High Levels of EBV-Encoded RNA 1 (EBER1) Trigger Interferon and Inflammation-Related Genes in Keratinocytes Expressing HPV16 E6/E7

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.

Epstein‐Barr virus, but not human cytomegalovirus, is associated with a high‐grade human papillomavirus–associated cervical lesions among women in North Carolina

“…14,26 However, some studies have shown that EBV does not reside in the cervical epithelium but rather within infiltrating lymphoid cells adjacent to the tumor epithelium, potentially influencing the microenvironment. 27,28 Although cellular localization of EBV remains contested, previous reports are in agreement that EBV can infect cervical tissues and potentially influence the risk of progression to high-grade cervical lesions and cervical cancer. 15,16,29,30 Our present findings confirm previous studies including a metaanalysis which found that EBV infection was significantly and positively associated with lesion grade in cervical epithelia and was more prevalent in malignant lesions.…”

“…EBV has been shown to be sexually transmitted, and replicates in cervical epithelial cells with eventual shedding . However, some studies have shown that EBV does not reside in the cervical epithelium but rather within infiltrating lymphoid cells adjacent to the tumor epithelium, potentially influencing the microenvironment . Although cellular localization of EBV remains contested, previous reports are in agreement that EBV can infect cervical tissues and potentially influence the risk of progression to high‐grade cervical lesions and cervical cancer …”

Tracking EBV-encoded RNAs (EBERs) from the nucleus to the excreted exosomes of B-lymphocytes

“…More recently, it was shown that the uptake of EBER1 containing exosomes by dendritic cells, results in the induction of IFN-related genes and inflammation mediated tumorigenesis. This effect was largely dependent on the level of EBER1 released in exosomes 40 . Our preliminary data indicates that exosomes released from EBER1 transfected cells induce cell proliferation 19 .…”

Role of Epstein-Barr Virus and Human Papillomavirus Coinfection in Cervical Cancer: Epidemiology, Mechanisms and Perspectives

“…However, a significant reduction in EBV immediate early (BZLF1, BRLF1) and early (BALF5, BMRF1) gene expression with increased expression of EBER1 was evidenced in HPV+/EBV+ human foreskin keratinocytes (HFK) compared to that in HPV−/EBV+ rafts [ 106 ]. In cervical cells, increased levels of EBER1 may contribute to the transition from inflammation to oncogenesis of HPV-associated cervical cancer by modulating innate immune responses [ 107 ]. In addition, LMP1 reduces the presence of HPV16 with no expression change in the EBNA1 and EBNA2 latent genes, suggesting that EBV latency is favored over lytic replication in HPV16+ cells.…”