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

2017 ◽  
Vol 17 (12) ◽  
pp. 825-835 ◽  
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

scholarly journals Molecular Changes in Dengue Envelope Protein Domain III upon Interaction with Glycosaminoglycans

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 935
Author(s):  
James G. Hyatt ◽  
Sylvain Prévost ◽  
Juliette M. Devos ◽  
Courtney J. Mycroft-West ◽  
Mark A. Skidmore ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Envelope Protein ◽  
Quaternary Structure ◽  
Chondroitin Sulphate ◽  
Viral Envelope ◽  
Protein Domain ◽  
Viral Envelope Protein ◽  
Oligomeric State ◽  
Molecular Changes ◽  
Viral Attachment

Dengue fever is a rapidly emerging vector-borne viral disease with a growing global burden of approximately 390 million new infections per annum. The Dengue virus (DENV) is a flavivirus spread by female mosquitos of the aedes genus, but the mechanism of viral endocytosis is poorly understood at a molecular level, preventing the development of effective transmission blocking vaccines (TBVs). Recently, glycosaminoglycans (GAGs) have been identified as playing a role during initial viral attachment through interaction with the third domain of the viral envelope protein (EDIII). Here, we report a systematic study investigating the effect of a range of biologically relevant GAGs on the structure and oligomeric state of recombinantly generated EDIII. We provide novel in situ biophysical evidence that heparin and chondroitin sulphate C induce conformational changes in EDIII at the secondary structure level. Furthermore, we report the ability of chondroitin sulphate C to bind EDIII and induce higher-order dynamic molecular changes at the tertiary and quaternary structure levels which are dependent on pH, GAG species, and the GAG sulphation state. Lastly, we conducted ab initio modelling of Small Angle Neutron Scattering (SANS) data to visualise the induced oligomeric state of EDIII caused by interaction with chondroitin sulphate C, which may aid in TBV development.

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

Biomolecules ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 309 ◽  
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.

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.

scholarly journals The YXXL Sequences of a Transmembrane Protein of Bovine Leukemia Virus Are Required for Viral Entry and Incorporation of Viral Envelope Protein into Virions

1999 ◽  
Vol 73 (2) ◽  
pp. 1293-1301 ◽  
Author(s):  
Kazunori Inabe ◽  
Masako Nishizawa ◽  
Shigeru Tajima ◽  
Kazuyoshi Ikuta ◽  
Yoko Aida
Keyword(s):  
Viral Entry ◽  
Envelope Protein ◽  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Transient Transfection ◽  
Viral Envelope ◽  
Tyrosine Residue ◽  
Wild Type ◽  
Viral Envelope Protein

ABSTRACT The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV.

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