Ebola VP40 in Exosomes Can Cause Immune Cell Dysfunction

Abstract

Ebola virus (EBOV) is an enveloped, ssRNA virus from the family Filoviridae capable 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.

The Role of Exosomal VP40 in Ebola Virus Disease

“…Furthermore, when these VP40-containing exosomes were placed on recipient immune cells (T cells and monocytes), cell death was readily observed (Pleet et al, 2016). This is similar to previous observations that exosomes from cells infected with HIV-1, HTLV-1, and Rift Valley Fever virus have been shown to negatively impact the viability of naive recipient immune cells (Lenassi et al, 2010;Jaworski et al, 2014a;Ahsan et al, 2016;Sampey et al, 2016).…”

“…Along these lines, both intracellular and concentrated exosomal preparation levels of exosomal marker CD63 were also found to be increased in cells transfected with VP40 plasmid (Pleet et al, 2016). The upregulation of various host molecules derived from exosomes (i.e., CD81, CD63, and CD9) by several viruses and incorporation into the viral membrane for various purposes have been previously described (Dongen et al, 2016).…”

“…This is not unique to Ebola, as other viruses, including HIV-1, have likewise been shown to use ESCRT proteins such as TSG101 for this purpose (Garrus et al, 2001;Martin-Serrano et al, 2001). In addition, 293T cells transfected with VP40 increase in intracellular levels of TSG101 (ESCRT I), and EAP20 and EAP45 (ESCRT II) proteins (Pleet et al, 2016). Increases in ESCRT components may suggest an increase in exosomal biogenesis in the presence of VP40.…”

“…In the case of Ebola, the viral matrix protein VP40 has recently been shown to be packaged into exosomes, which in turn can decrease the viability of recipient immune cells (Pleet et al, 2016). In this study, we review the recent findings regarding exosomal VP40 and the potential role of these exosomes in EVD pathogenesis.…”

“…Interestingly, it was observed that components of the miRNA machinery, including Dicer, Drosha, and Ago proteins, were downregulated in both donor 293T cells transfected with VP40 plasmids and naive recipient T cells (Pleet et al, 2016). Dicer, Drosha, and Ago are integral RNAi pathway proteins involved in the production of miRNAs and siRNAs for the silencing or degradation of target mRNA transcripts (Novina and Sharp, 2004).…”