Extracellular Vesicles and Ebola Virus: A New Mechanism of Immune Evasion

Abstract

Ebola virus (EBOV) disease can result in a range of symptoms anywhere from virtually asymptomatic to severe hemorrhagic fever during acute infection. Additionally, spans of asymptomatic persistence in recovering survivors is possible, during which transmission of the virus may occur. In acute infection, substantial cytokine storm and bystander lymphocyte apoptosis take place, resulting in uncontrolled, systemic inflammation in affected individuals. Recently, studies have demonstrated the presence of EBOV proteins VP40, glycoprotein (GP), and nucleoprotein (NP) packaged into extracellular vesicles (EVs) during infection. EVs containing EBOV proteins have been shown to induce apoptosis in recipient immune cells, as well as contain pro-inflammatory cytokines. In this manuscript, we review the current field of knowledge on EBOV EVs including the mechanisms of their biogenesis, their cargo and their effects in recipient cells. Furthermore, we discuss some of the effects that may be induced by EBOV EVs that have not yet been characterized and highlight the remaining questions and future directions.

Keywords: Ebola virus; GP; NP; VP40; cytokine; exosome; extracellular vesicles.

Figure 1

Separation of extracellular vesicles (EVs) from virus for downstream analysis. An optimized a workflow for the separation of EVs away from virus from a variety of backgrounds, including from cell culture supernatants (A) and patient samples (B). Expected profiles of Ebola virus (EBOV) vs. exosomal proteins when extracellular components (i.e., EV-containing cell culture supernatants or biofluids) are separated by size with qEV (IZON) columns, as outlined in the panel A workflow (C). Positive control virus-like particle (VLP) (containing VP40, nucleoprotein (NP) and GP) profiles are shown in lane 1, whereas a typical profile of EV-containing fractions of supernatants that were either unfiltered (top panel) or filtered through 0.22 µm (bottom panel) from cells expressing VP40, NP and GP, followed by separation on qEV size exclusion columns are shown. Expected profiles of mature human immunodeficiency virus (HIV) virions (as indicated by p24 capsid protein) vs. exosomal marker proteins when cell culture supernatants are separated by density, such as by iodixanol or sucrose gradients, as outlined in the panel A workflow (D). Profiles demonstrated here are the expected results for iodixanol fractions in increments of 1.2% from 6.0%–18.0%. Fractions where either virions or VLPs are known to elute or sediment are indicated by red boxes. Fractions containing only exosomes and no viral particles are enclosed by green boxes.

Figure 2

Possible mechanisms of unconventional EBOV protein secretion. Three proposed mechanisms of packaging of EBOV proteins VP40, GP, and NP into EVs are illustrated along with hypothesized times of release. (I) Incorporation as viral uncoated on entry: Entry of infectious virions or VLPs into host cells through endosomes results in cleavage of the outer portions of GP_1,2, binding to the host receptor, and fusion with the endosomal membrane to release the viral nucleocapsid into the cytoplasm. Viral GP and VP40 proteins remaining within the endosome after nucleocapsid release may become packaged into exosomes as a result of inward budding of the endosomal membrane, resulting in extracellular release an estimated 1–6 h post-infection. (II) Exosome packaging: classical loading of cargo into exosomes through endosomal sorting complexes required for transport (ESCRT) pathway (potentially ubiquitinated VP40; Ub-VP40), Golgi vesicle transport (GP_1,2 and soluble GP (sGP)), and other mechanisms (NP). Resulting hypothesized exosomes would be released approximately 24–30+ h post-infection (after viral translation of new protein components), and contain GP_1,2 on the surface, and VP40 and NP on the inside. The secretion of free sGP may also take place through fusion of multivesicular bodies (MVBs) with the plasma membrane. (III) Microvesicle budding: The outward blebbing of microvesicles from the plasma membrane containing nontraditionally assembled EBOV components. GP_1,2 is estimated to be oriented on the surface, with VP40 and NP on the inside. Microvesicles are estimated to be released approximately 24–30+ h post-infection. Additional details of these models are discussed in the text.