The microRNAs of Epstein–Barr Virus are expressed at dramatically differing levels among cell lines

Abstract

Epstein-Barr Virus (EBV) encodes multiple microRNAs (miRNAs) from two primary transcripts, BHRF1 and the BARTs. The expression of BHRF1 miRNAs is dependent on the type of viral latency, whereas the BART miRNAs are expressed in cells during all forms of latency. It is not known how these miRNAs are otherwise regulated, though. We have used quantitative, stem-loop, real-time PCR to measure the expression of EBV’s miRNAs and found them to differ nearly 50- and 25-fold among all tested cell lines and among EBV-positive Burkitt’s lymphomas, respectively. In addition, the expression of individual BART miRNAs within a cell can differ by 50-fold or more despite the fact these miRNAs are likely transcribed together as a single primary transcript. These measurements are illuminating: they indicate that few of EBV’s miRNAs are expressed at levels comparable to those of cellular miRNAs in most cell lines and therefore likely function interdependently.

A Cluster of Virus-Encoded MicroRNAs Accelerates Acute Systemic Epstein-Barr Virus Infection but Does Not Significantly Enhance Virus-Induced Oncogenesis In Vivo

“…For cDNA from miRNA, 10 ng of total RNA was used for reverse transcription using miRNA-specific primers (Applied Biosystems, Carlsbad, CA) and a TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Carlsbad, CA). BHRF1 miRNAs were detected via qPCR using TaqMan chemistry, a StepOnePlus machine, and miRNA-specific primers (Applied Biosystems, Carlsbad, CA) (27,28). RNU48 snoRNA was used as an internal reference to calculate the relative levels of BHRF1 miRNA expression.…”

Circulating Epstein–Barr virus microRNAs miR‐BART7 and miR‐BART13 as biomarkers for nasopharyngeal carcinoma diagnosis and treatment

“…The primer used for stem-loop reverse transcription (RT) in this study was based on one described in a previous report. 45 We used a miRNA specific forward primer and stem-loop specific universal reverse primer to perform the PCR. The primer and probe sets used for BART miRNA detection were modified from previously described (Supporting Information Table 2).…”

“…The primer and probe sets used for BART miRNA detection were modified from previously described (Supporting Information Table 2). 45 RT of the total RNA was performed using a multiplex RT method using reagents from the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems). A pool of RT stem-loop primers was made by mixing 100 nM of each of three specific stem-loop RT primers.…”

Functional delivery of viral miRNAs via exosomes

“…EBV-miRNAs are separated into three clusters of the viral genome: BHRF1 and cluster 1 and cluster 2 BARTs (13,14), which are abundantly expressed in EBV-associated tumors (13,15) and EBV-transformed lymphoblastoid B cells (LCL) (13). Although the expression pattern of EBV-miRNAs in vivo is unexplored, and their targets are largely unknown, comprehensive studies in vitro indicate that their expression pattern is linked to viral latency stage (13,14,16). LCL display latency type III, phenotypically resemble proliferating activated B-cell blasts (12), and secrete large quantities of immunoregulatory exosomes (17,18).…”

“…LCL display latency type III, phenotypically resemble proliferating activated B-cell blasts (12), and secrete large quantities of immunoregulatory exosomes (17,18). LCL express both BHRF1 and BART miRNAs, albeit at different levels (11,14,16). Demonstration of functional transfer of BHRF1 and/or BART miRNAs to noninfected cells via exosomes may have implications specifically for EBV infection but also generically for miRNA-mediated biological processes including embryogenesis, tissue homeostasis, and immunomodulation or in pathologies such as oncogenesis.…”