E-Protein Protonation Titration-induced Single Particle Chemical Force Spectroscopy for Microscopic Understanding and pI Estimation of Infectious DENV

The ionization state of amino acids on the outer surface of a virus regulates its physicochemical properties toward the sorbent surface. Serologically different strain of dengue virus (DENV) shows different extents of infectivity depending upon their interactions with a receptor on the host cell. To understand the structural dependence of E-protein protonation over its sequence dependence, we have followed E-protein titration kinetics both experimentally and theoretically for two differentially infected dengue serotypes, namely DENV-2 and DENV-4. We have performed an E-protein protonation titration-induced single particle chemical force spectroscopy using an atomic force microscope (AFM) to measure the surface chemistry of DENV in physiological aqueous solutions not only to understand the charge distribution dynamics on virus surface but also to estimate the isoelectric point (pI) accurately for infectious dengue viruses. Cryo-EM structure-based theoretical pI calculations of DENV-2 surface protein were shown to be consistent with the evaluated pI value from force spectroscopy measurements. This is a comprehensive study to understand how the cumulative charge distribution on the outer surface of a specific serotype of DENV regulates a prominent role of infectivity over minute changes at the genetic level.