title: 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.
authors.affiliation: 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.
authors.forename: Chantelle L
authors.initials: CL
authors.lastname: Hood
authors.forename: Jonathan
authors.initials: J
authors.lastname: Abraham
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Boyington
authors.forename: Kwanyee
authors.initials: K
authors.lastname: Leung
authors.forename: Peter D
authors.initials: PD
authors.lastname: Kwong
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Nabel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D056668:Cathepsin L / Q000378:metabolism*; D002460:Cell Line; D029043:Ebolavirus / Q000276:immunology / Q000502:physiology; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization
chemical_list: D057134:Antibodies, Neutralizing; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D056668:Cathepsin L