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.

Figure 1. Reverse transcriptase PCR for EBERs on genomic RNA.

RT- PCR was carried out on genomic RNA extracted from three EBV positive cell lines (B95-8, EBV-LCL and BL30-B95-8), 293T cells stably transfected with EBER-1 plasmid (293T-pHEBo-E1), and two EBV negative cell lines (BL30 and 293T stably cells transfected with empty plasmid (293T-pHEBo)). cDNA from these cells was subjected to 30 rounds of PCR amplification for (A) EBER-1 and (B) EBER-2 and the amplified products were visualized on a 2% agarose gel. Positive (+) (EBER-1 or EBER-2 plasmid DNA) and negative (−) (sterile water) controls are indicated. All three EBV positive cell lines showed specific amplification of EBER-1 and EBER-2. BL30 and 293T-pHEBo cells were clearly negative. Furthermore, EBER-1 specific amplification was also seen in EBER-1 transfected 293T-pHEBo-E1 cells. (C) To ensure that the EBER-amplification seen in Figure 1A and 1B was not due to EBV DNA contamination, PCR was performed for EBER-1 and EBER-2 on DNase treated RNA samples prior to reverse transcription. No amplification was seen, clearly indicating the absence of any contaminating DNA (results shown for EBER-1 only).

Figure 2. Reverse transcriptase PCR for EBERs on culture supernatants.

RNA was extracted from 1-PCR was carried out on RNA extracted from culture supernatant of EBV positive cell lines (EBV-LCL, B95-8), 293T cells stably transfected with EBER-1 (293T-pHEBo-E1) and two EBV negative cell lines (BL30 and 293T cells stably transfected with empty plasmid (293T-pHEBo)). cDNA was subjected to 30 rounds of PCR amplification for (A) EBER-1 and (B) EBER-2. Positive (+) (EBER-1 or EBER-2 plasmid DNA) and negative (−) (sterile water) controls are indicated. Both EBER-1 and EBER-2 were amplified from EBV positive cell lines (B95-8 and EBV-LCL) whilst the EBV negative cell lines BL30 and 293T-pHEBo were negative. EBER-1 transfected 293T-pHEBo-E1 also showed specific amplification.

Figure 3. Transmission electron microscopy and western blot for CD63 on exosomal fractions.

Exosomes were isolated using differential ultracentrifugation and examined using transmission electron microscopy. (A) Nanovesicles with typical size (50–120 nm) and morphology resembling exosomes were observed in isolates from both EBV positive (EBV-LCL) and negative (293T) cells. (B) Western blotting for the exosomal marker CD63, confirmed the identity of these nanovesicles to be exosomes.

Figure 4. Reverse transcriptase PCR for EBERs on exosomal RNA.

RT-PCR for (A) EBER-1 and (B) EBER-2 on the purified exosomes from EBV-positive and negative cells, gave positive amplification only in EBV infected cell lines. EBER-1 stably transfected 293T cells (293T-pHEBo-E1) were also positive for EBER-1, but the amplification signal was weaker than that seen with EBV-infected cell lines. (C) DNase treated RNA samples prior to reverse transcription consistently gave negative results, indicating that the amplification signals seen in Figure A and B were not due to DNA contamination (results shown for EBER-1 only). Positive (+) (EBER-1 or EBER-2 plasmid DNA) and negative (−) (sterile water) controls are indicated.

Figure 5. Detection of EBERs and EBER binding protein La in exosomal fractions.

(A) RNase A treatment of purified exosomes prior to RNA extraction and RT-PCR did not abolish EBER amplification signal, suggesting that EBERs are present in exosomes and not in the extra-exosomal fraction. (B) To determine if the EBER-1 binding protein La was present in exosomes, 25 µg of exosomal protein fraction was separated by 10% SDS PAGE and immunoblotted using anti-La monoclonal antibodies. Exosomal fractions from all cell lines clearly showed presence of La protein, irrespective of whether they were EBV infected or not.