Functional delivery of viral miRNAs via exosomes

TLDR

MicroRNAs regulate gene expression and are actively secreted in exosomes, which protect them from degradation and suggest extracellular functional roles. The study aims to show that EBV‑encoded miRNAs secreted by infected cells are transferred to and function in uninfected recipient cells. Using quantitative RT‑PCR, the authors demonstrate that mature EBV miRNAs are released by infected B cells in exosomes. The transferred EBV miRNAs are taken up by dendritic cells and peripheral blood mononuclear cells, where they dose‑dependently repress target genes such as CXCL11/ITAC, accumulate via exosome transfer, and localize to subcellular sites of gene silencing, supporting a role for miRNA‑mediated intercellular communication in EBV infection.

Abstract

Noncoding regulatory microRNAs (miRNAs) of cellular and viral origin control gene expression by repressing the translation of mRNAs into protein. Interestingly, miRNAs are secreted actively through small vesicles called "exosomes" that protect them from degradation by RNases, suggesting that these miRNAs may function outside the cell in which they were produced. Here we demonstrate that miRNAs secreted by EBV-infected cells are transferred to and act in uninfected recipient cells. Using a quantitative RT-PCR approach, we demonstrate that mature EBV-encoded miRNAs are secreted by EBV-infected B cells through exosomes. These EBV-miRNAs are functional because internalization of exosomes by MoDC results in a dose-dependent, miRNA-mediated repression of confirmed EBV target genes, including CXCL11/ITAC, an immunoregulatory gene down-regulated in primary EBV-associated lymphomas. We demonstrate that throughout coculture of EBV-infected B cells EBV-miRNAs accumulate in noninfected neighboring MoDC and show that this accumulation is mediated by transfer of exosomes. Thus, the exogenous EBV-miRNAs transferred through exosomes are delivered to subcellular sites of gene repression in recipient cells. Finally, we show in peripheral blood mononuclear cells from patients with increased EBV load that, although EBV DNA is restricted to the circulating B-cell population, EBV BART miRNAs are present in both B-cell and non-B-cell fractions, suggestive of miRNA transfer. Taken together our findings are consistent with miRNA-mediated gene silencing as a potential mechanism of intercellular communication between cells of the immune system that may be exploited by the persistent human gamma-herpesvirus EBV.