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Alan D T Barrett - One of the best experts on this subject based on the ideXlab platform.
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characterization of a dengue type specific epitope on dengue 3 Virus Envelope Protein domain iii
Journal of General Virology, 2010Co-Authors: Kiyohiko Matsui, Gregory D Gromowski, Alan D T BarrettAbstract:Dengue Virus (DENV) is a mosquito-borne disease caused by four genetically and serologically related Viruses termed DENV-1, -2, -3 and -4. The DENV Envelope (E) Protein ectodomain can be divided into three structural domains designated ED1, ED2 and ED3. The ED3 domain contains DENV type-specific and DENV complex-reactive antigenic sites. To date, nearly all antigenic studies on the E Protein have focused on DENV-2. In this study, the epitope recognized by a DENV-3 type-specific monoclonal antibody (mAb 14A4-8) was mapped to the DENV-3 ED3 domain using a combination of physical and biological techniques. Epitope mapping revealed that amino acid residues V305, L306, K308, E309, V310, K325, A329, G381 and I387 were critical for the binding of mAb 14A4-8 and amino acid residues T303, K307, K386, W389 and R391 were peripheral residues for this epitope. The location of the mAb 14A4-8 epitope overlaps with the DENV complex-reactive antigenic site in the DENV-3 ED3 domain.
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characterization of dengue complex reactive epitopes on dengue 3 Virus Envelope Protein domain iii
Virology, 2009Co-Authors: Kiyohiko Matsui, Gregory D Gromowski, Amy J Schuh, Ching J Lee, Alan D T BarrettAbstract:Abstract The disease dengue (DEN) is caused by four genetically and serologically related Viruses termed DENV-1, -2, -3, and -4. The DENV Envelope (E) Protein ectodomain can be divided into three structural domains designated ED1, ED2, and ED3. The ED3 contains the DENV type-specific and DENV complex-reactive (epitopes shared by DENV 1–4) antigenic sites. In this study the epitopes recognized by four DENV complex-reactive monoclonal antibodies (MAbs) with neutralizing activity were mapped on the DENV-3 ED3 using a combination of physical and biological techniques. Amino acid residues L306, K308, G381, I387, and W389 were critical for all four MAbs, with residues V305, E309, V310, K325, D382, A384, K386, and R391 being critical for various subsets of the MAbs. A previous study by our group (Gromowski, G.D., Barrett, N.D., Barrett, A.D., 2008. Characterization of dengue complex-specific neutralizing epitopes on the Envelope Protein domain III of dengue 2 Virus. J. Virol 82, 8828–8837) characterized the same panel of MAbs with DENV-2. The location of the DENV complex-reactive antigenic site on the DENV-2 and DENV-3 ED3s is similar; however, the critical residues for binding are not identical. Overall, this indicates that the DENV complex-reactive antigenic site on ED3 may be similar in location, but the surprising result is that DENV 2 and 3 exhibit unique sets of residues defining the energetics of interaction to the same panel of MAbs. These results imply that the amino acid sequences of DENV define a unique interaction network among these residues in spite of the fact that all flaviVirus ED3s to date assume the same structural fold.
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structure of the Envelope Protein domain iii of omsk hemorrhagic fever Virus
Virology, 2006Co-Authors: David E Volk, Leonard Chavez, Michael R Holbrook, David W. C. Beasley, Alan D T Barrett, David G. GorensteinAbstract:We have solved the NMR solution structure of domain III from the Omsk hemorrhagic fever Virus Envelope Protein and report the first sequencing of the Guriev strain of this Virus. Important structural differences between tick-borne flaviViruses, such as OHFV and TBE, and mosquito-borne flaviViruses, such as West Nile Virus, are discussed.
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identification of neutralizing epitopes within structural domain iii of the west nile Virus Envelope Protein
Journal of Virology, 2002Co-Authors: David W. C. Beasley, Alan D T BarrettAbstract:Using a panel of neutralizing monoclonal antibodies, we have mapped epitopes in domain III of the Envelope Protein of the New York strain of West Nile Virus. The ability of monoclonal antibodies that recognize these epitopes to neutralize Virus appeared to differ between lineage I and II West Nile Virus strains, and epitopes were located on the upper surface of domain III at residues E307, E330, and E332.
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amino acid substitution s in the stem anchor region of langat Virus Envelope Protein attenuates mouse neurovirulence
Virology, 2001Co-Authors: Michael R Holbrook, Robert E Shope, Alan D T BarrettAbstract:Abstract The identification of variants that are unable to bind membrane receptor preparations (MRPs) has previously been shown to select attenuated yellow fever and Japanese encephalitis Viruses. In this study, this methodology has been extended to the tick-borne serocomplex of flaviViruses. Langat (LGT) Virus strain TP21 was bound to mouse or human brain MRPs and Viruses that escaped binding were isolated and characterized. In addition, variant Viruses escaping neutralization by the monoclonal antibody (MAb) 9F9 were also isolated. All of the variant Viruses were attenuated for mouse neurovirulence (≥13-fold). Sequence analysis of the prM/E region of the variant Viruses identified mutations within the stem-anchor region of the E Protein in variants isolated following incubation with mouse or human brain MRPs at a pH ≥ 7.0. The MAb 9F9 variants and MRP variants isolated at pH 5.0, which should induce a conformational shift in the viral E Protein, had nearly identical mutations in the prM/M Protein immediately N-terminal to the prM/E cleavage site. MAb 9F9 neutralized none of the variant Viruses and hemagglutination inhibition assays suggest that the variant Virus surface Proteins have slightly different conformations compared to the parental Virus. These data support previous work indicating that the stem-anchor region of the E Protein is important to the surface architecture of the tick-borne flaviViruses. In addition, this study demonstrates that the M Protein is at least partially solvent accessible on the virion surface and that the M Protein plays a role in maintaining the conformation of the M/E surface complex.
Lukas K Tamm - One of the best experts on this subject based on the ideXlab platform.
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structure of the ebola Virus Envelope Protein mper tm domain and its interaction with the fusion loop explains their fusion activity
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Jinwoo Lee, David A Nyenhuis, Elizabeth A Nelson, David S Cafiso, Judith M White, Lukas K TammAbstract:EbolaVirus (EBOV), an Enveloped filamentous RNA Virus causing severe hemorrhagic fever, enters cells by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the EBOV Envelope glycoProtein GP, which consists of subunits GP1 and GP2. GP1 binds to cellular receptors, including Niemann-Pick C1 (NPC1) Protein, and GP2 is responsible for low pH-induced membrane fusion. Proteolytic cleavage and NPC1 binding at endosomal pH lead to conformational rearrangements of GP2 that include exposing the hydrophobic fusion loop (FL) for insertion into the cellular target membrane and forming a six-helix bundle structure. Although major portions of the GP2 structure have been solved in pre- and postfusion states and although current models place the transmembrane (TM) and FL domains of GP2 in close proximity at critical steps of membrane fusion, their structures in membrane environments, and especially interactions between them, have not yet been characterized. Here, we present the structure of the membrane proximal external region (MPER) connected to the TM domain: i.e., the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry, highlighting the functional importance of the MPER/TM–FL interaction in EBOV entry and fusion.
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structure of the ebola Virus Envelope Protein mper tm domain and its interaction with the fusion loop explains their fusion activity
Biophysical Journal, 2017Co-Authors: Jinwoo Lee, David A Nyenhuis, Elizabeth A Nelson, David S Cafiso, Judith M White, Lukas K TammAbstract:EbolaVirus (EBOV), an Enveloped filamentous RNA Virus causing severe hemorrhagic fever, enters cells by macropinocytosis and releases its genetic material into the cytoplasm after membrane fusion in a late endosomal compartment. Membrane fusion is governed by the EBOV surface Envelope glycoProtein (GP), which consists of subunits GP1 and GP2. GP1 binds to cellular receptors including Niemann-Pick C1 (NPC1) Protein and GP2 is responsible for membrane fusion at low pH. GP1 undergoes multiple steps of proteolytic cleavage and binds to NPC1 at endosomal pH. GP2 is rearranged in a fashion that exposes the hydrophobic fusion loop (FL) of GP2, which is then inserted into the cellular target membrane, ultimately forming a six-helix bundle structure and resulting in the formation of the fusion pore. Although major portions of the GP2 structure that have been solved in pre- and post-fusion states and the current model places the transmembrane (TM) and FL domains of GP2 in close proximity to each other at critical steps of membrane fusion, their structures in membrane environments and especially interactions between TM and FL have not yet been characterized. Here we present the structure of the membrane proximal external region (MPER) connected to the TM domain, i.e. the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region, not TM region, is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry highlighting the functional importance of the MPER/TM - FL interaction in EBOV entry and fusion.
Franz X Heinz - One of the best experts on this subject based on the ideXlab platform.
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a dna immunization model study with constructs expressing the tick borne encephalitis Virus Envelope Protein e in different physical forms
Journal of Immunology, 1999Co-Authors: Judith H Aberle, Steven L Allison, Karin Stiasny, Christian W Mandl, Stephan W Aberle, Michael Ecker, Rudolf Berger, Franz X HeinzAbstract:We have conducted a DNA immunization study to evaluate how the immune response is influenced by the physical structure and secretion of the expressed Ag. For this purpose, we used a series of plasmid constructs encoding different forms of the Envelope glycoProtein E of the flaviVirus tick-borne encephalitis Virus. These included a secreted recombinant subviral particle, a secreted carboxyl -terminally truncated soluble homodimer, a nonsecreted full-length form, and an inefficiently secreted truncated form. Mice were immunized using both i.m. injection and Gene Gun-mediated application of plasmids. The functional immune response was evaluated by determining specific neutralizing and hemagglutination-inhibiting Ab activities and by challenging the mice with a lethal dose of the Virus. As a measure for the induction of a Th1 and/or Th2 response, we determined specific IgG subclasses and examined IFN-γ, Il-4, and Il-5 induction. The plasmid construct encoding a secreted subviral particle, which carries multiple copies of the protective Ag on its surface, was superior to the other constructs in terms of extent and functionality of the Ab response as well as protection against Virus challenge. As expected, the type of Th response was largely dependent on the mode of application (i.m. vs Gene Gun), but our data show that it was also strongly influenced by the properties of the Ag. Most significantly, the plasmid encoding the particulate form was able to partially overcome the Th2 bias imposed by the Gene Gun, resulting in a balanced Th1/Th2 response.
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mapping of functional elements in the stem anchor region of tick borne encephalitis Virus Envelope Protein e
Journal of Virology, 1999Co-Authors: Steven L Allison, Karin Stiasny, Konrad Stadler, Christian W Mandl, Franz X HeinzAbstract:Envelope Protein E of the flaviVirus tick-borne encephalitis Virus mediates membrane fusion, and the structure of the N-terminal 80% of this 496-amino-acid-long Protein has been shown to differ significantly from that of other viral fusion Proteins. The structure of the carboxy-terminal 20%, the stem-anchor region, is not known. It contains sequences that are important for membrane anchoring, interactions with prM (the precursor of membrane Protein M) during virion assembly, and low-pH-induced structural changes associated with the fusion process. To identify specific functional elements in this region, a series of C-terminal deletion mutants were constructed and the properties of the resulting truncated recombinant E Proteins were examined. Full-length E Proteins and Proteins lacking the second of two predicted transmembrane segments were secreted in a particulate form when coexpressed with prM, whereas deletion of both segments resulted in the secretion of soluble homodimeric E Proteins. Sites located within a predicted α-helical region of the stem (amino acids 431 to 449) and the first membrane-spanning region (amino acids 450 to 472) were found to be important for the stability of the prM-E heterodimer but not essential for prM-mediated intracellular transport and secretion of soluble E Proteins. A separate site in the stem, also corresponding to a predicted α-helix (amino acids 401 to 413), was essential for the conversion of soluble Protein E dimers to a homotrimeric form upon low-pH treatment, a process resembling the transition to the fusogenic state in whole virions. This functional mapping will aid in the understanding of the molecular mechanisms of membrane fusion and Virus assembly.
Daved H Fremont - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the japanese encephalitis Virus Envelope Protein
Journal of Virology, 2012Co-Authors: Vincent C Luca, Jad P Abimansour, Christopher A Nelson, Daved H FremontAbstract:Japanese encephalitis Virus (JEV) is the leading global cause of viral encephalitis. The JEV Envelope Protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-A resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E Protein possesses the three domains characteristic of flaviVirus Envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flaviVirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flaviVirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.
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antibody recognition and neutralization determinants on domains i and ii of west nile Virus Envelope Protein
Journal of Virology, 2006Co-Authors: Theodore Oliphant, Christopher A Nelson, Michael Engle, Grant E Nybakken, Qing Xu, Soila Sukupolvipetty, Anantha Marri, Batel Lachmi, Udy Olshevsky, Daved H FremontAbstract:Previous studies have demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral surface of domain III (DIII) of the West Nile Virus (WNV) Envelope (E) strongly protect against infection in animals. Herein, we observed significantly less efficient neutralization by 89 MAbs that recognized domain I (DI) or II (DII) of WNV E Protein. Moreover, in cells expressing Fc γ receptors, many of the DI- and DII-specific MAbs enhanced infection over a broad range of concentrations. Using yeast surface display of E Protein variants, we identified 25 E Protein residues to be critical for recognition by DI- or DII-specific neutralizing MAbs. These residues cluster into six novel and one previously characterized epitope located on the lateral ridge of DI, the linker region between DI and DIII, the hinge interface between DI and DII, and the lateral ridge, central interface, dimer interface, and fusion loop of DII. Approximately 45% of DI-DII-specific MAbs showed reduced binding with mutations in the highly conserved fusion loop in DII: 85% of these (34 of 40) cross-reacted with the distantly related dengue Virus (DENV). In contrast, MAbs that bound the other neutralizing epitopes in DI and DII showed no apparent cross-reactivity with DENV E Protein. Surprisingly, several of the neutralizing epitopes were located in solvent-inaccessible positions in the context of the available pseudoatomic model of WNV. Nonetheless, DI and DII MAbs protect against WNV infection in mice, albeit with lower efficiency than DIII-specific neutralizing MAbs.
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antibody recognition and neutralization determinants on domains i and ii of west nile Virus Envelope Protein
Journal of Virology, 2006Co-Authors: Theodore Oliphant, Christopher A Nelson, Daved H Fremont, Michael Engle, Grant E Nybakken, Soila Sukupolvipetty, Anantha Marri, Batel Lachmi, Udy Olshevsky, Theodore C PiersonAbstract:Previous studies have demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral surface of domain III (DIII) of the West Nile Virus (WNV) Envelope (E) strongly protect against infection in animals. Herein, we observed significantly less efficient neutralization by 89 MAbs that recognized domain I (DI) or II (DII) of WNV E Protein. Moreover, in cells expressing Fc γ receptors, many of the DI- and DII-specific MAbs enhanced infection over a broad range of concentrations. Using yeast surface display of E Protein variants, we identified 25 E Protein residues to be critical for recognition by DI- or DII-specific neutralizing MAbs. These residues cluster into six novel and one previously characterized epitope located on the lateral ridge of DI, the linker region between DI and DIII, the hinge interface between DI and DII, and the lateral ridge, central interface, dimer interface, and fusion loop of DII. Approximately 45% of DI-DII-specific MAbs showed reduced binding with mutations in the highly conserved fusion loop in DII: 85% of these (34 of 40) cross-reacted with the distantly related dengue Virus (DENV). In contrast, MAbs that bound the other neutralizing epitopes in DI and DII showed no apparent cross-reactivity with DENV E Protein. Surprisingly, several of the neutralizing epitopes were located in solvent-inaccessible positions in the context of the available pseudoatomic model of WNV. Nonetheless, DI and DII MAbs protect against WNV infection in mice, albeit with lower efficiency than DIII-specific neutralizing MAbs.
Jian Song - One of the best experts on this subject based on the ideXlab platform.
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Molecular Basis of Arthritogenic AlphaVirus Receptor MXRA8 Binding to Chikungunya Virus Envelope Protein.
Cell, 2019Co-Authors: Hao Song, Xiyue Jin, Changyao Li, Zhengrong Gao, Haiyuan Wang, Zhennan Zhao, Yan Chai, Fei Yuan, Sheng Liu, Jian SongAbstract:Arthritogenic alphaViruses, such as Chikungunya Virus (CHIKV), cause severe and debilitating rheumatic diseases worldwide, resulting in severe morbidity and economic costs. Recently, MXRA8 was reported as an entry receptor. Here, we present the crystal structures of the mouse MXRA8, human MXRA8 in complex with the CHIKV E Protein, and the cryo-electron microscopy structure of human MXRA8 and CHIKV Virus-like particle. MXRA8 has two Ig-like domains with unique structural topologies. This receptor binds in the "canyon" between two protomers of the E spike on the surface of the virion. The atomic details at the interface between the two binding entities reveal that both the two domains and the hinge region of MXRA8 are involved in interaction with CHIKV E1-E2 residues from two protomers. Notably, the stalk region of MXRA8 is critical for CHIKV Virus entry. This finding provides important information regarding the development of therapeutic countermeasures against those arthritogenic alphaViruses.
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structures of the zika Virus Envelope Protein and its complex with a flaviVirus broadly protective antibody
Cell Host & Microbe, 2016Co-Authors: Lianpan Dai, Hao Song, Jian Song, Yongqiang Deng, Abednego Moki Musyoki, Huijun Cheng, Yanfang Zhang, Yuan Yuan, Joel Haywood, Haixia XiaoAbstract:Zika Virus (ZIKV), a mosquito-borne flaviVirus, is a current global public health concern. The flaviVirus Envelope (E) glycoProtein is responsible for Virus entry and represents a major target of neutralizing antibodies for other flaviViruses. Here, we report the structures of ZIKV E Protein at 2.0 A and in complex with a flaviVirus broadly neutralizing murine antibody 2A10G6 at 3.0 A. ZIKV-E resembles all the known flaviVirus E structures but contains a unique, positively charged patch adjacent to the fusion loop region of the juxtaposed monomer, which may influence host attachment. The ZIKV-E-2A10G6 complex structure reveals antibody recognition of a highly conserved fusion loop. 2A10G6 binds to ZIKV-E with high affinity in vitro and neutralizes currently circulating ZIKV strains in vitro and in mice. The E Protein fusion loop epitope represents a potential candidate for therapeutic antibodies against ZIKV.
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structures of the zika Virus Envelope Protein and its complex with a flaviVirus broadly protective antibody
Cell Host & Microbe, 2016Co-Authors: Lianpan Dai, Hao Song, Jian Song, Yongqiang Deng, Abednego Moki Musyoki, Huijun Cheng, Yanfang Zhang, Yuan Yuan, Xishan Lu, Joel HaywoodAbstract:Zika Virus (ZIKV), a mosquito-borne flaviVirus, is a current global public health concern. The flaviVirus Envelope (E) glycoProtein is responsible for Virus entry and represents a major target of neutralizing antibodies for other flaviViruses. Here, we report the structures of ZIKV E Protein at 2.0 A and in complex with a flaviVirus broadly neutralizing murine antibody 2A10G6 at 3.0 A. ZIKV-E resembles all the known flaviVirus E structures but contains a unique, positively charged patch adjacent to the fusion loop region of the juxtaposed monomer, which may influence host attachment. The ZIKV-E-2A10G6 complex structure reveals antibody recognition of a highly conserved fusion loop. 2A10G6 binds to ZIKV-E with high affinity in vitro and neutralizes currently circulating ZIKV strains in vitro and in mice. The E Protein fusion loop epitope represents a potential candidate for therapeutic antibodies against ZIKV.
