PMID- 32211339 OWN - NLM STAT- MEDLINE DCOM- 20210617 LR - 20210617 IS - 2235-2988 (Electronic) IS - 2235-2988 (Linking) VI - 10 DP - 2020 TI - N-Glycans Mediate the Ebola Virus-GP1 Shielding of Ligands to Immune Receptors and Immune Evasion. PG - 48 LID - 10.3389/fcimb.2020.00048 [doi] LID - 48 AB - The Ebola Virus (EBOV) glycoprotein (GP) sterically shields cell-membrane ligands to immune receptors such as human leukocyte antigen class-1 (HLA-I) and MHC class I polypeptide-related sequence A (MICA), thus mediating immunity evasion. It was suggested that the abundant N-glycosylation of the EBOV-GP is involved in this steric shielding. We aimed to characterize (i) the GP N-glycosylation sites contributing to the shielding, and (ii) the effect of mutating these sites on immune subversion by the EBOV-GP. The two highly glycosylated domains of GP are the mucin-like domain (MLD) and the glycan cap domain (GCD) with three and six N-glycosylation sites, respectively. We mutated the N-glycosylation sites either in MLD or in GCD or in both domains. We showed that the glycosylation sites in both the MLD and GCD domains contribute to the steric shielding. This was shown for the steric shielding of either HLA-I or MICA. We then employed the fluorescence resonance energy transfer (FRET) method to measure the effect of N-glycosylation site removal on the distance in the cell membrane between the EBOV-GP and HLA-I (HLA.A(*)0201 allele). We recorded high FRET values for the interaction of CFP-fused HLA.A(*)0201 and YFP-fused EBOV-GP, demonstrating the very close distance (<10 nm) between these two proteins on the cell membrane of GP-expressing cells. The co-localization of HLA-I and Ebola GP was unaffected by the disruption of steric shielding, as the removal of N-glycosylation sites on Ebola GP revealed similar FRET values with HLA-I. However, these mutations directed to N-glycosylation sites had restored immune cell function otherwise impaired due to steric shielding over immune cell ligands by WT Ebola GP. Overall, we showed that the GP-mediated steric shielding aimed to impair immune function is facilitated by the N-glycans protruding from its MLD and GCD domains, but these N-glycans are not controlling the close distance between GP and its shielded proteins. CI - Copyright (c) 2020 Iraqi, Edri, Greenshpan, Kundu, Bolel, Cahana, Ottolenghi, Gazit, Lobel, Braiman and Porgador. FAU - Iraqi, Muhammed AU - Iraqi M AD - The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel. FAU - Edri, Avishay AU - Edri A AD - The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel. FAU - Greenshpan, Yariv AU - Greenshpan Y AD - The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel. FAU - Kundu, Kiran AU - Kundu K AD - The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel. AD - National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er Sheva, Israel. FAU - Bolel, Priyanka AU - Bolel P AD - The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel. FAU - Cahana, Avishag AU - Cahana A AD - The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel. FAU - Ottolenghi, Aner AU - Ottolenghi A AD - The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel. FAU - Gazit, Roi AU - Gazit R AD - The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel. FAU - Lobel, Leslie AU - Lobel L AD - The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel. FAU - Braiman, Alex AU - Braiman A AD - The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel. FAU - Porgador, Angel AU - Porgador A AD - The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences, Ben-Gurion University of the Negev, Be'er Sheva, Israel. AD - National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Be'er Sheva, Israel. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. DEP - 20200306 PL - Switzerland TA - Front Cell Infect Microbiol JT - Frontiers in cellular and infection microbiology JID - 101585359 RN - 0 (Ligands) RN - 0 (Polysaccharides) RN - 0 (Viral Envelope Proteins) SB - IM MH - *Ebolavirus MH - *Hemorrhagic Fever, Ebola MH - Humans MH - Immune Evasion MH - Ligands MH - Polysaccharides MH - Viral Envelope Proteins/genetics PMC - PMC7068452 OTO - NOTNLM OT - Ab-antibody OT - EBOV-ebola virus OT - GP-glycoprotein OT - N-glycosylation OT - immune evasion OT - steric shielding EDAT- 2020/03/27 06:00 MHDA- 2021/06/22 06:00 PMCR- 2020/01/01 CRDT- 2020/03/27 06:00 PHST- 2019/08/27 00:00 [received] PHST- 2020/01/24 00:00 [accepted] PHST- 2020/03/27 06:00 [entrez] PHST- 2020/03/27 06:00 [pubmed] PHST- 2021/06/22 06:00 [medline] PHST- 2020/01/01 00:00 [pmc-release] AID - 10.3389/fcimb.2020.00048 [doi] PST - epublish SO - Front Cell Infect Microbiol. 2020 Mar 6;10:48. doi: 10.3389/fcimb.2020.00048. eCollection 2020.