Hot-spots and their contribution to the self-assembly of the viral capsid: in-vitro validation

Abstract

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.