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1.  Crystal Structure of Marburg Virus VP24 
Journal of Virology  2014;88(10):5859-5863.
The VP24 protein plays an essential, albeit poorly understood role in the filovirus life cycle. VP24 is only 30% identical between Marburg virus and the ebolaviruses. Furthermore, VP24 from the ebolaviruses is immunosuppressive, while that of Marburg virus is not. The crystal structure of Marburg virus VP24, presented here, reveals that although the core is similar between the viral genera, Marburg VP24 is distinguished by a projecting β-shelf and an alternate conformation of the N-terminal polypeptide.
PMCID: PMC4019143  PMID: 24574400
2.  Crystal Structure of the Nipah Virus Phosphoprotein Tetramerization Domain 
Journal of Virology  2014;88(1):758-762.
The Nipah virus phosphoprotein (P) is multimeric and tethers the viral polymerase to the nucleocapsid. We present the crystal structure of the multimerization domain of Nipah virus P: a long, parallel, tetrameric, coiled coil with a small, α-helical cap structure. Across the paramyxoviruses, these domains share little sequence identity yet are similar in length and structural organization, suggesting a common requirement for scaffolding or spatial organization of the functions of P in the virus life cycle.
PMCID: PMC3911761  PMID: 24155387
3.  Ebolavirus VP35 Coats the Backbone of Double-Stranded RNA for Interferon Antagonism 
Journal of Virology  2013;87(18):10385-10388.
Recognition of viral double-stranded RNA (dsRNA) activates interferon production and immune signaling in host cells. Crystal structures of ebolavirus VP35 show that it caps dsRNA ends to prevent sensing by pattern recognition receptors such as RIG-I. In contrast, structures of marburgvirus VP35 show that it primarily coats the dsRNA backbone. Here, we demonstrate that ebolavirus VP35 also coats the dsRNA backbone in solution, although binding to the dsRNA ends probably constitutes the initial binding event.
PMCID: PMC3753998  PMID: 23824825
4.  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.
PMCID: PMC3485981  PMID: 23076242
Ebola virus; IFNα; IFNβ; IFNγ; STAT1; VP24; VP35; X-ray crystallography; crystal structures; ebolavirus; interferon antagonist; karyopherin α proteins
5.  Neutralizing Ebolavirus: structural insights into the envelope glycoprotein and antibodies targeted against it 
The ebolavirus (EBOV) envelope glycoprotein (GP) is solely responsible for viral attachment to, fusion with, and entry of new host cells, and consequently is a major target of vaccine design efforts. Recently determined crystal structures of key antibodies in complex with their EBOV epitopes have provided insights into the molecular architecture of GP and defined likely hotspots for viral neutralization. In this review, we discuss the structural basis for antibody-mediated neutralization of ebolavirus and its implications for novel therapeutic or vaccine strategies.
PMCID: PMC2759674  PMID: 19559599
6.  Complement Is Activated by IgG Hexamers Assembled at the Cell Surface 
Science (New York, N.Y.)  2014;343(6176):1260-1263.
Complement activation by antibodies bound to pathogens, tumors, and self antigens is a critical feature of natural immune defense, a number of disease processes, and immunotherapies. How antibodies activate the complement cascade, however, is poorly understood. We found that specific noncovalent interactions between Fc segments of immunoglobulin G (IgG) antibodies resulted in the formation of ordered antibody hexamers after antigen binding on cells. These hexamers recruited and activated C1, the first component of complement, thereby triggering the complement cascade. The interactions between neighboring Fc segments could be manipulated to block, reconstitute, and enhance complement activation and killing of target cells, using all four human IgG isotypes. We offer a general model for understanding antibody-mediated complement activation and the design of antibody therapeutics with enhanced efficacy.
PMCID: PMC4250092  PMID: 24626930
7.  Filovirus RefSeq Entries: Evaluation and Selection of Filovirus Type Variants, Type Sequences, and Names 
Kuhn, Jens H. | Andersen, Kristian G. | Bào, Yīmíng | Bavari, Sina | Becker, Stephan | Bennett, Richard S. | Bergman, Nicholas H. | Blinkova, Olga | Bradfute, Steven | Brister, J. Rodney | Bukreyev, Alexander | Chandran, Kartik | Chepurnov, Alexander A. | Davey, Robert A. | Dietzgen, Ralf G. | Doggett, Norman A. | Dolnik, Olga | Dye, John M. | Enterlein, Sven | Fenimore, Paul W. | Formenty, Pierre | Freiberg, Alexander N. | Garry, Robert F. | Garza, Nicole L. | Gire, Stephen K. | Gonzalez, Jean-Paul | Griffiths, Anthony | Happi, Christian T. | Hensley, Lisa E. | Herbert, Andrew S. | Hevey, Michael C. | Hoenen, Thomas | Honko, Anna N. | Ignatyev, Georgy M. | Jahrling, Peter B. | Johnson, Joshua C. | Johnson, Karl M. | Kindrachuk, Jason | Klenk, Hans-Dieter | Kobinger, Gary | Kochel, Tadeusz J. | Lackemeyer, Matthew G. | Lackner, Daniel F. | Leroy, Eric M. | Lever, Mark S. | Mühlberger, Elke | Netesov, Sergey V. | Olinger, Gene G. | Omilabu, Sunday A. | Palacios, Gustavo | Panchal, Rekha G. | Park, Daniel J. | Patterson, Jean L. | Paweska, Janusz T. | Peters, Clarence J. | Pettitt, James | Pitt, Louise | Radoshitzky, Sheli R. | Ryabchikova, Elena I. | Saphire, Erica Ollmann | Sabeti, Pardis C. | Sealfon, Rachel | Shestopalov, Aleksandr M. | Smither, Sophie J. | Sullivan, Nancy J. | Swanepoel, Robert | Takada, Ayato | Towner, Jonathan S. | van der Groen, Guido | Volchkov, Viktor E. | Volchkova, Valentina A. | Wahl-Jensen, Victoria | Warren, Travis K. | Warfield, Kelly L. | Weidmann, Manfred | Nichol, Stuart T.
Viruses  2014;6(9):3663-3682.
Sequence determination of complete or coding-complete genomes of viruses is becoming common practice for supporting the work of epidemiologists, ecologists, virologists, and taxonomists. Sequencing duration and costs are rapidly decreasing, sequencing hardware is under modification for use by non-experts, and software is constantly being improved to simplify sequence data management and analysis. Thus, analysis of virus disease outbreaks on the molecular level is now feasible, including characterization of the evolution of individual virus populations in single patients over time. The increasing accumulation of sequencing data creates a management problem for the curators of commonly used sequence databases and an entry retrieval problem for end users. Therefore, utilizing the data to their fullest potential will require setting nomenclature and annotation standards for virus isolates and associated genomic sequences. The National Center for Biotechnology Information’s (NCBI’s) RefSeq is a non-redundant, curated database for reference (or type) nucleotide sequence records that supplies source data to numerous other databases. Building on recently proposed templates for filovirus variant naming [ ()////-], we report consensus decisions from a majority of past and currently active filovirus experts on the eight filovirus type variants and isolates to be represented in RefSeq, their final designations, and their associated sequences.
PMCID: PMC4189044  PMID: 25256396
Bundibugyo virus; cDNA clone; cuevavirus; Ebola; Ebola virus; ebolavirus; filovirid; Filoviridae; filovirus; genome annotation; ICTV; International Committee on Taxonomy of Viruses; Lloviu virus; Marburg virus; marburgvirus; mononegavirad; Mononegavirales; mononegavirus; Ravn virus; RefSeq; Reston virus; reverse genetics; Sudan virus; Taï Forest virus; virus classification; virus isolate; virus nomenclature; virus strain; virus taxonomy; virus variant
8.  Structural basis for ebolavirus matrix assembly and budding; protein plasticity allows multiple functions 
Cell  2013;154(4):763-774.
Proteins, particularly viral proteins, can be multifunctional, but the mechanism(s) behind this trait are not fully understood. Here, we illustrate through multiple crystal structures, biochemistry and cellular microscopy that VP40 rearranges into different structures, each with a distinct function required for the ebolavirus life cycle. A butterfly-shaped VP40 dimer trafficks to the cellular membrane. There, electrostatic interactions trigger rearrangement of the polypeptide into a linear hexamer. These hexamers construct a multi-layered, filamentous matrix structure that is critical for budding and resembles tomograms of authentic virions. A third structure of VP40, formed by a different rearrangement, is not involved in virus assembly, but instead uniquely binds RNA to regulate viral transcription inside infected cells. These results provide a functional model for ebolavirus matrix assembly and the other roles of VP40 in the virus life cycle, and demonstrate how a single, wild-type, unmodified polypeptide can assemble into different structures for different functions.
PMCID: PMC4138722  PMID: 23953110
Archives of virology  2013;158(6):1425-1432.
The International Committee on Taxonomy of Viruses (ICTV) organizes the classification of viruses into taxa, but is not responsible for the nomenclature for taxa members. International experts groups, such as the ICTV Study Groups, recommend the classification and naming of viruses and their strains, variants, and isolates. The ICTV Filoviridae Study Group has recently introduced an updated classification and nomenclature for filoviruses. Subsequently, and together with numerous other filovirus experts, a consistent nomenclature for their natural genetic variants and isolates was developed that aims at simplifying the retrieval of sequence data from electronic databases. This is a first important step toward a viral genome annotation standard as sought by the US National Center for Biotechnology Information (NCBI). Here, this work is extended to include filoviruses obtained in the laboratory by artificial selection through passage in laboratory hosts. The previously developed template for natural filovirus genetic variant naming ( ///-) is retained, but it is proposed to adapt the type of information added to each field for laboratory animal-adapted variants. For instance, the full-length designation of an Ebola virus Mayinga variant adapted at the State Research Center for Virology and Biotechnology “Vector” to cause disease in guinea pigs after seven passages would be akin to “Ebola virus VECTOR/C.porcellus-lab/COD/1976/Mayinga-GPA-P7”. As was proposed for the names of natural filovirus variants, we suggest using the full-length designation in databases, as well as in the method section of publications. Shortened designations (such as “EBOV VECTOR/C.por/COD/76/May-GPA-P7”) and abbreviations (such as “EBOV/May-GPA-P7”) could be used in the remainder of the text depending on how critical it is to convey information contained in the full-length name. “EBOV” would suffice if only one EBOV strain/variant/isolate is addressed.
PMCID: PMC3669655  PMID: 23358612
cuevavirus; Ebola; Ebola virus; ebolavirus; filovirid; Filoviridae; filovirus; genome annotation; ICTV; International Committee on Taxonomy of Viruses; Lloviu virus; Marburg virus; marburgvirus; mononegavirad; Mononegavirales; mononegavirus; virus classification; virus isolate; virus nomenclature; virus strain; virus taxonomy; virus variant
10.  Structural insights into RNA encapsidation and helical assembly of the Toscana virus nucleoprotein 
Nucleic Acids Research  2014;42(9):6025-6037.
Toscana virus is an emerging bunyavirus in Mediterranean Europe where it accounts for 80% of pediatric meningitis cases during the summer. The negative-strand ribonucleic acid (RNA) genome of the virus is wrapped around the virally encoded nucleoprotein N to form the ribonucleoprotein complex (RNP). We determined crystal structures of hexameric N alone (apo) and in complex with a nonameric single-stranded RNA. RNA is sequestered in a sequence-independent fashion in a deep groove inside the hexamer. At the junction between two adjacent copies of Ns, RNA binding induced an inter-subunit rotation, which opened the RNA-binding tunnel and created a new assembly interface at the outside of the hexamer. Based on these findings, we suggest a structural model for how binding of RNA to N promotes the formation of helical RNPs, which are a characteristic hallmark of many negative-strand RNA viruses.
PMCID: PMC4027202  PMID: 24688060
11.  Structure of the Reston ebolavirus VP30 C-terminal domain 
The crystal structure of the Reston ebolavirus VP30 C-terminal domain shows a rotated interface in comparison to the previous structure of the Zaire ebolavirus VP30 C-terminal domain.
The ebolaviruses can cause severe hemorrhagic fever. Essential to the ebolavirus life cycle is the protein VP30, which serves as a transcriptional cofactor. Here, the crystal structure of the C-terminal, NP-binding domain of VP30 from Reston ebolavirus is presented. Reston VP30 and Ebola VP30 both form homodimers, but the dimeric interfaces are rotated relative to each other, suggesting subtle inherent differences or flexibility in the dimeric interface.
PMCID: PMC3976061  PMID: 24699737
ebolaviruses; Reston ebolavirus; VP30 C-terminal domain
12.  Lassa Fever in Post-Conflict Sierra Leone 
Lassa fever (LF), an often-fatal hemorrhagic disease caused by Lassa virus (LASV), is a major public health threat in West Africa. When the violent civil conflict in Sierra Leone (1991 to 2002) ended, an international consortium assisted in restoration of the LF program at Kenema Government Hospital (KGH) in an area with the world's highest incidence of the disease.
Methodology/Principal Findings
Clinical and laboratory records of patients presenting to the KGH Lassa Ward in the post-conflict period were organized electronically. Recombinant antigen-based LF immunoassays were used to assess LASV antigenemia and LASV-specific antibodies in patients who met criteria for suspected LF. KGH has been reestablished as a center for LF treatment and research, with over 500 suspected cases now presenting yearly. Higher case fatality rates (CFRs) in LF patients were observed compared to studies conducted prior to the civil conflict. Different criteria for defining LF stages and differences in sensitivity of assays likely account for these differences. The highest incidence of LF in Sierra Leone was observed during the dry season. LF cases were observed in ten of Sierra Leone's thirteen districts, with numerous cases from outside the traditional endemic zone. Deaths in patients presenting with LASV antigenemia were skewed towards individuals less than 29 years of age. Women self-reporting as pregnant were significantly overrepresented among LASV antigenemic patients. The CFR of ribavirin-treated patients presenting early in acute infection was lower than in untreated subjects.
Lassa fever remains a major public health threat in Sierra Leone. Outreach activities should expand because LF may be more widespread in Sierra Leone than previously recognized. Enhanced case finding to ensure rapid diagnosis and treatment is imperative to reduce mortality. Even with ribavirin treatment, there was a high rate of fatalities underscoring the need to develop more effective and/or supplemental treatments for LF.
Author Summary
Lassa fever (LF) is a major public health threat in West Africa. After the violent civil conflict in Sierra Leone (1991 to 2002) ended, the LF research program at Kenema Government Hospital (KGH) was reestablished. Higher CFRs in LF patients were observed compared to studies conducted prior to the civil conflict. The criteria used for defining the stages of LF and differences in sensitivity of the assays used likely account for these differences. LF may be more widespread in Sierra Leone than recognized previously. Peak presentation of LF cases occurs in the dry season, which is consistent with previous studies. Our studies also confirmed reports conducted prior to the civil conflict that indicate that infants, children, young adults, and pregnant women are disproportionately impacted by LF. High fatality rates were observed among both ribavirin treated and untreated patients, which underscores then need for better LF treatments.
PMCID: PMC3961205  PMID: 24651047
13.  Virus nomenclature below the species level: a standardized nomenclature for natural variants of viruses assigned to the family Filoviridae 
Archives of virology  2012;158(1):301-311.
The task of international expert groups is to recommend the classification and naming of viruses. The ICTV Filoviridae Study Group and other experts have recently established an almost consistent classification and nomenclature for filoviruses. Here, further guidelines are suggested to include their natural genetic variants. First, this term is defined. Second, a template for full-length virus names (such as “Ebola virus H.sapiens-tc/COD/1995/Kikwit-9510621”) is proposed. These names contain information on the identity of the virus (e.g., Ebola virus), isolation host (e.g., members of the species Homo sapiens), sampling location (e.g., Democratic Republic of the Congo (COD)), sampling year, genetic variant (e.g., Kikwit), and isolate (e.g., 9510621). Suffixes are proposed for individual names that clarify whether a given genetic variant has been characterized based on passage zero material (-wt), has been passaged in tissue/cell culture (-tc), is known from consensus sequence fragments only (-frag), or does (most likely) not exist anymore (-hist). We suggest that these comprehensive names are to be used specifically in the methods section of publications. Suitable abbreviations, also proposed here, could then be used throughout the text, while the full names could be used again in phylograms, tables, or figures if the contained information aids the interpretation of presented data. The proposed system is very similar to the well-known influenzavirus nomenclature and the nomenclature recently proposed for rotaviruses. If applied consistently, it would considerably simplify retrieval of sequence data from electronic databases and be a first important step toward a viral genome annotation standard as sought by the National Center for Biotechnology Information (NCBI). Furthermore, adoption of this nomenclature would increase the general understanding of filovirus-related publications and presentations and improve figures such as phylograms, alignments, and diagrams. Most importantly, it would counter the increasing confusion in genetic variant naming due to the identification of ever more sequences through technological breakthroughs in high-throughput sequencing and environmental sampling.
PMCID: PMC3535543  PMID: 23001720
cuevavirus; Ebola; Ebola virus; ebolavirus; filovirid; Filoviridae; filovirus; genome annotation; ICTV; International Committee on Taxonomy of Viruses; Lloviu virus; Marburg virus; marburgvirus; mononegavirad; Mononegavirales; mononegavirus; virus classification; virus isolate; virus nomenclature; virus strain; virus taxonomy; virus variant
14.  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.
PMCID: PMC3866354
15.  Two Synthetic Antibodies that Recognize and Neutralize Distinct Proteolytic Forms of the Ebola Virus Envelope Glycoprotein 
Ebola Virus (EBOV) is a highly pathogenic member of the family Filoviridae of viruses that causes severe hemorrhagic fever. Infection proceeds through fusion of the host cell and viral membranes, a process that is mediated by the viral envelope glycoprotein (GP). Following endosomal uptake, a key step in viral entry is the proteolytic cleavage of GP by host endosomal cysteine proteases. Cleavage exposes a binding site for the host cell receptor Niemann-Pick C1 (NPC1) and may induce conformational changes in GP leading to membrane fusion. However, the precise details of the structural changes in GP associated with proteolysis and the role of these changes in viral entry have not been established. Here, we have employed synthetic antibody technology to identify antibodies targeting EBOV GP prior to and following proteolysis (i.e. in the “uncleaved” [GPUNCL] and “cleaved” [GPCL] forms). We identified antibodies with distinct recognition profiles: FabCL bound preferentially to GPCL (EC50 = 1.7 nM), whereas FabUNCL bound specifically to GPUNCL (EC50 = 75 nM). Neutralization assays with GP-containing pseudotyped viruses indicated that these antibodies inhibited GPCL or GPUNCL mediated viral entry with specificity matching their recognition profiles (IC50: 87 nM for IgGCL; 1 μM for FabUNCL). Competition ELISAs indicate that FabCL binds an epitope distinct from that of KZ52, a well-characterized EBOV GP antibody, and from that of the luminal domain of NPC1. The binding epitope of FabUNCL was also distinct from that of KZ52, suggesting that FabUNCL binds a novel neutralization epitope on GPUNCL. Furthermore, the neutralizing ability of FabCL suggests that there are targets on GPCL available for neutralization. This work showcases the applicability of synthetic antibody technology to the study of viral membrane fusion, and provides new tools for dissecting intermediates of EBOV entry.
PMCID: PMC3684266  PMID: 23111988
Ebola Virus; Filovirus; Viral Membrane Fusion; Synthetic Antibodies; Antibody Engineering; Phage Display
16.  Hiding the evidence: two strategies for innate immune evasion by hemorrhagic fever viruses 
Current opinion in virology  2012;2(2):151-156.
The innate immune system is one of the first lines of defense against invading pathogens. Pathogens have, in turn, evolved different strategies to counteract these responses. Recent studies have illuminated how the hemorrhagic fever viruses Ebola and Lassa fever prevent host sensing of double-stranded RNA (dsRNA), a key hallmark of viral infection. The ebolavirus protein VP35 adopts a unique bimodal configuration to mask key cellular recognition sites on dsRNA. Conversely, the Lassa fever virus nucleoprotein, NP, actually digests the dsRNA signature. Collectively, these structural and functional studies shed new light on the mechanisms of pathogenesis of these viruses and provide new targets for therapeutic intervention.
PMCID: PMC3758253  PMID: 22482712
17.  Marburg Virus VP35 Can Both Fully Coat the Backbone and Cap the Ends of dsRNA for Interferon Antagonism 
PLoS Pathogens  2012;8(9):e1002916.
Filoviruses, including Marburg virus (MARV) and Ebola virus (EBOV), cause fatal hemorrhagic fever in humans and non-human primates. All filoviruses encode a unique multi-functional protein termed VP35. The C-terminal double-stranded (ds)RNA-binding domain (RBD) of VP35 has been implicated in interferon antagonism and immune evasion. Crystal structures of the VP35 RBD from two ebolaviruses have previously demonstrated that the viral protein caps the ends of dsRNA. However, it is not yet understood how the expanses of dsRNA backbone, between the ends, are masked from immune surveillance during filovirus infection. Here, we report the crystal structure of MARV VP35 RBD bound to dsRNA. In the crystal structure, molecules of dsRNA stack end-to-end to form a pseudo-continuous oligonucleotide. This oligonucleotide is continuously and completely coated along its sugar-phosphate backbone by the MARV VP35 RBD. Analysis of dsRNA binding by dot-blot and isothermal titration calorimetry reveals that multiple copies of MARV VP35 RBD can indeed bind the dsRNA sugar-phosphate backbone in a cooperative manner in solution. Further, MARV VP35 RBD can also cap the ends of the dsRNA in solution, although this arrangement was not captured in crystals. Together, these studies suggest that MARV VP35 can both coat the backbone and cap the ends, and that for MARV, coating of the dsRNA backbone may be an essential mechanism by which dsRNA is masked from backbone-sensing immune surveillance molecules.
Author Summary
Filoviruses, Marburg virus and five ebolaviruses, cause severe hemorrhagic fever that is characterized by suppression of the innate immune system. Important to immunosuppression is the viral protein VP35, which binds to and masks double-stranded (ds)RNA, a key signature of virus infection that is recognized by host sentry proteins like RIG-I and MDA-5. Previous crystal structures of VP35 from two ebolaviruses showed it to form an asymmetric dimer to cap the ends of dsRNA molecules. However, the question remained whether VP35 could mask remaining lengths of dsRNA between the ends from immune surveillance. Here we present the crystal structure of the dsRNA-binding domain (RBD) of Marburg virus VP35, alone and in complex with dsRNA. This crystal structure presents a very different arrangement of VP35s on dsRNA. Rather than binding only the ends, the Marburg virus VP35s spiral around the dsRNA backbone, continuously coating it. Additional biochemical experiments indicate that this continuous coating occurs in solution, and that like the ebolaviruses, Marburg virus VP35 is also able to cap the dsRNA ends, even though this was not apparent in the crystal structure. Together, this work illustrates how Marburg virus VP35 prevents recognition of dsRNA by backbone-sensing immune sentry molecules and provides an additional avenue for antiviral development.
PMCID: PMC3441732  PMID: 23028316
18.  Structure of an Antibody in Complex with Its Mucin Domain Linear Epitope That Is Protective against Ebola Virus 
Journal of Virology  2012;86(5):2809-2816.
Antibody 14G7 is protective against lethal Ebola virus challenge and recognizes a distinct linear epitope in the prominent mucin-like domain of the Ebola virus glycoprotein GP. The structure of 14G7 in complex with its linear peptide epitope has now been determined to 2.8 Å. The structure shows that this GP sequence forms a tandem β-hairpin structure that binds deeply into a cleft in the antibody-combining site. A key threonine at the apex of one turn is critical for antibody interaction and is conserved among all Ebola viruses. This work provides further insight into the mechanism of protection by antibodies that target the protruding, highly accessible mucin-like domain of Ebola virus and the structural framework for understanding and characterizing candidate immunotherapeutics.
PMCID: PMC3302272  PMID: 22171276
19.  Structural Basis for the dsRNA Specificity of the Lassa Virus NP Exonuclease 
PLoS ONE  2012;7(8):e44211.
Lassa virus causes hemorrhagic fever characterized by immunosuppression. The nucleoprotein of Lassa virus, termed NP, binds the viral genome. It also has an additional enzymatic activity as an exonuclease that specifically digests double-stranded RNA (dsRNA). dsRNA is a strong signal to the innate immune system of viral infection. Digestion of dsRNA by the NP exonuclease activity appears to cause suppression of innate immune signaling in the infected cell. Although the fold of the NP enzyme is conserved and the active site completely conserved with other exonucleases in its DEDDh family, NP is atypical among exonucleases in its preference for dsRNA and its strict specificity for one substrate. Here, we present the crystal structure of Lassa virus NP in complex with dsRNA. We find that unlike the exonuclease in Klenow fragment, the double-stranded nucleic acid in complex with Lassa NP remains base-paired instead of splitting, and that binding of the paired complementary strand is achieved by “relocation” of a basic loop motif from its typical exonuclease position. Further, we find that just one single glycine that contacts the substrate strand and one single tyrosine that stacks with a base of the complementary, non-substrate strand are responsible for the unique substrate specificity. This work thus provides templates for development of antiviral drugs that would be specific for viral, rather than host exonucleases of similar fold and active site, and illustrates how a very few amino acid changes confer alternate specificity and biological phenotype to an enzyme.
PMCID: PMC3429428  PMID: 22937163
20.  Cathepsin Cleavage Potentiates the Ebola Virus Glycoprotein To Undergo a Subsequent Fusion-Relevant Conformational Change 
Journal of Virology  2012;86(1):364-372.
Cellular entry of Ebola virus (EBOV), a deadly hemorrhagic fever virus, is mediated by the viral glycoprotein (GP). The receptor-binding subunit of GP must be cleaved (by endosomal cathepsins) in order for entry and infection to proceed. Cleavage appears to proceed through 50-kDa and 20-kDa intermediates, ultimately generating a key 19-kDa core. How 19-kDa GP is subsequently triggered to bind membranes and induce fusion remains a mystery. Here we show that 50-kDa GP cannot be triggered to bind to liposomes in response to elevated temperature but that 20-kDa and 19-kDa GP can. Importantly, 19-kDa GP can be triggered at temperatures ∼10°C lower than 20-kDa GP, suggesting that it is the most fusion ready form. Triggering by heat (or urea) occurs only at pH 5, not pH 7.5, and involves the fusion loop, as a fusion loop mutant is defective in liposome binding. We further show that mild reduction (preferentially at low pH) triggers 19-kDa GP to bind to liposomes, with the wild-type protein being triggered to a greater extent than the fusion loop mutant. Moreover, mild reduction inactivates pseudovirion infection, suggesting that reduction can also trigger 19-kDa GP on virus particles. Our results support the hypothesis that priming of EBOV GP, specifically to the 19-kDa core, potentiates GP to undergo subsequent fusion-relevant conformational changes. Our findings also indicate that low pH and an additional endosomal factor (possibly reduction or possibly a process mimicked by reduction) act as fusion triggers.
PMCID: PMC3255896  PMID: 22031933
21.  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.
PMCID: PMC3230659  PMID: 22101933
22.  Structural Basis for Differential Neutralization of Ebolaviruses 
Viruses  2012;4(4):447-470.
There are five antigenically distinct ebolaviruses that cause hemorrhagic fever in humans or non-human primates (Ebola virus, Sudan virus, Reston virus, Taï Forest virus, and Bundibugyo virus). The small handful of antibodies known to neutralize the ebolaviruses bind to the surface glycoprotein termed GP1,2. Curiously, some antibodies against them are known to neutralize in vitro but not protect in vivo, whereas other antibodies are known to protect animal models in vivo, but not neutralize in vitro. A detailed understanding of what constitutes a neutralizing and/or protective antibody response is critical for development of novel therapeutic strategies. Here, we show that paradoxically, a lower affinity antibody with restricted access to its epitope confers better neutralization than a higher affinity antibody against a similar epitope, suggesting that either subtle differences in epitope, or different characteristics of the GP1,2 molecules themselves, confer differential neutralization susceptibility. Here, we also report the crystal structure of trimeric, prefusion GP1,2 from the original 1976 Boniface variant of Sudan virus complexed with 16F6, the first antibody known to neutralize Sudan virus, and compare the structure to that of Sudan virus, variant Gulu. We discuss new structural details of the GP1-GP2 clamp, thermal motion of various regions in GP1,2 across the two viruses visualized, details of differential interaction of the crystallized neutralizing antibodies, and their relevance for virus neutralization.
PMCID: PMC3347318  PMID: 22590681
Filovirus; Ebola; ebolavirus; Sudan virus; neutralization: glycoprotein; antibodies; structure
23.  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.
PMCID: PMC3285596  PMID: 22383882
24.  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.
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
25.  An efficient platform for screening expression and crystallization of glycoproteins produced in human cells 
Nature protocols  2009;4(4):592-604.
Glycoproteins mediate multiple, diverse and critical cellular functions, that are desirable to explore by structural analysis. However, structure determination of these molecules has been hindered by difficulties expressing milligram quantities of stable, homogeneous protein and in determining, which modifications will yield samples amenable to structural studies. We describe a platform proven effective for rapidly screening expression and crystallization of challenging glycoprotein targets produced in mammalian cells. Here, multiple glycoprotein constructs are produced in parallel by transient expression of adherent human embryonic kidney (HEK) 293T cells and subsequently screened in small quantities for crystallization by microfluidic free interface diffusion. As a result, recombinant proteins are produced and processed in a native, mammalian environment and crystallization screening can be accomplished with as little as 65 μg of protein. Moreover, large numbers of constructs can be screened for expression and crystallization and scaled up for structural studies in a matter of five weeks.
PMCID: PMC2911120  PMID: 19373230

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