRNAs co-target oncogenic and apoptotic viral and human genes during latency

Kasandra J Riley et al. EMBO J. .

Abstract

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.

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Conflict of interest statement

The authors delcare that they have no conflict of interest.

Figures

Figure 1
Figure 1
HITS-CLIP analysis of EBV and human miRNAs in Jijoye cells. (A) Northern blot analysis 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 LCL line infected with B95.8 EBV, which harbours a large deletion that eliminates expression of most BART miRNAs; Daudi and Jijoye are patient-derived, EBV+ BL cell 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 1 mismatch) were aligned to human (blue) or EBV (red) miRNAs in miRBASE v.17 and grouped into identical seed families. In all, 91% of total reads mapped to miRNA sequences, representing 295 unique seeds. (C) Individual miRNA sequencing reads for the 44 EBV miRNAs in miRBASE v.17 are depicted in their relative genomic locations on the Type 2 EBV RefSeq map (below). The miRNAs deleted in the B95.8 strain are noted (red bar).
Figure 2
Figure 2
Viral and human miRNAs co-regulate EBV genes. RefSeq (NC_009334.1) map of the Type 2 EBV genome (bottom, CDS grey, coordinates noted) aligned with HITS-CLIP deep sequencing reads (0 or 1 mismatch; ≥25 nts long) from six biological replicates of Ago-bound RNAs in Jijoye BL cells (unique reads, one colour per biological replicate). The EBV pre-miRNAs from the BART region are selectively and reproducibly Ago-bound. Inset boxes are expanded, scaled views of the three major mRNA peaks (A, B, C). (A) The EBV nuclear antigen 2 (EBNA2) reads overlap the entire EBNA2 CDS (grey). The precise coordinates of the 5′ and 3′UTRs (black and white, respectively) of Jijoye EBV EBNA2 and EBNA-LP are unknown, as indicated by jagged boundaries. (B) The BHRF1 (Bam HI fragment H rightward open reading frame 1) peak (coordinates are annotated ends of the UTRs) contains several robust, validated miRNA-binding sites designated below by vertical bars. (C) The latent membrane protein 1 (LMP1) reads map to the 3′UTR and indicate coordinate binding by at least two validated viral miRNAs and one human miRNA family.
Figure 3
Figure 3
EBV LMP1 is co-regulated by EBV and human miRNAs. (A) The full-length LMP1 3′UTR (1215 nts; top) was cloned downstream of firefly luciferase in the pmiRGLO dual-luciferase vector, with either wild-type Jijoye sequence or point mutations in putative miRNA-binding sequences (noted below, red nts). Relative locations of proposed binding sites for BART19-5p (B19), hsa-miR-18 (18), BART5-5p (B5), and BART11-5p (B11), and the human miR-17 miRNA family (17; blue) are noted. Sequences of WT LMP1 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 the miRNAs to the WT mRNA sequences indicated. (B) HEK293T cells were co-transfected with 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 the 17m mock-transfected reporter. (C) HEK293T cells were co-transfected with a firefly luciferase-LMP1 (WT or point mutants from A) reporter and either 40 nM total synthetic control miRNA duplex (CTL; scrambled sequence) or 1–2 EBV miRNAs predicted to base-pair with the LMP1 3′UTR. Firefly/Renilla luciferase ratios were normalized to the same reporter transfected with the negative control miRNA (CTL). In all luciferase assays, mean values were from at least four independent transfections. Error bars, s.d. P values from two-tailed Student’s t-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; lanes 3–6). Endogenous LMP1 was detected at 48 h post-nucleofection by western blot, and GAPDH was used as a loading control.
Figure 4
Figure 4
EBV BHRF1 is co-regulated by EBV and human miRNAs. (A) The full-length BHRF1 3′UTR (634 nts; top) with either the wild-type Jijoye sequence or mutations in putative miRNA-binding sequences (below, red nts) was cloned downstream of firefly luciferase in the pmiRGLO dual-luciferase vector. The relative locations of binding sites 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-binding site 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 tiny LNAs or synthetic miRNAs as in Figure 3B and C. ‘CTL’ is synthetic BART5-5p, which does not have predicted binding sites in BHRF1. In all luciferase assays, mean values were from at least four independent transfections. Error bars, s.d. P values from two-tailed Student’s t-tests of noted sample relative to CTL, ***P<0.0001. (D) HEK293T cells were co-transfected with pcDNA3-BHRF1 (full-length WT BHRF1, coordinates in Figure 2B) and the designated host or viral miRNA. BHRF1 protein levels were determined 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 tiny LNA (lane 5 is a 1:1:1 mix). Endogenous full-length/cleaved PARP and BHRF1 levels were determined 48 h post-nucleofection by western blot, and endogenous GAPDH was a loading control.
Figure 5
Figure 5
Human gene CAPRIN2 is co-regulated by EBV and human miRNAs. (A) RefSeq (hg18) map of the human CAPRIN2 3′UTR (349 nts; chromosome 12 coordinates) aligned with HITS-CLIP deep-sequencing reads from six biological replicates of Ago-bound RNAs in Jijoye B cells (raw unique reads, one colour per biological replicate). Relative locations of binding sites for human miR-17 family miRNAs and two EBV miRNA BART13-3p sites are denoted by black bars (drawn to scale below). (B) Sequence of CAPRIN2 miRNA-target sites with point mutations (red) in the seed-binding sites for miR-17-5p (17m) or one of two sites for BART13-3p binding (B13m1 and B13m2). The mutations in the BART13-3p-binding sites convert them into binding sites for an unrelated miRNA, ebv-miR-BART16-5p. (C, D) HEK293T cells were transfected with the designated luciferase-CAPRIN2 reporters and tiny LNA or synthetic miRNA as in Figures 3B and C. In all luciferase assays, mean values were from at least four independent transfections. Error bars, s.d. P values from two-tailed Student’s t-tests of noted pairs, ***P<0.001 or **P<0.01. (E) HEK293T cells were transfected with a control or test synthetic miRNA duplex (40 nM) or tiny LNA (5 nM). Western blots of extracts prepared 24 h post-transfection were probed for endogenous proteins with anti-CAPRIN2 or anti-GAPDH antibodies. (F) Jijoye cells were transfected with a control or test synthetic miRNA duplex (100 nM) and analysed as in E.
Figure 6
Figure 6
EBV miRNA and host miRNA targeting in EBV Latency III. (A) Overlap between the human 3′UTR targets for the 12 most highly expressed EBV miRNAs (green) and for the 56 most highly expressed human miRNAs (pink) in Jijoye cells (BC ≥3, PH ≥5 unique sequencing reads). Annotations are from RefSeq. (B) Overlap between the human 3′UTR targets for the 12 most highly expressed EBV miRNAs (green) and for the human miR-17∼92 miRNAs (pink) as in A. (C) Summary of HITS-CLIP-identified 3′UTR targets of human and EBV miRNAs in Jijoye cells. While a small fraction of the EBV miRNA sites in 3′UTRs are human transcripts targeted by EBV miRNAs alone (10% of all targets), 90% of human 3′UTR targets and at least two-third of the EBV mRNA targets of EBV miRNAs are co-targeted by one or more human miRNA. We validated co-targeting by EBV BARTs and human miR-17∼92 miRNAs of the LMP1, BHRF1, and CAPRIN2 mRNAs. Transcripts co-targeted by the miR-17∼92 and EBV miRNAs, representing 60% of all miR-17∼92 targets in Jijoye cells, are enriched in at least seven major GO categories (blue; also see Table I).
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