N-Glycans Mediate the Ebola Virus-GP1 Shielding of Ligands to Immune Receptors and Immune Evasion.

Muhammed Iraqi,Aner Ottolenghi,Alex Braiman,Kiran Kundu,Avishay Edri,Priyanka Bolel,Leslie Lobel,Roi Gazit,Yariv Greenshpan,Avishag Cahana,Angel Porgador

doi:10.3389/fcimb.2020.00048

Abstract

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.

Highlights

The Zaire Ebola Virus (EBOV) is one of the five known viruses within the EBOV genus (Kuhn et al, 2010)
We showed that EBOV-GP effectively shields ligands to activating NK receptors such as MHC class I polypeptide-related sequence A (MICA) and B7H6; overall, we showed that EBOV-GP expression by target cells favors the axis reducing NK activation, while not perturbing the NK inhibition axis (Braiman et al, 2006)
We demonstrated that the N-glycosylation of both the mucin-like domain (MLD) and glycan cap domain (GCD) domains of GP1 is imperative for the shielding effect and for evading NK cell function

Summary

Introduction

The Zaire Ebola Virus (EBOV) is one of the five known viruses within the EBOV genus (Kuhn et al, 2010). EBOV, along with three other EBOVs, causes a severe pathology in humans and other mammals, which manifests as a hemogenic fever known as Ebola Virus Disease (EVD). EBOV is the sole cause of practically all human deaths from EVD. It recently erupted in the EBOV epidemic that struck West Africa in the years 2014–2016 (Na et al, 2015), resulting in at least 28,000 suspected cases and ∼11,000 confirmed deaths in only 2 years (Leroy et al, 2009; Lori et al, 2015). EBOV has a negative-sense single-stranded RNA genome contained in filament-like viral particles composed of a viral envelope, matrix, and nucleocapsid components. Particles are ∼80 nm in diameter, and the viral envelope carries virally encoded glycoproteins (GPs) (Brinkmann et al, 2016) projecting as 7- to 10-nm-long spikes from their lipid bilayer surfaces

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