Epstein-Barr Virus-Encoded Small RNAs (EBERs) Are Present in Fractions Related to Exosomes Released by EBV-Transformed Cells

Abstract

Epstein-Barr virus (EBV) is an oncogenic herpesvirus associated with a number of human malignancies of epithelial and lymphoid origin. However, the mechanism of oncogenesis is unclear. A number of viral products, including EBV latent proteins and non-protein coding RNAs have been implicated. Recently it was reported that EBV-encoded small RNAs (EBERs) are released from EBV infected cells and they can induce biological changes in cells via signaling from toll-like receptor 3. Here, we investigated if these abundantly expressed non-protein coding EBV RNAs (EBER-1 and EBER-2) are excreted from infected cells in exosomal fractions. Using differential ultracentrifugation we isolated exosomes from three EBV positive cell lines (B95-8, EBV-LCL, BL30-B95-8), one EBER-1 transfected cell line (293T-pHEBo-E1) and two EBV-negative cell lines (BL30, 293T-pHEBo). The identity of purified exosomes was determined by electron microscopy and western blotting for CD63. The presence of EBERs in cells, culture supernatants and purified exosomal fractions was determined using RT-PCR and confirmed by sequencing. Purified exosomal fractions were also tested for the presence of the EBER-1-binding protein La, using western blotting. Both EBER-1 and EBER-2 were found to be present not only in the culture supernatants, but also in the purified exosome fractions of all EBV-infected cell lines. EBER-1 could also be detected in exosomal fractions from EBER-1 transfected 293T cells whilst the fractions from vector only transfectants were clearly negative. Furthermore, purified exosomal fractions also contained the EBER-binding protein (La), supporting the notion that EBERs are most probably released from EBV infected cells in the form of EBER-La complex in exosomes.

Sensing of latent EBV infection through exosomal transfer of 5′pppRNA

“…Although EBER-La complexes may be released in a vesicle-independent manner, we did not detect La in exosomes by proteomics or by Western blotting (9). We used sucrose densitybased purification of exosomes, which may explain why we do not detect La in exosomes preparations (23). In addition, in EBVinfected LCLs, but not in EBV − control cells, inhibiting small RNA sorting raises IFNB1 transcription (Fig.…”

“…Indeed, chronic activation of antiviral immunity by erroneous detection of extracellular RNA drives autoimmunity in predisposed individuals (20). Although binding of proinflammatory EBERs to cytoplasmic sensors has been reported in vitro (9)(10)(11)(12), there is little evidence that this may occur in latently infected B cells in a physiologically relevant context (21), but there are multiple indications that EBERs are secreted from infected cells (9,22,23).…”

141 Small RNA deep sequencing discriminates subsets of extracellular vesicles released by melanoma cells - evidence of unique microRNA cargos

“…In this study we identified 1041 miRNAs altogether in the extracellular vesicle and cell samples studied, which are more than most array systems would detect. [22][23][24][25][26][27]42,43 Further, comparison among expression of miRNAs is possible with deep sequencing approaches, if the absolute number of copies in the sample can be quantified. 44 In this study, by using the Quantile normalization method for all samples, miRNA expression showed positive relationship between the samples, although many detected miRNA are unique in the different subsets of vesicles ( Fig.…”

“…[17][18][19][20][21] Importantly, many small RNAs are released in the extracellular environment as EV cargo, which has been specifically documented for the exosomes. [22][23][24][25][26][27] Importantly, many cellular RNAs, including miRNAs are detected in the extracellular space, especially in extracellular vesicles. 28,29 Currently, extracellular RNA communication is also considered to be putatively important in animals, with important implications for biology, disease and medicine.…”

Exosomes as the Promising Biomarker for Epstein-Barr Virus (EBV)-Associated Cancers

“…Further studies are required to investigate and validate their roles in promoting EBV-derived cancers. The occurrence of exosomes carrying mRNAs encoding for oncogenic EBV proteins such as LMP1, LMP2, EBNA1 and EBNA2 [46,47] as well as EBERs has also been documented (Figure 1). While the functions of these RNAs in the EBV-infected cancer cells are well-described, whether or not the exosomal RNAs are transferable to the recipient cells and exert their tumorigenic effects remains a question.…”