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

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.

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Folding of the Ig-Like Domain of the Dengue Virus Envelope Protein Analyzed by High-Hydrostatic-Pressure NMR at a Residue-Level Resolution

Biomolecules ◽

10.3390/biom9080309 ◽

2019 ◽

Vol 9 (8) ◽

pp. 309 ◽

Cited By ~ 4

Author(s):

Saotome ◽

Doret ◽

Kulkarni ◽

Yang ◽

Barthe ◽

...

Keyword(s):

Hydrostatic Pressure ◽

Dengue Virus ◽

Envelope Protein ◽

High Hydrostatic Pressure ◽

Public Health Problem ◽

Viral Envelope ◽

Specific Information ◽

Pressure Jump ◽

Viral Envelope Protein ◽

Domain Iii

Dengue fever is a mosquito-borne endemic disease in tropical and subtropical regions, causing a significant public health problem in Southeast Asia. Domain III (ED3) of the viral envelope protein contains the two dominant putative epitopes and part of the heparin sulfate receptor binding region that drives the dengue virus (DENV)’s fusion with the host cell. Here, we used high-hydrostatic-pressure nuclear magnetic resonance (HHP-NMR) to obtain residue-specific information on the folding process of domain III from serotype 4 dengue virus (DEN4-ED3), which adopts the classical three-dimensional (3D) ß-sandwich structure known as the Ig-like fold. Interestingly, the folding pathway of DEN4-ED3 shares similarities with that of the Titin I27 module, which also adopts an Ig-like fold, but is functionally unrelated to ED3. For both proteins, the unfolding process starts by the disruption of the N- and C-terminal strands on one edge of the ß-sandwich, yielding a folding intermediate stable over a substantial pressure range (from 600 to 1000 bar). In contrast to this similarity, pressure-jump kinetics indicated that the folding transition state is considerably more hydrated in DEN4-ED3 than in Titin I27.

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West Nile virus is neutralized by HOCl-modified human serum albumin that binds to domain III of the viral envelope protein E

Virology ◽

10.1016/j.virol.2007.12.008 ◽

2008 ◽

Vol 373 (2) ◽

pp. 322-328 ◽

Cited By ~ 8

Author(s):

Markus Vossmann ◽

Martin Kirst ◽

Diana Ludolfs ◽

Michael Schreiber

Keyword(s):

West Nile Virus ◽

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Envelope Protein ◽

Viral Envelope ◽

Viral Envelope Protein ◽

West Nile ◽

Domain Iii

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SARS-CoV-2 envelope-protein corruption of homeostatic signaling mechanisms in mammalian cells

10.1101/2021.06.16.448640 ◽

2021 ◽

Author(s):

Tobias Schulze ◽

Andreas Hartel ◽

Sebastian Hoeler ◽

Clara Hemming ◽

Robert Lehn ◽

...

Keyword(s):

Envelope Protein ◽

Viral Protein ◽

Mammalian Cells ◽

Fluorescent Protein ◽

Ion Selectivity ◽

Cellular Protein ◽

Viral Envelope ◽

Host Cells ◽

Viral Envelope Protein ◽

Cell Protection

During a SARS-CoV2 infection, host cells produce large amounts of the viral envelope protein (Ep-CoV2). Ep-CoV2 is partially inserted into the membrane of nascent viral particles and into cellular membranes. To mimic the pathophysiological impact of the cellular protein fraction, Ep-CoV2 was overexpressed in mammalian cells and effects on key signaling parameters were monitored. By tagging with green fluorescent protein (GFP), we found that Ep-CoV2 protein is mostly present in the endoplasmic reticulum with additional trace amounts in the plasma membrane. We observed that wild-type Ep-CoV2 and, to a lesser extent, its mutants (N15A, V25F) corrupted some of the most important homeostatic mechanisms in cells. The same was observed with isolated transmembrane domains of the protein. The Ep-CoV2-evoked elevation of intracellular Ca2+ and pH as well as the induced membrane depolarization produced by the presence of the protein interfere with major signal transduction cascades in host cells. These functions of Ep-CoV2, which likely contribute to the pathogenesis of the viral protein, result from the ion-channel activity of the viral protein. Two independent assays, a functional reconstitution of Ep-CoV2 protein in artificial membranes and a rescue of K+-deficient yeast mutants, confirm that Ep-CoV2 generates a cation-conducting channel with a low unitary conductance and a complex ion selectivity. The data presented here suggest that specific channel function inhibitors of Ep-CoV2 can provide cell protection and virostatic effects.

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Characterization of Dengue Virus Resistance to Brequinar in Cell Culture

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00561-10 ◽

2010 ◽

Vol 54 (9) ◽

pp. 3686-3695 ◽

Cited By ~ 67

Author(s):

Min Qing ◽

Gang Zou ◽

Qing-Yin Wang ◽

Hao Ying Xu ◽

Hongping Dong ◽

...

Keyword(s):

Dengue Virus ◽

Envelope Protein ◽

Rna Synthesis ◽

De Novo ◽

Yellow Fever Virus ◽

Nonstructural Protein ◽

Viral Envelope ◽

Viral Envelope Protein ◽

Virion Assembly ◽

Virus Serotype

ABSTRACT Brequinar is an inhibitor of dihydroorotate dehydrogenase, an enzyme that is required for de novo pyrimidine biosynthesis. Here we report that brequinar has activity against a broad spectrum of viruses. The compound not only inhibits flaviviruses (dengue virus, West Nile virus, yellow fever virus, and Powassan virus) but also suppresses a plus-strand RNA alphavirus (Western equine encephalitis virus) and a negative-strand RNA rhabdovirus (vesicular stomatitis virus). Using dengue virus serotype 2 (DENV-2) as a model, we found that brequinar suppressed the viral infection cycle mainly at the step of RNA synthesis. Supplementing the culture medium with pyrimidines (cytidine or uridine) but not purines (adenine or guanine) could be used to reverse the inhibitory effect of the compound. Continuous culturing of DENV-2 in the presence of brequinar generated viruses that were partially resistant to the inhibitor. Sequencing of the resistant viruses revealed two amino acid mutations: one mutation (M260V) located at a helix in the domain II of the viral envelope protein and another mutation (E802Q) located at the priming loop of the nonstructural protein 5 (NS5) polymerase domain. Functional analysis of the mutations suggests that the NS5 mutation exerts resistance through enhancement of polymerase activity. The envelope protein mutation reduced the efficiency of virion assembly/release; however, the mutant virus became less sensitive to brequinar inhibition at the step of virion assembly/release. Taken together, the results indicate that (i) brequinar blocks DENV RNA synthesis through depletion of intracellular pyrimidine pools and (ii) the compound may also exert its antiviral activity through inhibition of virion assembly/release.

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Sulfated Escherichia coli K5 Polysaccharide Derivatives Inhibit Dengue Virus Infection of Human Microvascular Endothelial Cells by Interacting with the Viral Envelope Protein E Domain III

PLoS ONE ◽

10.1371/journal.pone.0074035 ◽

2013 ◽

Vol 8 (8) ◽

pp. e74035 ◽

Cited By ~ 25

Author(s):

Peter Vervaeke ◽

Marijke Alen ◽

Sam Noppen ◽

Dominique Schols ◽

Pasqua Oreste ◽

...

Keyword(s):

Escherichia Coli ◽

Endothelial Cells ◽

Envelope Protein ◽

Viral Envelope ◽

Microvascular Endothelial Cells ◽

Viral Envelope Protein ◽

Dengue Virus Infection ◽

Domain Iii ◽

Polysaccharide Derivatives ◽

K5 Polysaccharide

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Differential Diagnosis of Flavivirus Infections in Horses Using Viral Envelope Protein Domain III Antigens in Enzyme-Linked Immunosorbent Assay

Vector-Borne and Zoonotic Diseases ◽

10.1089/vbz.2017.2172 ◽

2017 ◽

Vol 17 (12) ◽

pp. 825-835 ◽

Cited By ~ 3

Author(s):

Thisun B.H. Piyasena ◽

Yin X. Setoh ◽

Jody Hobson-Peters ◽

Natalie A. Prow ◽

Helle Bielefeldt-Ohmann ◽

...

Keyword(s):

Differential Diagnosis ◽

Immunosorbent Assay ◽

Envelope Protein ◽

Viral Envelope ◽

Protein Domain ◽

Enzyme Linked Immunosorbent Assay ◽

Viral Envelope Protein ◽

Domain Iii

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Antiviral Peptides Targeting the West Nile Virus Envelope Protein

Journal of Virology ◽

10.1128/jvi.01840-06 ◽

2006 ◽

Vol 81 (4) ◽

pp. 2047-2055 ◽

Cited By ~ 67

Author(s):

Fengwei Bai ◽

Terrence Town ◽

Deepti Pradhan ◽

Jonathan Cox ◽

Ashish ◽

...

Keyword(s):

West Nile Virus ◽

Envelope Protein ◽

Phage Display Library ◽

Viral Envelope ◽

Host Cells ◽

Viral Envelope Protein ◽

Virus Envelope ◽

West Nile ◽

Inhibition Concentration

ABSTRACT West Nile virus (WNV) can cause fatal murine and human encephalitis. The viral envelope protein interacts with host cells. A murine brain cDNA phage display library was therefore probed with WNV envelope protein, resulting in the identification of several adherent peptides. Of these, peptide 1 prevented WNV infection in vitro with a 50% inhibition concentration of 67 μM and also inhibited infection of a related flavivirus, dengue virus. Peptide 9, a derivative of peptide 1, was a particularly potent inhibitor of WNV in vitro, with a 50% inhibition concentration of 2.6 μM. Moreover, mice challenged with WNV that had been incubated with peptide 9 had reduced viremia and fatality compared with control animals. Peptide 9 penetrated the murine blood-brain barrier and was found in the brain parenchyma, implying that it may have antiviral activity in the central nervous system. These short peptides serve as the basis for developing new therapeutics for West Nile encephalitis and, potentially, other flaviviruses.

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Structure of Acidic pH Dengue Virus Showing the Fusogenic Glycoprotein Trimers

Journal of Virology ◽

10.1128/jvi.02411-14 ◽

2014 ◽

Vol 89 (1) ◽

pp. 743-750 ◽

Cited By ~ 37

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.

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An External Loop Region of Domain III of Dengue Virus Type 2 Envelope Protein Is Involved in Serotype-Specific Binding to Mosquito but Not Mammalian Cells

Journal of Virology ◽

10.1128/jvi.78.1.378-388.2004 ◽

2004 ◽

Vol 78 (1) ◽

pp. 378-388 ◽

Cited By ~ 155

Author(s):

Jan-Jong Hung ◽

Meng-Ti Hsieh ◽

Ming-Jer Young ◽

Chuan-Liang Kao ◽

Chwan-Chuen King ◽

...

Keyword(s):

Dengue Virus ◽

Envelope Protein ◽

Mammalian Cells ◽

Host Cells ◽

Mammalian Cell Lines ◽

Loop Region ◽

Mosquito Cells ◽

Heparan Sulfates ◽

Domain Iii

ABSTRACT Dengue virus (DV) is a flavivirus and infects mammalian cells through mosquito vectors. This study investigates the roles of domain III of DV type 2 envelope protein (EIII) in DV binding to the host cell. Recombinant EIII interferes with DV infection to BHK21 and C6/36 cells by blocking dengue virion adsorption to these cells. Inhibition of EIII on BHK21 cells was broad with no serotype specificity; however, inhibition of EIII on C6/36 cells was relatively serotype specific. Soluble heparin completely blocks binding of EIII to BHK21 cells, suggesting that domain III binds mainly to cell surface heparan sulfates. This suggestion is supported by the observation that EIII binds very weakly to gro2C and sog9 mutant mammalian cell lines that lack heparan sulfate. In contrast, heparin does not block binding of EIII to mosquito cells. Furthermore, a synthetic peptide that includes amino acids (aa) 380 to 389 of EIII, IGVEPGQLKL, inhibits binding of EIII to C6/36 but not BHK21 cells. This peptide corresponds to a lateral loop region on domain III of E protein, indicating a possible role of this loop in binding to mosquito cells. In summary, these results suggest that EIII plays an important role in binding of DV type 2 to host cells. In addition, EIII interacts with heparan sulfates when binding to BHK21 cells, and a loop region containing aa 380 to 389 of EIII may participate in DV type 2 binding to C6/36 cells.

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