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1.  Correction: The Ebola Virus Interferon Antagonist VP24 Directly Binds STAT1 and Has a Novel, Pyramidal Fold 
PLoS Pathogens  2013;9(12):10.1371/annotation/360ddc68-0313-4eae-af24-043cc040c52d.
doi:10.1371/annotation/360ddc68-0313-4eae-af24-043cc040c52d
PMCID: PMC3866354
2.  The ebolavirus VP24 interferon antagonist 
Virulence  2012;3(5):440-445.
Suppression during the early phases of the immune system often correlates directly with a fatal outcome for the host. The ebolaviruses, some of the most lethal viruses known, appear to cripple initial stages of the host defense network via multiple distinct paths. Two of the eight viral proteins are critical for immunosuppression. One of these proteins is VP35, which binds double-stranded RNA and antagonizes several antiviral signaling pathways.1,2 The other protein is VP24, which binds transporter molecules to prevent STAT1 translocation.3 A more recent discovery is that VP24 also binds STAT1 directly,4 suggesting that VP24 may operate in at least two separate branches of the interferon pathway. New crystal structures of VP24 derived from pathogenic and nonpathogenic ebolaviruses reveal its novel, pyramidal fold, upon which can be mapped sites required for virulence and for STAT1 binding. These structures of VP24, and new information about its direct binding to STAT1, provide avenues by which we may explore its many roles in the viral life cycle, and reasons for differences in pathogenesis among the ebolaviruses.
doi:10.4161/viru.21302
PMCID: PMC3485981  PMID: 23076242
Ebola virus; IFNα; IFNβ; IFNγ; STAT1; VP24; VP35; X-ray crystallography; crystal structures; ebolavirus; interferon antagonist; karyopherin α proteins
3.  A Shared Structural Solution for Neutralizing Ebolaviruses 
Nature structural & molecular biology  2011;18(12):1424-1427.
Sudan virus (genus ebolavirus) is lethal, yet no monoclonal antibody is known to neutralize it. Here we describe antibody 16F6 that neutralizes Sudan virus and present its structure bound to the trimeric viral glycoprotein. Unexpectedly, the 16F6 epitope overlaps that of KZ52, the only other antibody against the GP1,2 core to be visualized. Further, both antibodies against this key GP1–GP2-bridging epitope neutralize at a post-internalization step, likely fusion.
doi:10.1038/nsmb.2150
PMCID: PMC3230659  PMID: 22101933
4.  The Ebola Virus Interferon Antagonist VP24 Directly Binds STAT1 and Has a Novel, Pyramidal Fold 
PLoS Pathogens  2012;8(2):e1002550.
Ebolaviruses cause hemorrhagic fever with up to 90% lethality and in fatal cases, are characterized by early suppression of the host innate immune system. One of the proteins likely responsible for this effect is VP24. VP24 is known to antagonize interferon signaling by binding host karyopherin α proteins, thereby preventing them from transporting the tyrosine-phosphorylated transcription factor STAT1 to the nucleus. Here, we report that VP24 binds STAT1 directly, suggesting that VP24 can suppress at least two distinct branches of the interferon pathway. Here, we also report the first crystal structures of VP24, derived from different species of ebolavirus that are pathogenic (Sudan) and nonpathogenic to humans (Reston). These structures reveal that VP24 has a novel, pyramidal fold. A site on a particular face of the pyramid exhibits reduced solvent exchange when in complex with STAT1. This site is above two highly conserved pockets in VP24 that contain key residues previously implicated in virulence. These crystal structures and accompanying biochemical analysis map differences between pathogenic and nonpathogenic viruses, offer templates for drug design, and provide the three-dimensional framework necessary for biological dissection of the many functions of VP24 in the virus life cycle.
Author Summary
Ebolaviruses cause severe hemorrhagic fever that is exacerbated by immediate suppression of host immune function. VP24, one of only eight proteins encoded by ebolaviruses, functions in virus replication and assembly, and is thought to contribute to immune suppression by binding to a certain class of molecules called karyopherins to prevent them from transporting a transcription factor termed STAT1. Here we report that VP24 is also able to directly bind STAT1 by itself, and thereby likely contributes to immune suppression by an additional mechanism. Analysis of these multiple roles of VP24 and design of drugs against them have been hindered by the lack of structural information on VP24 and its lack of homology to any other known protein. Hence, here we also present X-ray structures of VP24 derived from two different ebolavirus species that are pathogenic and nonpathogenic to humans. These structures and accompanying deuterium exchange mass spectrometry identify the likely binding site of STAT1 onto VP24, map sites that are conserved or differ between pathogenic and nonpathogenic species, and provide the critical 3D templates by which we may dissect and interpret the many roles that VP24 plays in the virus life cycle.
doi:10.1371/journal.ppat.1002550
PMCID: PMC3285596  PMID: 22383882
5.  Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein 
Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing of the trimeric ebolavirus glycoprotein are described.
The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is responsible for viral attachment, fusion and entry. Knowledge of its structure is important both for understanding ebolavirus entry and for the development of medical interventions. Crystal structures of viral glycoproteins, especially those in their metastable prefusion oligomeric states, can be difficult to achieve given the challenges in production, purification, crystallization and diffraction that are inherent in the heavily glycosylated flexible nature of these types of proteins. The crystal structure of ebolavirus GP in its trimeric prefusion conformation in complex with a human antibody derived from a survivor of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008 ▶), Nature (London), 454, 177–182]. Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing are described. Glycoproteins were produced in human embryonic kidney 293T cells, which allowed rapid screening of constructs and expression of protein in milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted crystallization and a series of deglycosylation strategies, including sugar mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic pathway inhibitors, were attempted to improve the weakly diffracting glyco­protein crystals. The signal-to-noise ratio of the search model for molecular replacement was improved by determining the structure of the uncomplexed Fab. Phase combination with Fab model phases and a selenium anomalous signal, followed by NCS-averaged density modification, resulted in a clear interpretable electron-density map. Model building was assisted by the use of B-value-sharpened electron-density maps and the proper sequence register was confirmed by building alternate sequences using N-linked glycan sites as anchors and secondary-structural predictions.
doi:10.1107/S0907444909032314
PMCID: PMC2777170  PMID: 19923712
glycoproteins; structure determination; difficult structures; antibody complexes; viral proteins; human proteins; tactics to improve diffraction; techniques for phase determination; deglycosylation; model building
6.  Complex of a protective antibody with its Ebola virus GP peptide epitope: unusual features of a Vλx-light chain 
Journal of molecular biology  2007;375(1):202-216.
13F6-1-2 is a murine monoclonal antibody that recognizes the heavily glycosylated mucin-like domain of the Ebola virus virion-attached glycoprotein (GP) and protects animals against lethal viral challenge. Here we present the crystal structure, at 2.0 Å, of 13F6-1-2 in complex with its Ebola virus GP peptide epitope. The GP peptide binds in an extended conformation, anchored primarily by interactions to the heavy chain. Two GP residues, Gln P406 and Arg P409, make extensive side chain hydrogen bond and electrostatic interactions to the antibody and are likely critical for recognition and affinity. The 13F6-1-2 antibody utilizes a rare Vλx light chain. The three light chain complementarity determining regions (CDRs) do not adopt canonical conformations and represent new classes of structures distinct from Vκ and other Vλ light chains. In addition, although Vλx had been thought to confer specificity, all light chain contacts are mediated through germline-encoded residues. This structure of an antibody that protects against the Ebola virus now provides a framework for humanization and development of a post-exposure immunotherapeutic.
doi:10.1016/j.jmb.2007.10.017
PMCID: PMC2173910  PMID: 18005986
Ebola virus; Vλx light chain; glycoprotein; neutralizing antibody; new canonical structures of immunoglobulins; hypervariable loops; complementarity determining region; Fab-peptide complex

Results 1-6 (6)