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1.
PLoS One ; 13(6): e0199397, 2018.
Article in English | MEDLINE | ID: mdl-29928016

ABSTRACT

The envelope glycoprotein 51 (gp51) is essential for bovine leukaemia virus (BLV) entry to bovine B-lymphocytes. Although the bovine adaptor protein 3 complex subunit delta-1 (boAP3D1) has been proposed as the potential receptor, the specific ligand-receptor interaction has not yet been completely defined and boAP3D1 receptor and gp51 3D structures have not been determined. This study was thus aimed at a functional annotation of boAP3D1 cellular adaptor protein and BLV gp51 and, proposing a reliable model for gp51-AP3D1 interaction using bioinformatics tools. The boAP3D1 receptor interaction patterns were calculated based on models of boAP3D1 receptor and gp51 complexes' 3D structures, which were constructed using homology techniques and data-driven docking strategy. The results showed that the participation of 6 key amino acids (aa) on gp51 (Asn170, Trp127, His115, Ala97, Ser98 and Glu128) and 4 aa on AP3D1 (Lys925, Asp807, Asp695 and Arg800) was highly probable in the interaction between gp51 and BLVR domains. Three gp51 recombinant peptides were expressed and purified to validate these results: the complete domain (rgp51), the N-terminal portion (rNgp51) and the C-terminal fragment (rCgp51); and binding assays to Madin-Darby bovine kidney (MDBK) cells were then carried out with each recombinant. It was found that rNgp51 preferentially bound to MDBK cells, suggesting this domain's functional role during invasion. The rNgp51-MDBK cell interaction was sensitive to trypsin (98% reduction) and chymotrypsin treatment (80% reduction). These results highlighted that the N-terminal portion of gp51 interacted in vitro with the AP3D1 receptor and provides a plausible in silico interaction model.


Subject(s)
Adaptor Protein Complex delta Subunits/metabolism , Computer Simulation , Leukemia Virus, Bovine/metabolism , Viral Envelope Proteins/metabolism , Adaptor Protein Complex delta Subunits/chemistry , Amino Acid Sequence , Animals , Cattle , Cell Line , Hydrophobic and Hydrophilic Interactions , Molecular Docking Simulation , Molecular Sequence Annotation , Protein Binding , Protein Interaction Mapping , Protein Structure, Secondary , Recombinant Proteins/metabolism , Reproducibility of Results , Thermodynamics , Viral Envelope Proteins/chemistry
2.
J Virol Methods ; 127(1): 33-9, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15893563

ABSTRACT

The purpose of the present study was to implement a fluorometric method for detecting and quantifying viral antigens in human meduloblastoma cells infected by two types of fixed rabies virus (CVS-MB and CVS-BHK) and a street virus using a cell-enzyme linked immunosorbent assay (cell-ELISA) technique; alkaline phosphatase was used as the antibody-marker enzyme and 4-methyl-umbelliferyl-phosphate as the fluorogenic substrate. The system was used for detecting up to 1:10,000 viral inoculums, followed by evaluating the effect of heparin on infection. Infected cultures were reliably differentiated from their respective negative controls in both assays allowing data to be analysed statistically. As reported in another study, heparin produces strong inhibition when the CVS-BHK viral strain is used for infection; it has thus been suggested that it binds to the neural cell adhesion molecule and could be blocked by using this drug. This fluorometric method is less time-consuming, has increased reproducibility and useful for quantitation of collected data and can therefore be considered as a useful tool for research.


Subject(s)
Antigens, Viral/isolation & purification , Enzyme-Linked Immunosorbent Assay/methods , Heparin/pharmacology , Rabies virus/isolation & purification , Antibody Specificity , Cell Line, Tumor , Dose-Response Relationship, Drug , Fluorometry , Humans , Hymecromone/analogs & derivatives , Neural Cell Adhesion Molecules/biosynthesis , Neural Cell Adhesion Molecules/chemistry , Rabies virus/drug effects , Rabies virus/immunology
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