Epstein-Barr virus-encoded microRNA miR-BART2 down-regulates the viral DNA polymerase BALF5

Abstract

MicroRNAs (miRNAs) have been implicated in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein–Barr virus (EBV) encodes 23 miRNAs of unknown function. Here we show that the EBV-encoded miRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. MiR-BART2 guides cleavage within the 3′-untranslated region (3′UTR) of BALF5 by virtue of its complete complementarity to its target. Induction of the lytic viral replication cycle results in a reduction of the level of miR-BART2 with a strong concomitant decrease of cleavage of the BALF5 3′UTR. Expression of miR-BART2 down-regulates the activity of a luciferase reporter gene containing the BALF5 3′UTR. Forced expression of miR-BART2 during lytic replication resulted in a 40–50% reduction of the level of BALF5 protein and a 20% reduction of the amount of virus released from EBV-infected cells. Our results are compatible with the notion that EBV-miR-BART2 inhibits transition from latent to lytic viral replication.

The MicroRNA Profile of Prostate Carcinoma Obtained by Deep Sequencing

“…Northern blotting analysis was carried out as described (18,19). The membranes were analyzed with a PhosphoImager (Amersham-Biosciences).…”

“…The first 1,350 bp of the MYO6-3′UTR were PCR amplified from genomic DNA using primers MYO6-AgeI 5′-ACCGGTCACAC-CAGCCTTACAGCTGGG-3′and MYO6-PmlI 5′-CACGTGGGCTCACAAACACTAGTCAGC-3′ and ligated into the modified pGL3 luciferase reporter plasmid (Promega; ref. 19). Mutation of the predicted target site seed sequences of pGL3-MYO6-3′UTR for miR-143 and miR-145 were carried out by site-directed mutagenesis according to the QuickChange Site Directed Mutagenesis kit (Stratagene) using the following primers: miR-145-1_for_EcoRV 5′GCACAGTACCATGCGATATCTC-TAATAAAACATGAGC-3′, miR-145-1_rev_EcoRV 5′ G CTCATG T T T TAT TAG AG ATATCG C ATGG -TACTGTGC-3′, miR-145-2_for_SacII 5′-GGCA-TAGTGGCTAGCCGCGGAGGAATTCAAATATTC-3′, miR-145 -2_re v_SacII 5′ G AATAT T TG A AT TC-C T C C G C G G C TA G C C A C TAT G C C -3 ′ , m i R143_for_EcoRV 5′-CAAATATTCTTTCAATCGA-TATCGTATAGTGATTTTTG-3′, and miR-143_rev_ EcoRV 5′-CAAAAATCACTATACGATATCGATT-GAAAGAATATTTG-3′.…”

“…Transfection efficiency was determined by fluorescence-activated cell sorting analysis and luciferase reporter assays were done 48 h after transfection as described (17,19).…”

NcRNA-microchip analysis

“…9 In addition, EBV-encoded miRNAs were found to target the 3'-untranslated region (3'-UTR) of viral mRNAs (BALF5, LMP1) as well as host mRNAs (PUMA, CXCL11). [10][11][12][13] Decreased expression levels of respective proteins are postulated to modulate EBV infection and persistence.…”

“…In addition, it has been demonstrated that v-snoRNA1 is processed into a miRNA-like species, which might target BALF5 mRNA, as it was also reported for EBV-encoded miRNA-BART2. 10 Thus, identification of regulatory ncRNAs, associated with EBV infection, will increase our knowledge on the complex interactions between EBV invasion and host defense and might also aid in the therapy of EBV infection and related diseases in the future.…”