title: Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction.
abstract: Ebola virus (EBOV) is an enveloped, ssRNA virus from the familyFiloviridaecapable of causing severe hemorrhagic fever with up to 80-90% mortality rates. The most recent outbreak of EBOV in West Africa starting in 2014 resulted in over 11,300 deaths; however, long-lasting persistence and recurrence in survivors has been documented, potentially leading to further transmission of the virus. We have previously shown that exosomes from cells infected with HIV-1, HTLV-1 and Rift Valley Fever virus are able to transfer viral proteins and non-coding RNAs to naïve recipient cells, resulting in an altered cellular activity. In the current manuscript, we examined the effect of Ebola structural proteins VP40, GP, NP and VLPs on recipient immune cells, as well as the effect of exosomes containing these proteins on naïve immune cells. We found that VP40-transfected cells packaged VP40 into exosomes, and that these exosomes were capable of inducing apoptosis in recipient immune cells. Additionally, we show that presence of VP40 within parental cells or in exosomes delivered to naïve cells could result in the regulation of RNAi machinery including Dicer, Drosha, and Ago 1, which may play a role in the induction of cell death in recipient immune cells. Exosome biogenesis was regulated by VP40 in transfected cells by increasing levels of ESCRT-II proteins EAP20 and EAP45, and exosomal marker proteins CD63 and Alix. VP40 was phosphorylated by Cdk2/Cyclin complexes at Serine 233 which could be reversed with r-Roscovitine treatment. The level of VP40-containing exosomes could also be regulated by treated cells with FDA-approved Oxytetracycline. Additionally, we utilized novel nanoparticles to safely capture VP40 and other viral proteins from Ebola VLPs spiked into human samples using SDS/reducing agents, thus minimizing the need for BSL-4 conditions for most downstream assays. Collectively, our data indicates that VP40 packaged into exosomes may be responsible for the deregulation and eventual destruction of the T-cell and myeloid arms of the immune system (bystander lymphocyte apoptosis), allowing the virus to replicate to high titers in the immunocompromised host. Moreover, our results suggest that the use of drugs such as Oxytetracycline to modulate the levels of exosomes exiting EBOV-infected cells may be able to prevent the devastation of the adaptive immune system and allow for an improved rate of survival.
authors.affiliation: Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas VA, USA.
authors.forename: Michelle L
authors.initials: ML
authors.lastname: Pleet
authors.affiliation: Department of Physiological Sciences, Eastern Virginia Medical School, Norfolk VA, USA.
authors.forename: Allison
authors.initials: A
authors.lastname: Mathiesen
authors.affiliation: Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas VA, USA.
authors.forename: Catherine
authors.initials: C
authors.lastname: DeMarino
authors.affiliation: Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas VA, USA.
authors.forename: Yao A
authors.initials: YA
authors.lastname: Akpamagbo
authors.affiliation: Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas VA, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Barclay
authors.affiliation: Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas VA, USA.
authors.forename: Angela
authors.initials: A
authors.lastname: Schwab
authors.affiliation: Research Center for Neglected Diseases of Poverty, Department of Microbiology, Immunology and Tropical Medicine, George Washington University School of Medicine and Health Sciences, Washington DC, USA.
authors.forename: Sergey
authors.initials: S
authors.lastname: Iordanskiy
authors.affiliation: Laboratory of Molecular Virology, School of Systems Biology, George Mason University, ManassasVA, USA; University of North Carolina HIV Cure Center, Department of Medicine, University of North Carolina at Chapel Hill School of Medicine, Chapel HillNC, USA.
authors.forename: Gavin C
authors.initials: GC
authors.lastname: Sampey
authors.affiliation: Ceres Nanosciences Inc., Manassas VA, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Lepene
authors.affiliation: Center for Sickle Cell Disease, Department of Medicine, Howard University, Washington DC, USA.
authors.forename: Sergei
authors.initials: S
authors.lastname: Nekhai
authors.affiliation: Integrated BioTherapeutics, Inc., Gaithersburg MD, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Aman
authors.affiliation: Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas VA, USA.
authors.forename: Fatah
authors.initials: F
authors.lastname: Kashanchi
keywords: EBOV; ESCRT; Ebola virus; VLP; VP40; apoptosis; exosomes; microRNA