Chapter 2b: The molecular antigenic structure of the TBEV

2021 ◽  
Author(s):  
Franz-Xaver Heinz ◽  
Karin Stiasny
Keyword(s):  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Lipid Membrane ◽  
Cell Entry ◽  
Antigenic Structure ◽  
E Proteins ◽  
Amino Acid Sequence Level ◽  
Endosomal Membrane ◽  
E Protein ◽  
Golgi Network

TBEV-particles are assembled in an immature, noninfectious form in the endoplasmic reticulum by the envelopment of the viral core (containing the viral RNA) by a lipid membrane associated with two viral proteins, prM and E. Immature particles are transported through the cellular exocytic pathway and conformational changes induced by acidic pH in the trans-Golgi network allow the proteolytic cleavage of prM by furin, a cellular protease, resulting in the release of mature and infectious TBE-virions. The E protein controls cell entry by mediating attachment to as yet ill-defined receptors as well as by low-pH-triggered fusion of the viral and endosomal membrane after uptake by receptor-mediated endocytosis. Because of its key functions in cell entry, the E protein is the primary target of virus neutralizing antibodies, which inhibit these functions by different mechanisms. Although all flavivirus E proteins have a similar overall structure, divergence at the amino acid sequence level is up to 60 percent (e.g. between TBE and dengue viruses), and therefore cross-neutralization as well as (some degree of) cross-protection are limited to relatively closely related flaviviruses, such as those constituting the tick-borne encephalitis serocomplex.

Chapter 2b: The molecular antigenic structure of the TBEV

2020 ◽  
Keyword(s):  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Lipid Membrane ◽  
Cell Entry ◽  
Antigenic Structure ◽  
E Proteins ◽  
Amino Acid Sequence Level ◽  
Endosomal Membrane ◽  
E Protein ◽  
Golgi Network

TBEV-particles are assembled in an immature, noninfectious form in the endoplasmic reticulum by the envelopment of the viral core (containing the viral RNA) by a lipid membrane associated with two viral proteins, prM and E. Immature particles are transported through the cellular exocytic pathway and conformational changes induced by acidic pH in the trans-Golgi network allow the proteolytic cleavage of prM by furin, a cellular protease, resulting in the release of mature and infectious TBE-virions. The E protein controls cell entry by mediating attachment to as yet ill-defined receptors as well as by low-pH-triggered fusion of the viral and endosomal membrane after uptake by receptor-mediated endocytosis. Because of its key functions in cell entry, the E protein is the primary target of virus neutralizing antibodies, which inhibit these functions by different mechanisms. Although all flavivirus E proteins have a similar overall structure, divergence at the amino acid sequence level is up to 60 percent (e.g. between TBE and dengue viruses), and therefore cross-neutralization as well as (some degree of) cross-protection are limited to relatively closely related flaviviruses, such as those constituting the tick-borne encephalitis serocomplex.

Chapter 2b: The molecular and antigenic structure of TBEV

2019 ◽  
Author(s):  
Franz-Xaver Heinz ◽  
Karin Stiasny
Keyword(s):  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Lipid Membrane ◽  
Cell Entry ◽  
Antigenic Structure ◽  
E Proteins ◽  
Amino Acid Sequence Level ◽  
Endosomal Membrane ◽  
E Protein ◽  
Golgi Network

• TBEV-particles are assembled in an immature, noninfectious form in the endoplasmic reticulum by the envelopment of the viral core (containing the viral RNA) by a lipid membrane associated with two viral proteins, prM and E. • Immature particles are transported through the cellular exocytic pathway and conformational changes induced by acidic pH in the trans-Golgi network allow the proteolytic cleavage of prM by furin, a cellular protease, resulting in the release of mature and infectious TBE-virions. • The E protein controls cell entry by mediating attachment to as yet ill-defined receptors as well as by low-pH-triggered fusion of the viral and endosomal membrane after uptake by receptor-mediated endocytosis. • Because of its key functions in cell entry, the E protein is the primary target of virus neutralizing antibodies, which inhibit these functions by different mechanisms. • Although all flavivirus E proteins have a similar overall structure, divergence at the amino acid sequence level is up to 60 percent (e.g. between TBE and dengue viruses), and therefore cross-neutralization as well as (some degree of) cross-protection are limited to relatively closely related flaviviruses, such as those constituting the tick-borne encephalitis serocomplex.

scholarly journals Structural and Thermodynamic Basis of Epitope Binding by Neutralizing and Nonneutralizing Forms of the Anti-HIV-1 Antibody 4E10

2015 ◽  
Vol 89 (23) ◽  
pp. 11975-11989 ◽  
Author(s):  
Edurne Rujas ◽  
Naveed Gulzar ◽  
Koldo Morante ◽  
Kouhei Tsumoto ◽  
Jamie K. Scott ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Membrane Lipids ◽  
Function Analysis ◽  
Antigenic Structure ◽  
Neutralizing Activity ◽  
Anti Hiv ◽  
Hiv 1 ◽  
4E10 Antibody

ABSTRACTThe 4E10 antibody recognizes the membrane-proximal external region (MPER) of the HIV-1 Env glycoprotein gp41 transmembrane subunit, exhibiting one of the broadest neutralizing activities known to date. The neutralizing activity of 4E10 requires solvent-exposed hydrophobic residues at the apex of the complementarity-determining region (CDR) H3 loop, but the molecular basis for this requirement has not been clarified. Here, we report the cocrystal structures and the energetic parameters of binding of a peptide bearing the 4E10-epitope sequence (4E10ep) to nonneutralizing versions of the 4E10 Fab. Nonneutralizing Fabs were obtained by shortening and decreasing the hydrophobicity of the CDR-H3 loop (termed ΔLoop) or by substituting the two tryptophan residues of the CDR-H3 apex with Asp residues (termed WDWD), which also decreases hydrophobicity but preserves the length of the loop. The analysis was complemented by the first crystal structure of the 4E10 Fab in its ligand-free state. Collectively, the data ruled out major conformational changes of CDR-H3 at any stage during the binding process (equilibrium or transition state). Although these mutations did not impact the affinity of wild-type Fab for the 4E10ep in solution, the two nonneutralizing versions of 4E10 were deficient in binding to MPER inserted in the plasma membrane (mimicking the environment faced by the antibodyin vivo). The conclusions of our structure-function analysis strengthen the idea that to exert effective neutralization, the hydrophobic apex of the solvent-exposed CDR-H3 loop must recognize an antigenic structure more complex than just the linear α-helical epitope and likely constrained by the viral membrane lipids.IMPORTANCEThe broadly neutralizing anti-HIV-1 4E10 antibody blocks infection caused by nearly all viral strains and isolates examined thus far. However, 4E10 (or 4E10-like) antibodies are rarely found in HIV-1-infected individuals or elicited through vaccination. Impediments to the design of successful 4E10 immunogens are partly attributed to an incomplete understanding of the structural and binding characteristics of this class of antibodies. Since the broadly neutralizing activity of 4E10 is abrogated by mutations of the tip of the CDR-H3, we investigated their impact on binding of the MPER-epitope at the atomic and energetic levels. We conclude that the difference between neutralizing and nonneutralizing antibodies of 4E10 is neither structural nor energetic but is related to the capacity to recognize the HIV-1 gp41 epitope inserted in biological membranes. Our findings strengthen the idea that to elicit similar neutralizing antibodies, the suitable MPER vaccine must be “delivered” in a membrane environment.

scholarly journals Impact of Quaternary Organization on the Antigenic Structure of the Tick-Borne Encephalitis Virus Envelope Glycoprotein E

2009 ◽  
Vol 83 (17) ◽  
pp. 8482-8491 ◽  
Author(s):  
Stefan Kiermayr ◽  
Karin Stiasny ◽  
Franz X. Heinz
Keyword(s):  
Neutralizing Antibodies ◽  
Encephalitis Virus ◽  
Antigenic Structure ◽  
Icosahedral Symmetry ◽  
Virus Envelope ◽  
E Protein ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Virion Surface ◽  
Subviral Particles

ABSTRACT The envelope protein E of flaviviruses mediates both receptor-binding and membrane fusion. At the virion surface, 180 copies of E are tightly packed and organized in a herringbone-like icosahedral structure, whereas in noninfectious subviral particles, 60 copies are arranged in a T=1 icosahedral symmetry. In both cases, the basic building block is an E dimer which exposes the binding sites for neutralizing antibodies at its surface. It was the objective of our study to assess the dependence of the antigenic structure of E on its quaternary arrangement, i.e., as part of virions, recombinant subviral particles, or soluble dimers. For this purpose, we used a panel of 11 E protein-specific neutralizing monoclonal antibodies, mapped to distinct epitopes in each of the three E protein domains, and studied their reactivity with the different soluble and particulate forms of tick-borne encephalitis virus E protein under nondenaturing immunoassay conditions. Significant differences in the reactivities with these forms were observed that could be related to (i) limited access of certain epitopes at the virion surface; (ii) limited occupancy of epitopes in virions due to steric hindrance between antibodies; (iii) differences in the avidity to soluble forms compared to the virion, presumably related to the flexibility of E at its domain junctions; and (iv) modulations of the external E protein surface through interactions with its stem-anchor structure. We have thus identified several important factors that influence the antigenicity of the flavivirus E protein and have an impact on the interaction with neutralizing antibodies.

scholarly journals A benzene-mapping approach for uncovering cryptic pockets in membrane-bound proteins

2020 ◽  
Author(s):  
Lorena Zuzic ◽  
Jan K Marzinek ◽  
Jim Warwicker ◽  
Peter J Bond
Keyword(s):  
Conformational Changes ◽  
Viral Entry ◽  
Lipid Membrane ◽  
Membrane Lipids ◽  
Neutralizing Antibody ◽  
Md Simulations ◽  
Viral Envelope ◽  
E Protein ◽  
Repulsive Potentials ◽  
Functional Interface

ABSTRACTMolecular dynamics (MD) simulations in combination with small organic probes present in the solvent have previously been used as a method to reveal cryptic pockets that may not have been identified in experimental structures. We report such a method implemented within the CHARMM forcefield to effectively explore cryptic pockets on the surfaces of membrane-embedded proteins using benzene as a probe molecule. This relies on modified non-bonded parameters in addition to repulsive potentials between membrane lipids and benzene molecules. The method was tested on part of the outer shell of the dengue virus (DENV), for which research into a safe and effective neutralizing antibody or drug molecule is still ongoing. In particular, the envelope (E) protein, associated with the membrane (M) protein, is a lipid membrane-embedded complex which forms a dimer in the mature viral envelope. Solvent mapping was performed for the full, membrane-embedded EM protein complex and compared with similar calculations performed for the isolated, soluble E protein ectodomain dimer in solvent. Ectodomain-only simulations with benzene exhibited unfolding effects not observed in the more physiologically relevant membrane-associated systems. A cryptic pocket which has been experimentally shown to bind n-octyl-β-D-glucoside detergent was consistently revealed in all benzene-containing simulations. The addition of benzene also enhanced the flexibility and hydrophobic exposure of cryptic pockets at a key, functional interface in the E protein, and revealed a novel, potentially druggable pocket that may be targeted to prevent conformational changes associated with viral entry into the cell.GRAPHICAL ABSTRACT

scholarly journals Non-structural proteins of dengue 2 virus offer limited protection to interferon-deficient mice after dengue 2 virus challenge

2006 ◽  
Vol 87 (2) ◽  
pp. 339-346 ◽  
Author(s):  
Amanda E. Calvert ◽  
Claire Y.-H. Huang ◽  
Richard M. Kinney ◽  
John T. Roehrig
Keyword(s):  
Neutralizing Antibodies ◽  
Protective Immunity ◽  
Structural Proteins ◽  
Wild Type ◽  
Survival Times ◽  
E Proteins ◽  
Virus Challenge ◽  
E Protein ◽  
Low Levels ◽  
Deficient Mice

Chimeric (D2/WN) viruses containing the pre-membrane (prM) and envelope (E) proteins of West Nile virus (WN virus) and the capsid (C) and non-structural proteins of dengue 2 (DEN2) virus were used to evaluate the protective immunity elicited by either the flaviviral E protein or non-structural proteins. AG129 interferon-deficient mice, previously shown to be protected against lethal DEN1 or DEN2 viral infection after vaccination with a wild-type or candidate vaccine strain of DEN1 or DEN2 virus, respectively, were immunized with chimeric D2/WN virus and then challenged with DEN2 virus. D2/WN chimeric viruses were non-pathogenic in AG129 mice. These viruses elicited little anti-DEN E antibody, high levels of anti-DEN NS1 antibody and no or very low levels of DEN2 virus-neutralizing antibodies. Only 15 % of D2/WN-immunized mice survived challenge with DEN2 virus. However, their mean survival time increased by 11–14 days over non-immunized controls. These results suggest that, whilst the non-structural proteins were able to enhance mean survival times of AG129 mice, this protection was not as effective as protection mediated by the E protein.

scholarly journals Structure of Acidic pH Dengue Virus Showing the Fusogenic Glycoprotein Trimers

2014 ◽  
Vol 89 (1) ◽  
pp. 743-750 ◽  
Author(s):  
Xinzheng Zhang ◽  
Ju Sheng ◽  
S. Kyle Austin ◽  
Tabitha E. Hoornweg ◽  
Jolanda M. Smit ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Conformational Changes ◽  
Lipid Membrane ◽  
Neutralizing Antibody ◽  
Intermediate Stage ◽  
Host Cells ◽  
Acidic Ph ◽  
E Proteins ◽  
Fab Fragments ◽  
Domain Iii

ABSTRACTFlaviviruses undergo large conformational changes during their life cycle. Under acidic pH conditions, the mature virus forms transient fusogenic trimers of E glycoproteins that engage the lipid membrane in host cells to initiate viral fusion and nucleocapsid penetration into the cytoplasm. However, the dynamic nature of the fusogenic trimer has made the determination of its structure a challenge. Here we have used Fab fragments of the neutralizing antibody DV2-E104 to stop the conformational change of dengue virus at an intermediate stage of the fusion process. Using cryo-electron microscopy, we show that in this intermediate stage, the E glycoproteins form 60 trimers that are similar to the predicted “open” fusogenic trimer.IMPORTANCEThe structure of a dengue virus has been captured during the formation of fusogenic trimers. This was accomplished by binding Fab fragments of the neutralizing antibody DV2-E104 to the virus at neutral pH and then decreasing the pH to 5.5. These trimers had an “open” conformation, which is distinct from the “closed” conformation of postfusion trimers. Only two of the three E proteins within each spike are bound by a Fab molecule at domain III. Steric hindrance around the icosahedral 3-fold axes prevents binding of a Fab to the third domain III of each E protein spike. Binding of the DV2-E104 Fab fragments prevents domain III from rotating by about 130° to the postfusion orientation and thus precludes the stem region from “zipping” together the three E proteins along the domain II boundaries into the “closed” postfusion conformation, thus inhibiting fusion.

scholarly journals Structural basis of a potent human monoclonal antibody against Zika virus targeting a quaternary epitope

2019 ◽  
Vol 116 (5) ◽  
pp. 1591-1596 ◽  
Author(s):  
Feng Long ◽  
Michael Doyle ◽  
Estefania Fernandez ◽  
Andrew S. Miller ◽  
Thomas Klose ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Zika Virus ◽  
Neutralizing Antibodies ◽  
Human Monoclonal Antibody ◽  
Structural Basis ◽  
E Proteins ◽  
Fab Fragment ◽  
Cryo Electron Microscopy ◽  
E Protein ◽  
Mouse Model Of Infection

Zika virus (ZIKV) is a major human pathogen and member of the Flavivirus genus in the Flaviviridae family. In contrast to most other insect-transmitted flaviviruses, ZIKV also can be transmitted sexually and from mother to fetus in humans. During recent outbreaks, ZIKV infections have been linked to microcephaly, congenital disease, and Guillain-Barré syndrome. Neutralizing antibodies have potential as therapeutic agents. We report here a 4-Å-resolution cryo-electron microscopy structure of the ZIKV virion in complex with Fab fragments of the potently neutralizing human monoclonal antibody ZIKV-195. The footprint of the ZIKV-195 Fab fragment expands across two adjacent envelope (E) protein protomers. ZIKV neutralization by this antibody is presumably accomplished by cross-linking the E proteins, which likely prevents formation of E protein trimers required for fusion of the viral and cellular membranes. A single dose of ZIKV-195 administered 5 days after virus inoculation showed marked protection against lethality in a stringent mouse model of infection.

scholarly journals Structural transition and antibody binding of Ebola GP and Zika E proteins from pre-fusion to fusion-initiation states

2018 ◽  
Author(s):  
A Lappala-Vernon ◽  
W Nishima ◽  
J C Miner ◽  
P W Fenimore ◽  
W Fischer ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Viral Entry ◽  
Fusion Proteins ◽  
Neutralizing Antibodies ◽  
Structural Information ◽  
Host Cells ◽  
Host Immune System ◽  
E Proteins ◽  
Disease Etiology ◽  
Viral Genomes

AbstractMembrane fusion proteins are responsible for viral entry into host cells– a crucial first step in viral infection. These proteins undergo large conformational changes from pre-fusion to fusion initiation structures, and, despite differences in viral genomes and disease etiology, many fusion proteins are arranged as trimers. Structural information for both pre-fusion and fusion initiation states is critical for understanding virus neutralization by the host immune system. In the case of Ebola glycoprotein (GP) and Zika envelope protein (Zika E), pre-fusion state structures have been identified experimentally, but only partial structures of fusion initiation states have been described. While the fusion initiation structure is in an energetically unfavorable state that is difficult to solve experimentally, the existing structural information combined with computational approaches enabled the modeling of fusion initiation state structures of both proteins. These structural models provide an improved understanding of four different neutralizing antibodies in the prevention of viral host entry.

scholarly journals Cryo-EM structures reveal two distinct conformational states in a picornavirus cell entry intermediate

2020 ◽  
Author(s):  
Pranav N.M. Shah ◽  
David J. Filman ◽  
Krishanthi S. Karunatilaka ◽  
Emma L. Hesketh ◽  
Elisabetta Groppelli ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Cell Entry ◽  
Viral Rna ◽  
Productive Infection ◽  
Human Pathogens ◽  
Temperature Dependent ◽  
Endosomal Membrane ◽  
N Terminus ◽  
Clear Identification ◽  
Membrane Interactive

ABSTRACTThe virions of enteroviruses such as poliovirus undergo a global conformational change after binding to the cellular receptor, characterized by a 4% expansion, and opening of holes at the two and quasi-three-fold symmetry axes of the capsid. The resultant particle is called a 135S particle or A-particle and is thought to be on the pathway to a productive infection. Previously published studies have concluded that the membrane interactive peptides, namely VP4 and the N-terminus of VP1, are irreversibly externalized in the 135S particle. However, using established protocols to produce the 135S particle, and single particle cryo-electron microscopy methods, we have identified at least two unique states that we call the early and late 135S particle. Surprisingly, only in the “late” 135S particles have detectable levels of the VP1 N-terminus trapped outside the capsid. Moreover, we observe a distinct density inside the capsid that can be accounted for by VP4 that remains associated with the genome. Taken together our results conclusively demonstrate that the 135S particle is not a unique conformation, but rather a family of conformations that could exist simultaneously.AUTHOR SUMMARYNonenveloped viruses need to provide mechanisms that allow their genomes to be delivered across membrane. This process remains poorly understood. For enterovirus such as poliovirus, genome delivery involves a program of conformational changes that include expansion of the particle and externalization of two normal internal peptides, VP4 and the VP1 N-terminus, which then insert into the cell membrane, triggering endocytosis and the creation of pores that facilitate the transfer of the viral RNA genome across the endosomal membrane. This manuscript describes five high-resolution cryo-EM structures of altered poliovirus particles that represent a number of intermediates along this pathway. The structures reveal several surprising findings, including the discovery of a new intermediate that is expanded but has not yet externalized the membrane interactive peptides, the clear identification of a unique exit site VP1 N-terminus, the demonstration that the externalized VP1 N-terminus partitions between two different sites in a temperature-dependent fashion, direct visualization of an amphipathic helix at the N-terminus of VP1 in an ideal position for interaction with cellular membranes, and the observation that a significant portion of VP4 remains inside the particle and accounts for a feature that had been previously ascribed to part of the viral RNA. These findings represent significant additions to our understanding of the cell entry process of an important class of human pathogens.

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