Biochemical and Structural Characterization of Cathepsin L-Processed Ebola Virus Glycoprotein: Implications for Viral Entry and Immunogenicity

Abstract

Ebola virus (EBOV) cellular attachment and entry is initiated by the envelope glycoprotein (GP) on the virion surface. Entry of this virus is pH dependent and associated with the cleavage of GP by proteases, including cathepsin L (CatL) and/or CatB, in the endosome or cell membrane. Here, we characterize the product of CatL cleavage of Zaire EBOV GP (ZEBOV-GP) and evaluate its relevance to entry. A stabilized recombinant form of the EBOV GP trimer was generated using a trimerization domain linked to a cleavable histidine tag. This trimer was purified to homogeneity and cleaved with CatL. Characterization of the trimeric product by N-terminal sequencing and mass spectrometry revealed three cleavage fragments, with masses of 23, 19, and 4 kDa. Structure-assisted modeling of the cathepsin L-cleaved ZEBOV-GP revealed that cleavage removes a glycosylated glycan cap and mucin-like domain (MUC domain) and exposes the conserved core residues implicated in receptor binding. The CatL-cleaved ZEBOV-GP intermediate bound with high affinity to a neutralizing antibody, KZ52, and also elicited neutralizing antibodies, supporting the notion that the processed intermediate is required for viral entry. Together, these data suggest that CatL cleavage of EBOV GP exposes its receptor-binding domain, thereby facilitating access to a putative cellular receptor in steps that lead to membrane fusion.Ebola virus (EBOV) is a member of the Filoviridae family and causes severe hemorrhagic fever in humans and nonhuman primates, with case fatality rates of up to 90%. Virus entry and attachment is mediated by a single envelope glycoprotein (GP) as a class I fusion protein, which is proteolytically processed during maturation into two subunits, GP1 and GP2. The GP1 N terminus contains a putative receptor-binding domain (RBD) (2,9,11,12), and the GP2 C terminus contains a fusion peptide, two heptad-repeat regions, and a transmembrane domain. GP1 and GP2 are linked by a disulfide bond (Cys 53 -Cys 609 ) and form trimers of heterodimers on the surface of virions. EBOV GP is also extensively glycosylated, especially within a region of GP1 termed the mucin-like domain (MUC domain), which contains multiple N-and O-linked glycans. We and others have previously shown the MUC domain of GP1 to be cytotoxic and to induce cell rounding (17, 21), and deletion of this region increases pseudovirus infectivity compared to that of full-length GP (11). The MUC domain, however, is also known to enhance cell binding through the human macrophage C-type lectin specific for galactose and N-acetylglucosamine (hMGL) (18), suggesting that glycans in this domain may be involved in the initial cellular attachment. Several other studies have identified factors that enhance cell binding and/or infectivity, including folate receptor ␣ (4), ␤ integrins (19), C-type lectins DC-SIGN and L-SIGN (1), and Tyro3 family members (16). However, the critical cellular receptor(s) thought to interact directly with the GP1 RBD have yet to be identified.Following virus uptake into host cel...

Camouflage and Misdirection: The Full-On Assault of Ebola Virus Disease

“…After uptake into macropinosomes, particles travel to low pH compartments of late endosomes and lysosomes where the viral envelope glycoprotein (GP) is proteolytically cleaved by endosomal cysteine proteases (i.e., cathepsin B and L). This cleavage removes a heavily glycosylated region from GP (Chandran et al, 2005; Dube et al, 2009; Hood et al, 2010; Misasi et al, 2012; Schornberg et al, 2006) and exposes a domain in GP that binds specifically to the endosomal/lysosomal resident filovirus receptor Niemann-Pick C1 protein (NPC1) (Carette et al, 2011; Côté et al, 2011). While current evidence suggests that NPC1 binding may be sufficient to trigger fusion of the viral and cellular membranes (Miller et al, 2012), it is as yet unclear whether additional host proteins or intracellular conditions are necessary (e.g., reducing conditions, altered pH, additional protease cleavage) (Brecher et al, 2011; Chandran et al, 2005).…”

Codon-optimized filovirus DNA vaccines delivered by intramuscular electroporation protect cynomolgus macaques from lethal Ebola and Marburg virus challenges

“…We considered whether the poorer protection from EBOV challenge in macaques given the combination vaccines as compared to only the EBOV vaccine might relate to a skewing of the response toward or against the GP mucin domains. Although correlates of protective immunity with regard to responses to the mucin domain are poorly defined, it has been shown that removal of the mucin domain reveals additional neutralizing epitopes 36,37 and that antibodies to the mucin domain can be protective in mice. 38 Of interest, the genome sequences reported for the EBOV strain causing the recent epidemic of Ebola hemorrhagic fever in West Africa were reported to be~97% homologous with earlier strains from the Democratic Republic of Congo and Gabon.…”

Ebola Virus Potential Drug Targets and Prospects for Small Molecule Drug Discovery

“…EBOV fusion glycoprotein (GP) consists of receptor binding subunit (GP1) and a fusion subunit (GP2) connected by a disulfide bond (Hood et al, 2010). Following receptor-mediated endocytosis (Bhattacharyya et al, 2010;Aleksandrowicz et al, 2011), the highly glycosylated domain is trimmed from the C-terminal of GP1 by endosomal cathepsins (B/L) (Kaletsky et al, 2007;Dube et al, 2009) under acidic environment.…”

“…Following receptor-mediated endocytosis (Bhattacharyya et al, 2010;Aleksandrowicz et al, 2011), the highly glycosylated domain is trimmed from the C-terminal of GP1 by endosomal cathepsins (B/L) (Kaletsky et al, 2007;Dube et al, 2009) under acidic environment. The cleavage exposes the N -terminal hypothetical binding domain of GP1, which binds to Nieman-Pick C1 (Hood et al, 2010;Kaletsky et al, 2007;Dube et al, 2009;Chandran et al, 2005;Schornberg et al, 2006;Côté et al, 2011). The lysosomal membrane cholesterol transporter protein Niemann-Pick C1 (NPC1) is essential for Ebola virus entry and infection.…”