RT Journal Article SR Electronic T1 Hot-spots and their contribution to the self-assembly of the viral capsid: in-vitro validation JF bioRxiv FD Cold Spring Harbor Laboratory SP 724146 DO 10.1101/724146 A1 Alejandra Gabriela Valdez-Lara A1 Mariana Andrade-Medina A1 Josué Alejandro Alemán-Vilis A1 Aldo Adrián Pérez-Montoya A1 Nayely Pineda-Aguilar A1 Eduardo Martínez-Guerra A1 Abigail Roldán-Salgado A1 Paul Gaytán A1 Mauricio Carrillo-Tripp YR 2019 UL http://biorxiv.org/content/early/2019/11/15/724146.abstract AB The viral capsid is a macromolecular complex formed by self-assembled proteins (CPs) which, in many cases, are biopolymers with an identical amino acid sequence. Specific CP-CP interactions drive the capsid self-assembly process. However, it is believed that only a small set of protein-protein interface residues significantly contribute to the formation of the capsid; the so-called “hot-spots”. Here, we investigate the effect of in-vitro point-mutations on the Bromoviridae family structure-conserved interface residues of the icosahedral Cowpea Chlorotic Mottle Virus, previously hypothesized as hot-spots. We study the self-assembly of those mutated recombinant CPs for the formation of capsids by Thermal Shift Assay (TSA). We show that the TSA biophysical technique is a reliable way to characterize capsid assembly. Our results show that point-mutations on non-conserved interface residues produce capsids indistinguishable from the wild-type. In contrast, a single mutation on structure-conserved residues E176 or V189 prevents the formation of the capsid while maintaining the tertiary fold of the CP. Our findings provide experimental evidence of the in-silico conservation-based hot-spot prediction accuracy. As a whole, our methodology provides a framework that could aid in the rational development of molecules to inhibit virus formation, or advance capsid bioengineering to design for their stability, function and applications.CCMVCowpea Chlorotic Mottle VirusCPcapsid proteinVLPvirus-like particlePPIprotein-protein interactionsWTwild-typeTSAThermal Shift AssayDSCDifferential Scanning Micro-calorimetryCOMcenter-of-mass