Multiple Protein–Protein Interactions Drive the Assembly and Budding of the Chikungunya Virion

ABSTRACT The assembly of enveloped viruses is a highly orchestrated process that depends on the coupling of multiple protein–protein interactions within a membrane environment. To gain mechanistic insight into this process, we use Chikungunya virus as a model system to study Alphavirus assembly, focusing on the interplay between core–spike and spike–spike interactions. We begin with coarse-grained molecular dynamics simulations to systematically explore how the symmetry of the nucleocapsid core, together with the relative strengths of spike–core and spike– spike interactions, influences budding efficiency and the emergence of icosahedral particle symmetry. Building on these computational predictions, we perform site-directed mutagenesis on Chikungunya virus 181/25 and examine the consequences for particle assembly and budding in cultured cells, as well as the impact of these mutations during in-cellulo assembly. Our results reveal that canonical core–spike interactions, while necessary, are not sufficient for successful assembly. Instead, lateral interactions among glycoproteins emerge as critical determinants of efficient budding, particle stability, and the maintenance of icosahedral symmetry. Together, these findings provide an integrated computational and experimental framework for understanding the molecular principles governing Alphavirus assembly.