PMID- 34319156 OWN - NLM STAT- MEDLINE DCOM- 20211021 LR - 20220328 IS - 1098-5514 (Electronic) IS - 0022-538X (Print) IS - 0022-538X (Linking) VI - 95 IP - 20 DP - 2021 Sep 27 TI - Ebola Virus Requires Phosphatidylserine Scrambling Activity for Efficient Budding and Optimal Infectivity. PG - e0116521 LID - 10.1128/JVI.01165-21 [doi] LID - e01165-21 AB - 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 DeltaXKR8 cells. Deletion of 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 Ebola virus was identified in 1976. While outbreaks are primarily attributed to zoonotic transfer events, new evidence is emerging outbreaks may be caused by a combination of spillover events and viral latency or persistence in survivors. The possibility that Ebola virus can remain dormant and then reemerge 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. FAU - Acciani, Marissa D AU - Acciani MD AD - Department of Infectious Diseases, College of Veterinary Medicine, University of Georgiagrid.213876.9, Athens, Georgia, USA. FAU - Lay Mendoza, Maria F AU - Lay Mendoza MF AD - Department of Infectious Diseases, College of Veterinary Medicine, University of Georgiagrid.213876.9, Athens, Georgia, USA. FAU - Havranek, Katherine E AU - Havranek KE AD - Department of Infectious Diseases, College of Veterinary Medicine, University of Georgiagrid.213876.9, Athens, Georgia, USA. FAU - Duncan, Avery M AU - Duncan AM AD - Department of Infectious Diseases, College of Veterinary Medicine, University of Georgiagrid.213876.9, Athens, Georgia, USA. FAU - Iyer, Hersha AU - Iyer H AD - Department of Infectious Diseases, College of Veterinary Medicine, University of Georgiagrid.213876.9, Athens, Georgia, USA. FAU - Linn, Olivia L AU - Linn OL AD - Department of Infectious Diseases, College of Veterinary Medicine, University of Georgiagrid.213876.9, Athens, Georgia, USA. FAU - Brindley, Melinda A AU - Brindley MA AUID- ORCID: 0000-0002-4929-8085 AD - Department of Infectious Diseases, Department of Population Health, College of Veterinary Medicine, University of Georgiagrid.213876.9, Athens, Georgia, USA. LA - eng GR - R01 AI139238/AI/NIAID NIH HHS/United States GR - R01AI139238/HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20210728 PL - United States TA - J Virol JT - Journal of virology JID - 0113724 RN - 0 (Glycoproteins) RN - 0 (Phosphatidylserines) RN - 0 (Phospholipid Transfer Proteins) RN - 0 (Viral Envelope Proteins) SB - IM MH - Cell Line MH - Ebolavirus/*metabolism/pathogenicity MH - Glycoproteins/metabolism MH - Hemorrhagic Fever, Ebola/virology MH - Humans MH - Phosphatidylserines/*metabolism/physiology MH - Phospholipid Transfer Proteins/metabolism/physiology MH - Viral Envelope Proteins/metabolism MH - Virion/metabolism MH - Virus Assembly/genetics/physiology MH - Virus Release/genetics/*physiology PMC - PMC8475530 OTO - NOTNLM OT - Ebola OT - XKR8 OT - budding OT - phosphatidylserine EDAT- 2021/07/29 06:00 MHDA- 2022/03/29 06:00 PMCR- 2022/03/27 CRDT- 2021/07/28 12:20 PHST- 2021/07/29 06:00 [pubmed] PHST- 2022/03/29 06:00 [medline] PHST- 2021/07/28 12:20 [entrez] PHST- 2022/03/27 00:00 [pmc-release] AID - 01165-21 [pii] AID - 10.1128/JVI.01165-21 [doi] PST - ppublish SO - J Virol. 2021 Sep 27;95(20):e0116521. doi: 10.1128/JVI.01165-21. Epub 2021 Jul 28.