Epstein-Barr viral microRNAs target caspase 3

Abstract

The Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that transforms B cells and causes several malignancies including Burkitt's lymphoma. EBV differentially expresses at least 49 mature microRNAs (miRNAs) during latency in various infected epithelial and B cells. Recent high-throughput studies and functional assays have begun to reveal the function of the EBV miRNAs suggesting roles in latency, cell cycle control, and apoptosis. In particular, the central executioner of apoptosis, Caspase 3 (CASP3), was proposed as a target of select EBV miRNAs. However, whether CASP3 is truly a target of EBV miRNAs, and if so, which specific miRNAs target CASP3 is still under debate. Based on previously published high-throughput biochemical data and a bioinformatic analysis of the entire CASP3 3'-UTR, we identified 12 EBV miRNAs that have one or more seed binding sites in the CASP3 3'-UTR. We individually tested all 12 miRNAs for repression of CASP3 in luciferase reporter assays, and nine showed statistically significant (P < 0.001) repression of a full-length CASP3 reporter. Further, three EBV miRNAs, including BART22, exhibited repression of endogenous CASP3 protein. These data confirm that CASP3 is a direct target of specific EBV BART miRNAs.

Keywords: Burkitt’s lymphoma; Caspase 3; Epstein-Barr virus; microRNA.

Fig. 1

Caspase 3 has 16 predicted sites for EBV miRNA base pairing, 15 of which align with HITS-CLIP data. HITS-CLIP high-throughput sequencing reads (0 or 1 mismatch; ≥25 nt long) from six biological replicates of Ago-bound RNAs in Jijoye BL cells (unique reads, one color per biological replicate) mapped to scale on the Caspase 3 3′-UTR (1576 nt; RefSeq ID: NM_004346.3). Predicted sites of miRNA seed sequence binding for EBV miRNAs (black) and human miRNAs (purple) are noted below, with non-verifiable EBV miRNA sites in gray. When two sites for a given miRNA are present, they are labeled “A” and “B” from 5′ to 3′, respectively. Sites for BARTs 4, 7, and 22B met our initial HITS-CLIP reproducibility thresholds (three or more experimental replicates, peak height of five or more; [18])

Fig. 2

EBV miRNAs repress a full-length luciferase-Caspase 3 3′-UTR reporter. HEK293T cells were co-transfected with the designated luciferase-Caspase 3 reporters and either total synthetic control miRNA duplex (CTL) or an EBV miRNA predicted to base pair with the Caspase 3′-UTR (Table 1 includes all WT and mutant sequences). Firefly/Renilla luciferase ratios were normalized to the same reporter transfected with the negative control miRNA (CTL). a Wild type (WT) Caspase 3 is repressed significantly by nine EBV miRNAs. b WT repression is compared to loss of repression of the relevant mutant for each of the miRNAs that showed the greatest WT repression. In all luciferase assays, mean values were from at least four independent transfections. Error bars, standard deviation; P values from two-tailed Student’s t-tests of noted pairs, *P < 0.0001, **P < 0.01

Fig. 3

Select EBV miRNAs repress endogenous Caspase 3 protein. HEK293T cells were transfected with the denoted control or EBV miRNA. Western blots of extracts prepared 24 h post-transfection were probed for endogenous proteins with anti-Caspase 3 or anti-tubulin antibodies. Normalized CASP3 levels from triplicate experiments are reported below with the S.E.M