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 Production of a neutralizing antibody against envelope protein of dengue virus type 2 using the linear array epitope technique

2014 ◽  
Vol 95 (10) ◽  
pp. 2155-2165 ◽  
Author(s):  
Peng-Yeh Lai ◽  
Chia-Tse Hsu ◽  
Shao-Hung Wang ◽  
Jin-Ching Lee ◽  
Min-Jen Tseng ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Envelope Protein ◽  
Linear Array ◽  
Polyclonal Antibodies ◽  
Academic Research ◽  
Neutralizing Antibody ◽  
Viral Envelope ◽  
Host Cells ◽  
Viral Envelope Protein ◽  
Domain Iii

Dengue virus (DENV; genus Flavivirus) contains a positive-stranded RNA genome. Binding of DENV to host cells is mediated through domain III of the viral envelope protein. Many therapeutic mAbs against domain III have been generated and characterized because of its high antigenicity. We have previously established a novel PCR method named the linear array epitope (LAE) technique for producing monoclone-like polyclonal antibodies. To prove this method could be utilized to produce antibody against epitopes with low antigenicity, a region of 10 aa (V365NIEAEPPFG374) from domain III of the envelope protein in DENV serotype 2 (DENV2) was selected to design the primers for the LAE technique. A DNA fragment encoding 10 directed repeats of these 10 aa for producing the tandem-repeated peptides was obtained and fused with glutathione S-transferase (GST)-containing vector. This fusion protein (GST-Den EIII10-His6) was purified from Escherichia coli and used as antigen for immunizing rabbits to obtain the polyclonal antibody. Furthermore, the EIII antibody could recognize envelope proteins either ectopically overexpressed or synthesized by DENV2 infection using Western blot and immunofluorescence assays. Most importantly, this antibody was also able to detect DENV2 virions by ELISA, and could block viral entry into BHK-21 cells as shown by immunofluorescence and quantitative real-time PCR assays. Taken together, the LAE technique could be applied successfully for the production of antibodies against antigens with low antigenicity, and shows high potential to produce antibodies with good quality for academic research, diagnosis and even therapeutic applications in the future.

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.

A synthetic peptide derived from domain III envelope glycoprotein of Dengue virus induces neutralizing antibody

Virus Genes ◽  
2017 ◽  
Vol 54 (1) ◽  
pp. 25-32 ◽  
Author(s):  
J. Asnet Mary ◽  
Akanitt Jittmittraphap ◽  
Siriporn Chattanadee ◽  
Pornsawan Leaungwutiwong ◽  
R. Shenbagarathai
Keyword(s):  
Dengue Virus ◽  
Envelope Glycoprotein ◽  
Synthetic Peptide ◽  
Neutralizing Antibody ◽  
Domain Iii

scholarly journals Crystal Structure of Dengue Virus Type 1 Envelope Protein in the Postfusion Conformation and Its Implications for Membrane Fusion

2009 ◽  
Vol 83 (9) ◽  
pp. 4338-4344 ◽  
Author(s):  
Vinod Nayak ◽  
Moshe Dessau ◽  
Kaury Kucera ◽  
Karen Anthony ◽  
Michel Ledizet ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dengue Virus ◽  
Conformational Change ◽  
Virus Type ◽  
Envelope Protein ◽  
Lipid Membrane ◽  
Ph Sensor ◽  
Endosomal Membrane ◽  
Domain Iii

ABSTRACT Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the “pH sensor” that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.

scholarly journals An epitope-resurfaced virus-like particle can induce broad neutralizing antibody against four serotypes of dengue virus

2018 ◽  
Author(s):  
Wen-Fan Shen ◽  
Jedhan Ucat Galula ◽  
Jyung-Hurng Liu ◽  
Mei-Ying Liao ◽  
Chang-Hao Huang ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Quaternary Structure ◽  
Neutralizing Antibody ◽  
Icosahedral Symmetry ◽  
E Proteins ◽  
Cell Repertoire ◽  
Virus Like Particles ◽  
B Cell Repertoire ◽  
Protein Dimers ◽  
Virus Like Particle

AbstractDengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein we show for the first time that mD2VLP particles possess a T=1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes through cryo-electron microscopy reconstruction. Mice vaccinated with highly matured virus-like particles derived from DENV serotype 2 (mD2VLP) can generate higher cross reactive (CR) neutralization antibodies (NtAbs) and were protected against all 4 serotypes of DENV through clonal expansion supported by hybridoma and B-cell repertoire analysis. Our results revealed that a “epitope-resurfaced” mature-form dengue VLP has the potential to induce quaternary structure-recognizing broad CR NtAbs.

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.

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 Characterization of Dengue Virus Complex-Specific Neutralizing Epitopes on Envelope Protein Domain III of Dengue 2 Virus

2008 ◽  
Vol 82 (17) ◽  
pp. 8828-8837 ◽  
Author(s):  
Gregory D. Gromowski ◽  
Nicholas D. Barrett ◽  
Alan D. T. Barrett
Keyword(s):  
Dengue Virus ◽  
Critical Role ◽  
Antigenic Site ◽  
Protein Domain ◽  
Virus Infectivity ◽  
E Proteins ◽  
Mutant Proteins ◽  
Virus Complex ◽  
Domain Iii

ABSTRACT The surface of the mature dengue virus (DENV) particle is covered with 180 envelope (E) proteins arranged as homodimers that lie relatively flat on the virion surface. Each monomer consists of three domains (ED1, ED2, and ED3), of which ED3 contains the critical neutralization determinant(s). In this study, a large panel of DENV-2 recombinant ED3 mutant proteins was used to physically and biologically map the epitopes of five DENV complex-specific monoclonal antibodies (MAbs). All five MAbs recognized a single antigenic site that includes residues K310, I312, P332, L389, and W391. The DENV complex antigenic site was located on an upper lateral surface of ED3 that was distinct but overlapped with a previously described DENV-2 type-specific antigenic site on ED3. The DENV complex-specific MAbs required significantly higher occupancy levels of available ED3 binding sites on the virion, compared to DENV-2 type-specific MAbs, in order to neutralize virus infectivity. Additionally, there was a great deal of variability in the neutralization efficacy of the DENV complex-specific MAbs with representative strains of the four DENVs. Overall, the differences in physical binding and potency of neutralization observed between DENV complex- and type-specific MAbs in this study demonstrate the critical role of the DENV type-specific antibodies in the neutralization of virus infectivity.

scholarly journals Epitope resurfacing on dengue virus-like particle vaccine preparation to induce broad neutralizing antibody

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Wen-Fan Shen ◽  
Jedhan Ucat Galula ◽  
Jyung-Hurng Liu ◽  
Mei-Ying Liao ◽  
Cheng-Hao Huang ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Quaternary Structure ◽  
Neutralizing Antibody ◽  
Icosahedral Symmetry ◽  
E Proteins ◽  
Protein Dimers ◽  
Virus Like Particle ◽  
Arboviral Diseases ◽  
Potential Vaccine ◽  
Microscopy Examination

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein VLP derived from DENV serotype-2 were engineered becoming highly matured (mD2VLP) and showed variable size distribution with diameter of ~31 nm forming the major population under cryo-electron microscopy examination. Furthermore, mD2VLP particles of 31 nm diameter possess a T = 1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Mice vaccinated with mD2VLP generated higher cross-reactive (CR) neutralization antibodies (NtAbs) and were fully protected against all 4 serotypes of DENV. Our results highlight the potential of ‘epitope-resurfaced’ mature-form D2VLPs in inducing quaternary structure-recognizing broad CR NtAbs to guide future dengue vaccine design.

scholarly journals Engineered Dengue Virus Domain III Proteins Elicit Cross-Neutralizing Antibody Responses in Mice

2018 ◽  
Vol 92 (18) ◽  
Author(s):  
Julia C. Frei ◽  
Ariel S. Wirchnianski ◽  
Jennifer Govero ◽  
Olivia Vergnolle ◽  
Kimberly A. Dowd ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Neutralizing Antibodies ◽  
Primary Infection ◽  
Vaccine Development ◽  
Secondary Infection ◽  
Severe Disease ◽  
Febrile Illness ◽  
Neutralizing Antibody ◽  
Immunogen Design ◽  
Domain Iii

ABSTRACTDengue virus is the most globally prevalent mosquito-transmitted virus. Primary infection with one of four cocirculating serotypes (DENV-1 to -4) causes a febrile illness, but secondary infection with a heterologous serotype can result in severe disease, due in part to antibody-dependent enhancement of infection (ADE). In ADE, cross-reactive but nonneutralizing antibodies, or subprotective levels of neutralizing antibodies, promote uptake of antibody-opsonized virus in Fc-γ receptor-positive cells. Thus, elicitation of broadly neutralizing antibodies (bNAbs), but not nonneutralizing antibodies, is desirable for dengue vaccine development. Domain III of the envelope glycoprotein (EDIII) is targeted by bNAbs and thus is an attractive immunogen. However, immunization with EDIII results in sera with limited neutralization breadth. We developed “resurfaced” EDIII immunogens (rsDIIIs) in which the A/G strand epitope that is targeted by bNAb 4E11 is maintained but less desirable epitopes are masked. RsDIIIs bound 4E11, but not serotype-specific or nonneutralizing antibodies. One rsDIII and, unexpectedly, wild-type (WT) DENV-2 EDIII elicited cross-neutralizing antibody responses against DENV-1 to -3 in mice. While these sera were cross-neutralizing, they were not sufficiently potent to protect AG129 immunocompromised mice at a dose of 200 μl (50% focus reduction neutralization titer [FRNT50], ∼1:60 to 1:130) against mouse-adapted DENV-2. Our results provide insight into immunogen design strategies based on EDIII.IMPORTANCEDengue virus causes approximately 390 million infections per year. Primary infection by one serotype causes a self-limiting febrile illness, but secondary infection by a heterologous serotype can result in severe dengue syndrome, which is characterized by hemorrhagic fever and shock syndrome. This severe disease is thought to arise because of cross-reactive, non- or poorly neutralizing antibodies from the primary infection that are present in serum at the time of secondary infection. These cross-reactive antibodies enhance the infection rather than controlling it. Therefore, induction of a broadly and potently neutralizing antibody response is desirable for dengue vaccine development. Here, we explore a novel strategy for developing immunogens based on domain III of the E glycoprotein, where undesirable epitopes (nonneutralizing or nonconserved) are masked by mutation. This work provides fundamental insight into the immune response to domain III that can be leveraged for future immunogen design.

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