Abstract
Different types of cells infected with Epstein-Barr virus (EBV) can release exosomes containing viral components that functionally affect neighboring cells. Previously, we found that EBV was localized mostly in infiltrating lymphocytes within the stromal layer of cervical lesions. In this study, we aimed to determine effects of exosome-transferred EBV-encoded RNAs (EBERs) on keratinocytes expressing human papillomavirus (HPV) 16 E6/E7 (DonorI-HPV16 HFKs). Lipid transfection of in vitro-transcribed EBER1 molecules (ivt EBER1) into DonorI-HPV16 HFKs caused strong induction of interferon (IFN)-related genes and interleukin 6 (IL-6). To gain insights into the physiological situation, monocyte-derived dendritic cells (moDCs), low passage DonorI-HPV16 HFKs and primary keratinocytes were used as recipient cells for internalization of exosomes from wild-type EBV (wt EBV) or B95-8 EBV-infected lymphoblastoid cell lines (LCLs). qRT-PCR was used to determine the expression of EBER1, HPV16 E6/E7, IFN-related genes and IL-6 in recipient cells. The secretion of inflammatory cytokines was investigated using cytometric bead array. Wt EBV-modified exosomes induced both IFN-related genes and IL-6 upon uptake into moDCs, while exosomes from B95-8 EBV LCLs induced only IL-6 in moDCs. Internalization of EBV–modified exosomes was demonstrated in DonorI-HPV16 HFKs, yielding only EBER1 but not EBER2. However, EBER1 transferred by exosomes did not induce IFN-related genes or IL-6 expression and inflammatory cytokine secretion in DonorI-HPV16 HFKs and primary keratinocytes. EBER1 copy numbers in exosomes from wt EBV-infected LCLs were 10-fold higher than in exosomes from B95-8 LCLs (equal cell equivalent), whereas ivt EBER1 was used at approximately 100-fold higher concentration than in exosomes. These results demonstrated that the induction of IFN-related genes and IL-6 by EBER1 depends on quantity of EBER1 and type of recipient cells. High levels of EBER1 in cervical cells or infiltrating dendritic cells may play a role in the inflammation-to-oncogenesis transition of HPV-associated cervical cancer through modulation of innate immune signals.
Highlights
Epstein-Barr virus (EBV) is a DNA gammaherpesvirus which can establish latent infection in host cells
The results showed that all five IFN-related genes and interleukin 6 (IL-6) were strongly induced by lipid delivery of in vitro transcribed EBER1 (EBER1) transcripts (Fig 1)
The results showed that Retinoic acid-inducible gene I (RIG-I), interferon-induced transmembrane protein 1 (IFITM1), OAS2, interferon-induced GTP-binding protein Mx1 (Mx1) and IL-6 were induced by IK140508 exosomes whereas RN exosomes had an effect only on the induction of IL-6 but not IFN-related genes in monocyte-derived dendritic cells (moDCs) (Fig 3A)
Summary
Epstein-Barr virus (EBV) is a DNA gammaherpesvirus which can establish latent infection in host cells. It enters the human host via a mucosal route, infects and induces proliferation of latently-infected cells. Infection of a Bcell by EBV leads to EBV latent gene expression which drives B-cell proliferation [1]. Despite its predominantly benign character, EBV is classified as a group 1 carcinogen according to the International Agency for Research on Cancer (IARC). It is causally associated with a wide range of malignancies including Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal carcinoma and gastric carcinoma [2]
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