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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    • "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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    Microbes and Infection 12/2014; 17(2). DOI:10.1016/j.micinf.2014.11.012 · 2.86 Impact Factor
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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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    ABSTRACT: Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes.
    PLoS ONE 04/2013; 8(4):e60838. DOI:10.1371/journal.pone.0060838 · 3.23 Impact Factor
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    • "Three GP1-S–S-GP2 units come together to form the homotrimer that protrudes from the virion surface [120,121]. It was reported that the GP1 cleavage is a two-step event: first cathepsin L cleaved GP1 into 20 kDa fragment and after the cleavage with cathepsin B 19 kDa fragment is generated [122]. The translocation of pseudovirions bearing 20 kDa GP into cytoplasm was strongly inhibited by cathepsin B inhibitors, while the entry of pseudovirions bearing 19 kDa GP1 was not [123,124]. "
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    ABSTRACT: Proteinases and their inhibitors play essential functional roles in basic biological processes in both hosts and pathogens. Endo/lysosomal cathepsins participate in immune response in pathogen recognition and elimination. They are essential for both antigen processing and presentation (host adaptive immune response) and activation of endosomal toll like receptors (innate immune response). Pathogens can produce proteases and also natural inhibitors to subvert the host immune response. Several pathogens are sensed through the intracellular pathogen recognition receptors, but only some of them use the host proteolytic system to escape into the cytosol. In this review, I provide an update on the most recent developments regarding the role of proteinases and their inhibitors in the initiation and regulation of immune responses.
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