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

Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health,Room 4502, Building 40, MSC-3005, 40 Convent Drive, Bethesda, Maryland 20892-3005, USA.
Journal of Virology (Impact Factor: 4.44). 03/2010; 84(6):2972-82. DOI: 10.1128/JVI.02151-09
Source: PubMed


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.

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    • "In vitro cleavage of the trimeric GP spike derived from both ebolaviruses and marburgviruses by endosomal cysteine cathepsins, or by surrogate enzymes such as thermolysin, results in the generation of a GP trimer lacking GP1 sequences corresponding to the Muc domain and the glycan cap (Chandran et al., 2005; Dube et al., 2009; Hood et al., 2010; J. E. Lee et al., 2008; Schornberg et al., 2006). This cleaved GP intermediate (GPCL), which is competent to bind to the filovirus receptor NPC1 (Côté et al., 2011; Miller et al., 2012), is proposed to resemble one or more GP species generated within the endocytic pathway during viral entry. "
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    • "The specific or nonspecific interactions between GP1 and cell surface host factors, such as T-cell immunoglobulin mucin domain-1 (TIM-1), facilitate virus attachment and endocytosis without changing the conformation of GP trimers [31]. While the whole virion is endocytosed and transported into mature endosome, GP1 is cleaved by endosomal proteases Cathepsin L and B (CatL/CatB) to remove the hyperglycosylated region [32] [33]. Then the exposed RBD interacts with endosomal lumen receptor Niemann-Pick C1 (NPC1) to transform the conformation of GP1 and GP2 at low pH [29]. "
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    • "The mechanisms of EBOV attachment and internalization have been studied extensively [17], [19], [45], [47], [50], [53], [70], [71], [72], [73], [74], [75]. EBOV attaches to various putative attachment factors on the cell surface [13] where they undergo either clathrin/caveolae/dynamin-independent, actin/lipid raft-dependent macropinocytosis-like endocytosis [17], [70], [74] or clathrin/caveolae/dynamin-dependent endocytosis [75] depending on type of target cells involved. "
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