title: Secreted Oral Epithelial Cell Membrane Vesicles Induce Epstein-Barr Virus Reactivation in Latently Infected B Cells.
abstract: In the oral epithelium, peripheral stores of Epstein-Barr virus (EBV) are transmitted from infiltrating B cells to epithelial cells. Once the virus is transmitted to epithelial cells, the highly permissive nature of this cell type for lytic replication allows virus amplification and exchange to other hosts. Since the initial transfer of EBV from B cells to epithelial cells requires transitioning of the B-cell to a state that induces virus reactivation, we hypothesized that there might be epithelium-specific signals that allow the infiltrating B cells to sense the appropriate environment to initiate reactivation and begin this exchange process. We previously found that the epithelium-specific miR-200 family of microRNAs promotes EBV lytic replication. Here we show that there are high levels of miR-200 family members in oral and tonsillar epithelia and in saliva. Analysis of cultured oral epithelial cells (OKF6) showed that they actively secrete membrane vesicles (exosomes) that are enriched with miR-200 family members. Coculturing of EBV-positive B cells with OKF6 cells induced viral reactivation. Further, treatment of EBV-positive B cells with OKF6 cell-derived membrane vesicles promoted reactivation. Using a cell system that does not naturally express miR-200 family members, we found that enforced expression of a miR-200 family member produced membrane vesicles that were able to induce the lytic cascade in EBV-positive B cells. We propose that membrane vesicles secreted by oral and tonsillar epithelial cells may serve as a tissue-specific environmental cue that initiates reactivation in B cells, promoting the transfer of virus from peripheral B-cell stores to the oral epithelium to facilitate virus amplification and exchange to other hosts.
  
  
  OBJECTIVE
  Epstein-Barr virus (EBV) is an important human pathogen that is causally associated with several lymphomas and carcinomas. The switch from latency to the lytic cycle is critical for successful host infection and for EBV pathogenesis. Although the EBV lytic cycle can be triggered by certain agents in vitro, the mechanisms that signal reactivation in vivo are poorly understood. We previously reported that endogenously expressed miR-200 family members likely play a role in facilitating the lytic tendencies of EBV in epithelial cells. Here we show that membrane vesicles secreted from oral epithelial cells contain miR-200 family members and that they can be transmitted to proximal EBV-positive B cells, where they trigger reactivation. We propose that this intercellular communication pathway may serve as a sensor mechanism for infiltrating B cells to recognize an appropriate environment to initiate reactivation, thereby allowing the exchange of virus to the oral epithelium.
authors.affiliation: Tulane University Health Sciences Center and Tulane Cancer Center, New Orleans, Louisiana, USA.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Lin
authors.affiliation: Department of Medicine, Section of OB/GYN, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Swan
authors.affiliation: Institute of Translational Research, Ochsner Clinic Foundation, New Orleans, Louisiana, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Tulane University Health Sciences Center and Tulane Cancer Center, New Orleans, Louisiana, USA.
authors.forename: Subing
authors.initials: S
authors.lastname: Cao
authors.affiliation: Department of Psychiatry and Behavioral Sciences, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Zoe
authors.initials: Z
authors.lastname: Brett
authors.affiliation: Department of Psychiatry and Behavioral Sciences, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Stacy
authors.initials: S
authors.lastname: Drury
authors.affiliation: Tulane University Health Sciences Center and Tulane Cancer Center, New Orleans, Louisiana, USA.
authors.forename: Michael J
authors.identifier: http://orcid.org/0000-0001-9930-3578
authors.initials: MJ
authors.lastname: Strong
authors.affiliation: Tulane University Health Sciences Center and Tulane Cancer Center, New Orleans, Louisiana, USA.
authors.forename: Claire
authors.initials: C
authors.lastname: Fewell
authors.affiliation: Department of Medicine, Section of Gastroenterology, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Adriane
authors.initials: A
authors.lastname: Puetter
authors.affiliation: Tulane University Health Sciences Center and Tulane Cancer Center, New Orleans, Louisiana, USA.
authors.forename: Xia
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Microbiology & Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: MaryBeth
authors.initials: M
authors.lastname: Ferris
authors.affiliation: Department of Microbiology & Immunology, Tulane University School of Medicine, New Orleans, Louisiana, USA.
authors.forename: Deborah E
authors.initials: DE
authors.lastname: Sullivan
authors.affiliation: Institute of Translational Research, Ochsner Clinic Foundation, New Orleans, Louisiana, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.affiliation: Tulane University Health Sciences Center and Tulane Cancer Center, New Orleans, Louisiana, USA erik@tulane.edu.
authors.forename: Erik K
authors.initials: EK
authors.lastname: Flemington
mesh_terms: D001402:B-Lymphocytes / Q000821:virology*; D004847:Epithelial Cells / Q000821:virology*; D000067128:Extracellular Vesicles / Q000821:virology*; D057809:HEK293 Cells; D004854:Herpesvirus 4, Human / Q000502:physiology*; D054884:Host-Pathogen Interactions / Q000502:physiology; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D046529:Microscopy, Electron, Transmission; D009061:Mouth Mucosa / Q000166:cytology / Q000821:virology*; D014066:Palatine Tonsil / Q000166:cytology / Q000821:virology; D012463:Saliva / Q000821:virology; D014775:Virus Activation / Q000502:physiology*; D014779:Virus Replication
chemical_list: C533896:MIRN200 microRNA, human; C546363:MIRN429 microRNA, human; D035683:MicroRNAs