Identification And Evaluation Of DENV2-EDâ…¢ And Potential Target Receptor Molecules Of Anti-dengue Virus

Dengue virus (DENV) belongs to family Flaviviridae and genus Flavivirus. which is the causative agent of the most common mosquito-borne viral disease. The consequences of DENV infection in humans ranges from a self-limiting illness known as dengue fever(DF) to a severe hemorrhagic fever(DHF) which can progress to dengue shock syndrome(DSS).Approximately2.5-3billion people are at risk for DENV transmission and each year there are estimated to be50～100million new infections resulting in around1～5%death rate. Most of death are children. In recent years, the areas and frequency of dengue epidemics widely spread due to global warming, environmental degrading and population movement increasing. In our country, dengue outbreak have been repeatedly appeared in Hainan, Guangdong, Guangxi, Fujian and other areas. Now WHO has already put DHF/DSS as one of global public health problems.Vaccination is an economical and effective way to prevent of infectious diseases. Although dengue vaccine research history has been50years, dengue vaccine development still faces serious challenges:(1) DENV vaccine need to cover four serotypes designated as DENV-1to-4.(2) DENV vaccine should offer long time protection. Studies have reported that re-infection will occur after the initial infection20years later.(3) No suitable animal models are designed for replication of dengue disease.(4)Although the protective effect of neutralizing antibodies has been widely accepted, but its relationship with the actual protective effect remains to be confirmed; (5)Dengue vaccine need to be re-evaluated when DENV virus changes. Therefore, effectivity and safety are the main point of the reliable dengue vaccine. It is very urgent for the current virology to deeply research dengue pathogenesis and explore the treatment strategies and approaches.Dengue virus is internalized by receptor-mediated endocytosis into host cell and complete a series of replication, translation, assembly and proliferation and release process at the endoplasmic reticulum membrane.Virus enters the cell for replication and a series of reaction ultimately mediated by receptors. Therefore, binding between dengue virus and receptors of host cell surface is not only the most important initial step for virus infection. But also provides a guideline for where to find a suitable potential target molecule anti-dengue virus.The virion is characterized as a small enveloped particle containing a single positive sense polarity strand of RNA of approximately11kb in length, which encodes three structural proteins (C, M/prM and E) and seven nonstructural proteins (NS1, NS2a/b, NS3, NS4a/b, NS5) involved in viral RNA replication. Envelope E compose approximately500amino acid residues with a molecular weight of55-60kDa. DENV utilizes its envelope E protein which is the main structure responsible for host cell binding and fusing to interact with hose cell receptors.The crystal structure analysis of envelope protein revealed that it includes three domains (Ⅰ-Ⅲ):Domain I organizes into an eight-stranded central P-barrel structure. Domain Ⅱ contains the highly conserved hydrophobic fusion peptide which is involved with dimerization of envelop protein monomers on the surface of mature virion. Domain Ⅲ has an immunoglobulin-like fold which exposed and accessible on the virion surface. It contains epitopes which can induce type-specific and subtype-specific neutralizing antibodies that may block the entry of virus into host cells. Thus, domain Ⅲ has emerged as a promising important target for vaccine candidate.Bio-informatics analysis further showed that domain Ⅲ is stable protein, the relative molecular weight is10790.5. The target molecules can be focus from full-length RNA dengue virus, to the major surface protein structural protein E, and then Domain Ⅲ. It may improve the success rate of the virus receptor screening and accuracy using the truncated viral.Dengue virus receptor research has been paid widely attention.This mosquito-borne virus can infect a variety of cells which come from different original tissues. The binding process is complex and multistep process between dengue virus and receptors of host cell surface. It may involve different viral attachment protein and a plurality of target cell receptor. This have been approved by results from many studies recent years. Like40kDa and45kDa glycoprotein found from the embryonic stage of Aedes albopictus C6/36cells. Receptor protein identified as microtubule-like protein, laminin, and other heat shock protein; Receptor protein found in vascular endothelial cells (VECs), liver cells, Monocytes/macrophages and DC cells from human,etc.This study attempts to find related receptor from mammalian cells. BHK-21cells is Baby Hamster Syrian Kindey cells. It is widely used in the vector virus proliferation and vaccine research. For BHK-21cells is susceptible by dengue virus, there may exist a DENV infection mediated by its membrane receptor. But until now it is still lack of detailed report on it. Therefore, Screening and identification of DENV receptor on BHK-21cell membrane may not only give more information of the pathogenesis of DENV, but also can develope great potential value of antiviral drugs.VOPBA is virus overlay protein binding assay which is the "classic" method to screen virus-associated cell factors. It is based on Western blot. In VOPBA, the proteins of the target cells are extracted, separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane is incubated with virus particles and tested by virus-specific antibodies. The proteins that bind the virus particles may serve as the virus-targeted molecules. PVDF membrane is hydrophobic, the membrane pore size has a great influence on the experimental results. Using VOPBA, a set of cell proteins related to DENV infection was successfully identified. In this study, a recombinant domain Ⅲ of the DENV-2-enveloped EDⅢ protein(DENV-2EDIII) was used for immunity rabbits to get anti-DENV serum. Screening and identification of potential DENV-2-binding proteins of the BHK-21cells and the DENV-2-binding proteins were verified by mass spectrometry and western blot analysis. The binding modes and binding residues of DENV-2-binding proteins and DENV-2EDIII protein were simulated by bio-informatics softwares, which may lay down the great foundation for further understanding dengue pathogenesis, developing receptor-specific antagonist and controlling of DENV infection and prevalence.Objective1screening potential target molecules from dengue viruses1.1To amplify and clone DENV Ⅱ envelope protein gene and construct pMD18-T-DV2-Eplasmid;1.2To amplify and clone domain Ⅲ gene and construct expression plasmid pET-32a(+)-DV2-EDⅢ;1.3To induce the expression of domain Ⅲ in Escherichia coli;1.4To purify recombinant protein and immunize rabbit to obtain polyclonal antibod and then evaluate its function.2screening potential target molecules from susceptible cells2.1Screening receptors of Dengue virus in BHK-21cell by One-dimensional electrophoresis and VOPBA;2.2Screening specific binding molecules with the utilization of two-dimensional electrophoresis and VOPBA and then mass spectrometry analysis.2.3bio-informatics softwares analyse the binding modes and residues of DENV-2EDIII protein and potential target molecules.Methods1. PCR amplify the domain Ⅲ DNA fragment of the envelope protein with pMD18-T-DV2-E. 2. By digestion connected the ED III into the prokaryotic expression vector pET-32a (+), and then transformed into E. coli DH5a;3. Transform the recombinant pET-32a(+)-DV2-EDlII plasmids into BL21(DE3) and induce by IPTG, analyze the expressed products by SDS-PAGE;4. Optimize expression conditions such as induce temperature, induce time and IPTG concentration;5. Lysis the induced bacteria by sonication to analyze the solubility of recombinant protein;6. Analyze the immunoreactivity of the recombinant protein with His·Tag mAbs and DENV(I-IV) mAbs by Western blot;7. Purify the recombinant protein with Ni-NTA and electroelution from SDS-PAGE gels;8. purified recombinant protein was used to immunize rabbits to obtain polyclonal antiserum;9. Determine the titer of polyclonal antibody by ELISA;10. Competitive analysis was put in different concentrations of purified fusion protein of dengue virus in BHK-21cells, with the different concentrations of BSA protein as a negative control;11. Continuous double dilution method and analyzed for the test plaque were carried out by prepared Dengue virus EDIII polyclonal rabbit serum proteins;12. Passage dengue virus in BHK-21cell, purify and titer virus;13. Dengue virus were proliferated in BHK-21cells and collected. Monolayer cells titration method (Reed-Muench) was used to determine virus titer.,i.e. monolayer cells titration method for determination of the highest dilution when half cell culture appeared lesions.14. Prepare total protein and membrane protein of BHK-21cells; The proteins were separated by SDS-PAGE, screened specificity of binding molecules using VOPBA.15. Screen the binding molecules in BHK-21cell by Virus overlay protein binding assay; mass spectrometry analysis of the amino acid sequence of the specific proteins.16. The binding modes and residues of screened connection molecules and DENV-2EIII protein were predicted by bioinformatics softwares, which may lay down the great foundation for further designing receptor antagonist protein and deeply understanding dengue pathogenesis.Results:1ED Ⅲ protein is a potential receptor binding molecules.a)EDIII was successfully constructed and amplified; The expression plasmid pET-32a(+)-DV2-EDⅢwere successfully constructed;b)After induction with IPTG, a specific soluble protein was expressed from Escherichia coli which contains the expression plasmid pET-32a(+)-DV2-EDⅢ;c) The optimize expression condition were induced with lmmol/L IPTG at25℃for6h;d) The purity of recombinant protein can reach98.6%after the initial Ni-NTA purification and second gel electro-osmosis purification.e) Purified protein to immunize New Zealand rabbits, harvest anti-dengue virus E immunosorbent assay titer greater than1:51200, Western blot shows recombinant protein has a specific reaction.f) Competitive assays show that the purified recombinant protein can be effectively bound to the BHK-21cells, while may prevent viral entry into susceptible cells.g) The DENV-2EDⅢ polyclonal antibody can neutralize90%DENV-2virus when dilution of1:16and80%at a dilution of1:1024.2A45kDa potential target molecules of BHK-21cells was screened by VOPBA2.1Dengue virus was successfully passaged in BHK-21cell and the purified virus titer was4.52×107PFU/ml;2.2Extract BHK-21cells membrane proteins as references. There were no proteins degradation after separation by SDS-PAGE gel protein bar with, which showed the proteins could be used for downstream experiments.2.3A specific band of45kDa occurred after VOPBA of the proteins from BHK-21cells incubated with the virus, while the negative control group did not show this specific band.2.4Mass spectrometry analysis of amino acids on the protein was cytokeratin.2.5Bioinformatics analysis of molecular interactions and potential connections with dengue virus.Conclusion:1. The DENV-2EDⅢ polyclonal antibody can neutralize DENV-2virus and prevent dengue virus into susceptible cells.2. A specific45kDa binding molecules maybe receptor of dengue virus in BHK-21cell by VOPBA.3. Mass spectrometry suggests the protein is cytokeratin, which need to further study.4. Simulation and prediction by bio-informatics analysis of potential target molecules with dengue virus EDIII possible binding modes and protein binding sites.In summary, EDIII was successfully constructed and amplified; pET-32a(+)-DV2-EDIII were successfully constructed; After induction with IPTG, a specific soluble protein was expressed from Escherichia coli which contains the expression plasmid pET-32a(+)-DV2-EDⅢ; Purified protein to immunize New Zealand rabbits, harvest polyclonal anti-dengue virus antibody. Competitive assays and plaque neutralization demonstrated a promising result of the recombinant protein.A45kDa protein of BHK-21cells was screened and identified by VOPBA. Mass spectrometry suggests this protein may be cytokeratin, which may work for Dengue virus entering the cell. Besides, the binding modes and residues of cytokeratin and DENV-2EDIII protein were simulated by bioinformatics software, which may lay down the great foundation for further designing mutants and understanding dengue pathogenesis. However, the exact nature of the interactions and the identification of various signal transduction pathways involved remain to be explored.