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

Abstract

Epstein-Barr virus-encoded RNAs (EBER1 and EBER2) are two highly abundant, non-protein coding RNAs consistently expressed in all EBV infected cells, but their function remains poorly understood. Conventional in situ hybridization studies have indicated that these RNAs are present exclusively in the nucleus. We have recently demonstrated that EBERs can be excreted from infected cells via exosomes. However, the details of the steps involved in their excretion remain unknown. In this study, we aimed to directly track the journey of EBERs from the nucleus to the excretory exosomes of EBV immortalized B-lymphocytes. Using a combination of molecular and novel immuno-gold labelled electron microscopy (EM) based techniques, we demonstrate the presence of EBERs, not only in the nucleus, but also in the cytoplasm of EBV infected B cell lines. EBERs were also seen in exosomes shed from infected cells along with the EBER binding protein La. Our results show, for the first time, that at least a proportion of EBERs are transported from the nucleus to the cytoplasm where they appear to be loaded into multi-vesicular bodies for eventual excretion via exosomes.

Immune Escape by Non-coding RNAs of the Epstein Barr Virus

“…EBERs have been suggested to also influence immune responses by being sensed via pathogen associated molecular pattern (PAMP) recognition receptors (PRRs) that detect viral RNAs, including toll-like receptor 3 (TLR3) and 8 (TLR8), RIG-I ( Figure 1 ) and PKR ( Vuyisich et al, 2002 ; McKenna et al, 2006 ; Samanta et al, 2008 ; Iwakiri et al, 2009 ; Li et al, 2019 ). Such immune stimulation by EBERs might also be transferred via EBER containing exosomes to dendritic cells ( Ahmed et al, 2014 , 2018 ; Baglio et al, 2016 ). BART miRNA 6-3p down-regulates this recognition by attenuating the RIG-I pathway ( Figure 1 ; Lu et al, 2017 ).…”

New insights into Epstein‑Barr virus‑associated tumors: Exosomes (Review)

“…Lupus antigen (La) is an abundant RNA binding protein in the nucleus of latently infected B cells that binds nascent viral Pol III transcripts, protecting the 3′ ends from degradation by exonucleases ( 74 ). EBERs are transported from the nucleus to the cytoplasm and shed in exosomes by binding to La ( 16 ). Based on these studies, Baglio et al ( 75 ) proposed that by interacting with La and loading into exosomes, EBV nuclear 5′pppEBER1 escapes cytosolic detection in cells with established latent infection.…”

“…As an important component of the tumor microenvironment (TME), exosomes are vectors by which tumor cells, including EBV-associated tumor cells, can transfer oncogenic cargo that can act on target cells ( 15 ). To date, a few studies have addressed exosomes derived from EBV + tumors, and these studies suggest that the oncogenic molecules encoded by EBV not only display tumorigenic effects on uninfected cells via transfer through exosomes, but also exert potential immunosuppressive effects ( 14 , 16 , 17 ). Moreover, these exosomes can enter circulating body fluids and be transported throughout the body ( 10 ).…”

Epstein-Barr Virus in Multiple Sclerosis

“…In MS, these GC-like aggregates, triggered by EBV infection of the brain, could be responsible for recruiting, activating and sustaining B and T-cells [119,118] that inadvertently react to auto-antigens, such as myelin basic protein (MBP) and GDP-L-fucose synthase, expressed on oligodendrocytes (Figure 1) [98,99,101]. Moreover, cellular and viral components such miRNAs and EBERs, secreted in exosomes could also promote inflammatory and pathological changes that contribute to CNS injury in MS [108,131].…”