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Ebola virus (EBOV) interacts with cells using two categories of cell surface receptors, C-type lectins and phosphatidylserine (PS) receptors. PS receptors typically bind to apoptotic cell membrane PS and orchestrate the uptake and clearance of apoptotic bodies. Many viruses coated with PS-containing lipid envelopes, acquired during budding from host cells, can also exploit these receptors for internalization. PS is restricted to the inner leaflet of the plasma membrane in homeostatic cells, an orientation that would be unfavorable for PS receptor-mediated uptake if conserved on the viral envelope. Therefore, it is theorized that viral infection induces host cell PS externalization to the outer leaflet during replication. Cells have several membrane scramblase enzymes that enrich outer leaflet PS when activated. Here, we investigate two scramblases, TMEM16F and XKR8, as possible mediators of cellular and viral envelope surface PS levels during recombinant VSV/EBOV-GP replication and EBOV virus-like particle (VLP) production. We found that rVSV/EBOV-GP and EBOV VLPs produced in XKR8 knockout cells contain decreased levels of PS in their outer leaflets. ΔXKR8-made rVSV/EBOV-GP is 70% less efficient at infecting cells through apoptotic mimicry compared to viruses made in parental cells. Our data suggest that virion surface PS acquisition requires XKR8 activity, whereas TMEM16F activity is not essential. Unexpectedly, we observed defective rVSV/G, rVSV/EBOV-GP, and EBOV VLP budding in ΔXKR8 cells, suggesting that phospholipid scrambling via XKR8 enhances both Ebola infectivity and budding efficiency. Overexpression of XKR8 dramatically increased budding activity, suggesting outer leaflet PS is required for both particle production and increased infectivity.ImportanceThe Democratic Republic of the Congo experienced its deadliest Ebola outbreak from 2018 to 2020, with 3,444 confirmed cases and 2,264 deaths (as of March 12, 2020). Owing to the extensive damage that these outbreaks have caused in Africa, as well as its future epidemic potential, Ebola virus (EBOV) ranks among the top eight priority pathogens outlined by the WHO in 2018. A comprehensive understanding of Ebola entry pathways into target cells is critical for antiviral development and outbreak control. Thus far, host-cell scramblases TMEM16F and XKR8 have each been named as the sole mediator of Ebola envelope surface phosphatidylserine (PS). We assessed the contributions of these proteins using CRISPR knockout cells and two EBOV models: rVSV/EBOV-GP and EBOV VLPs. We observed that XKR8 is required for optimal EBOV envelope PS levels, PS receptor engagement, and particle budding across all viral models, whereas TMEM16F did not play a major role.

The distribution of phospholipid types between the two leaflets of a membrane bilayer is a controlled feature of membrane structure. One of the two membrane catalytic activities governing this distribution randomizes the composition of the two leaflets–-the phospholipid scramblases. Two proteins (Xkr8 and TMEM16F) required for the activation of these activities have been identified. One of these proteins (TMEM16F) is quite clearly a scramblase itself and provides insight into the mechanism by which transbilayer phospholipid movement is facilitated.