ABSTRACT
Members of the low-density lipoprotein receptor family possess various numbers of ligand binding repeats that non-equally contribute to binding of minor group human rhinoviruses. Using an artificial concatemer of five copies of repeat 3 of the human very-low density lipoprotein receptor, we demonstrate protection of HRV2 against low-pH mediated uncoating and inhibition of penetration of an RNA-specific fluorescent dye into the intact virion. This indicates that the recombinant receptor inhibits viral breathing and irreversible conformational modifications of the capsid that precede RNA release, providing a new mechanism for rhinovirus neutralization by soluble receptor molecules.
Subject(s)
Anti-Retroviral Agents/pharmacology , RNA, Viral/drug effects , Receptors, LDL/chemistry , Recombinant Fusion Proteins/pharmacology , Rhinovirus/drug effects , Anti-Retroviral Agents/chemistry , Fluorescent Dyes/analysis , Humans , RNA, Viral/analysis , Receptors, LDL/metabolism , Virion/chemistry , Virion/drug effectsABSTRACT
Labeling of iron-free human serum transferrin by an amine-reactive probe, fluorescein isothiocyanate (FITC) was monitored with different dye to protein ratios. The degree of labeling was followed and determined by capillary electrophoresis. Depending on the number of the bound FITC, a shift in the electropherogram was observed, but the conjugated-transferrin forms were resolved from the unbound FITC in zone electrophoresis. The lowest protein concentration that resulted in detectable transferrin conjugate was 0.13 microM, and the limit of detection of the conjugated protein was 0.13 nM (10 ng/ml) with a signal to noise ratio of one to three.
Subject(s)
Electrophoresis, Capillary/methods , Fluorescein-5-isothiocyanate/chemistry , Transferrin/metabolism , Endocytosis , HumansABSTRACT
The capsid of human rhinovirus serotype 2, consisting of four viral proteins, was fluorescence-labeled with fluorescein isothiocyanate and analyzed by capillary electrophoresis using UV and laser-induced fluorescence detection. Heat denaturation, proteolytic digestion, and receptor binding were applied for confirmation of the identity of the peak with the labeled virus. Incomplete derivatization with the fluorophore preserved the affinity of the virus for its receptor, indicating that its cell entry pathway is unperturbed by this chemical modification; indeed, an infectivity assay confirms that the labeled virus samples are infectious. The results show that fluorescence labeling of the viral capsid might lead to a valuable probe for studying infection processes in the living cell.
Subject(s)
Capsid Proteins/analysis , Electrophoresis, Capillary/methods , Fluorescein-5-isothiocyanate/chemistry , Fluorescent Dyes , Rhinovirus/chemistry , Capsid/chemistry , Capsid/metabolism , Capsid Proteins/genetics , Capsid Proteins/metabolism , HumansABSTRACT
The crystallographic T = 1 (pseudo T = 3) icosahedral symmetry of the human rhinovirus capsid dictates the presence of 60 identical, symmetry related surface structures that are available for antibody and receptor binding. X-ray crystallography has shown that 60 individual very-low density lipoprotein receptor (VLDLR) modules bind to HRV2. Their arrangement around the fivefold axes of the virion suggested that tandem oligomers of such modules could attach simultaneously to symmetry-related sites. By resolving virus particles carrying various numbers of artificial recombinant concatemers of VLDLR repeat 3 (V33333) by capillary electrophoresis and extrapolation of the measured mobilities to that at saturation of all binding sites, we present evidence for up to 12 molecules of the concatemer to bind one single virion.
Subject(s)
Receptors, LDL/metabolism , Receptors, Virus/metabolism , Rhinovirus/metabolism , Virion/metabolism , Electrophoresis, Capillary , HumansABSTRACT
This contribution deals with comparative studies on the chiral separation of thiazide diuretics using cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD-RH), cellulose tris(4-methylbenzoate) (Chiralcel OJ-R) and teicoplanin (Chirobiotic T) phases. All columns showed good chiral recognition ability for this class of compounds. Out of seven compounds investigated, six were resolved with baseline resolution with at least one of the three columns.