Ebola Virus Requires Phosphatidylserine Scrambling Activity for Efficient Budding and Optimal Infectivity

Abstract

Ebola virus (EBOV) attaches to target 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 debris. Many enveloped viruses also contain exposed PS and can therefore exploit these receptors for cell entry. Viral infection can induce PS externalization in host cells, resulting in increased outer PS levels on budding virions. Scramblase enzymes carry out cellular PS externalization, thus, we targeted these proteins in order to manipulate viral envelope PS levels. We investigated two scramblases previously identified to be involved in EBOV PS levels, transmembrane protein 16F and Xk-related protein 8 (XKR8), as possible mediators of cellular and viral envelope surface PS levels during the replication of recombinant vesicular stomatitis virus containing its native glycoprotein (rVSV/G) or the EBOV glycoprotein (rVSV/EBOV-GP). We found that rVSV/G and rVSV/EBOV-GP virions produced in XKR8 knockout cells contain decreased levels of PS on their surfaces, and the PS-deficient rVSV/EBOV-GP virions are 70% less efficient at infecting cells through PS receptors. We also observed reduced rVSV and EBOV virus-like particle (VLP) budding in ΔXKR8 cells. Deleting XKR8 in HAP1 cells reduced rVSV/G and rVSV/EBOV-GP budding by 60% and 65% respectively, and reduced Ebola VLP budding more than 60%. We further demonstrated that caspase cleavage of XKR8 is required to promote budding. This suggests that XKR8, in addition to mediating virion PS levels, may also be critical for enveloped virus budding at the plasma membrane. Importance Within the last decade, countries in western and central Africa have experienced the most widespread and deadly Ebola outbreaks since the virus was identified in 1976. While outbreaks are primarily attributed to zoonotic transfer events, new evidence is emerging that outbreaks may be caused by a combination of spillover events and viral latency or persistence in survivors. The possibility that Ebola can remain dormant then re-emerge in survivors highlights the critical need to prevent the virus from entering and establishing infection in human cells. Thus far, host-cell scramblases TMEM16F and XKR8 have been implicated in Ebola envelope surface phosphatidylserine (PS) and cell entry using PS receptors. 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 and infectivity, and particle budding across all viral models.

The use of viral vectors in vaccine development

“…VSV represents a valuable vaccine platform thanks to its ability to replicate at high titers, its low seroprevalence, and little pre-existing immunity in humans. VSV-based vaccines are produced through transfection of mammalian cells including baby hamster kidney cells stably expressing T7 polymerase (BHK-T7) or HEK293T with a recombinant VSV plasmid expressing the transgene of interest in place of the virus’ G gene 55 (Fig. 3 ).…”

Scrambled or flipped: 5 facts about how cellular phosphatidylserine localization can mediate viral replication

“…Some viruses also modulate cellular calcium signaling pathways to increase cytosolic calcium levels, which would trigger calcium-activated scramblases [36]. Studies examining EBOV PtdSer levels have implicated both apoptotic scramblase XKR8 [37,38] and calcium-activated scramblase TMEM16F [39] in EBOV envelope PtdSer and infectivity.…”

“…While investigating the role of scramblase XKR8 in EBOV entry through apoptotic mimicry, our group observed that XKR8-knockout human haploid HAP1 cells consistently released fewer recombinant vesicular stomatitis virus particles and EBOV virus-like particles (VLPs) [38]. VLP release was restored by transfecting XKR8-knockout HAP1 cells with exogenous XKR8.…”

“…Furthermore, several studies reviewed here illuminate potential new tools that may be useful for improving upon current and future vaccines. The groups investigating EBOV apoptotic mimicry manipulated (specifically reduced) viral envelope PtdSer distribution and infectivity in vitro by inhibiting cellular XKR8 or TMEM16F PtdSer scrambling activity [37][38][39]. Biochemical studies also show that basal and apoptotic surface PtdSer levels can be elevated by overexpressing TMEM16F and XKR8 [67][68][69].…”

RNA Viruses, Pregnancy and Vaccination: Emerging Lessons from COVID-19 and Ebola Virus Disease

“…Phosphatidylserine (PS) receptors are demonstrated to facilitate infection of SARS-CoV-2 and hence have been suggested as a therapeutic target [ 37 ]. EBOV is also known to make use of the host apoptotic clearance mechanism to augment its entry into target cells by externalizing PS on its surface [ 38 , 39 , 40 ]. In both SARS-CoV-2 [ 37 ] and EBOV [ 41 , 42 ], the T cell Ig and mucin domain (TIM) family of proteins is shown to be involved in the above mentioned mechanism.…”