title: Optical biosensor analysis in studying herpes simplex virus glycoprotein D binding to target nectin1 receptor.
abstract: Studies on molecular interactions between cellular receptors of herpes simplex virus (HSV) and the viral glycoproteins showing receptor-binding activity are of great relevance for understanding the molecular basis of virus entry. Information on such interactions further provides the basis for a rational design of antiviral drugs. A variety of biochemical and biophysical methodologies are used for determining the binding parameters of interacting biomolecules. Most of them require relatively high amounts of the analyzed compounds, or the use of labeled target molecules. Here, we report the study of the binding of two recombinant forms of HSV glycoprotein D, gD(Delta 290-299t) and gD(305t), and a recombinant form of the human cellular receptor for HSV, nectin1-Fc, by using an optical biosensor (IAsys Plus, Affinity Sensors, UK). This device detects and quantifies the changes in refractive index in the vicinity of the surface of sensor chips to which ligands are immobilized. The changes in the refractive index are proportional to the change in the absorbed mass, thus the analysis allows the monitoring of the interaction process in real-time and the determination of the binding parameters. HSV cellular receptor has been immobilized on the surface of the biosensor cuvette, bearing a carboxymethyl dextran layer. The immobilized receptor cuvette was then used for the binding experiments of the two glycoproteins. A significant difference in their dissociation constants was determined, showing for the gD(Delta 290-299t) protein a much higher affinity (K(D), 2.8 x 10(-7) M) with respect to gD(305t) (K(D), 2.8 x 10(-6) M). The active ligand concentration decreased on time, however the binding properties of the immobilized receptor were maintained over 5 weeks.
authors.affiliation: Department of Pharmaceutical Sciences, University of Bologna, via Belmeloro 6, Bologna 40126, Italy. bertucci@alma.unibo.it
authors.forename: Carlo
authors.initials: C
authors.lastname: Bertucci
authors.forename: Samanta
authors.initials: S
authors.lastname: Cimitan
authors.forename: Laura
authors.initials: L
authors.lastname: Menotti
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D015815:Cell Adhesion Molecules / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D000075983:Nectins; D055095:Optics and Photonics; D011485:Protein Binding / Q000502:physiology; D014759:Viral Envelope Proteins / Q000032:analysis* / Q000378:metabolism*
chemical_list: D015815:Cell Adhesion Molecules; C000619010:NECTIN1 protein, human; C000619015:Nectin1 protein, mouse; D000075983:Nectins; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1