Highly Conserved Core Residues Define Old-World Alphaviruses and Trace Early Evolutionary Divergence.

Alphaviruses are positive-sense, single-stranded RNA viruses that assemble into striking double-icosahedral particles. During budding, the nucleocapsid core forms in the cytoplasm and adopts a T = 4 icosahedral symmetry, a hallmark characteristic of Alphaviruses. Here, we combine structural, evolutionary, and phylogenetic analyses to identify the amino acids most likely to govern capsomer formation (pentamers and hexamers) and core organization. We find a small network of residues associated with capsid protein-protein and capsomer-capsomer interactions is highly conserved in present-day old-world alphaviruses but diverges in New-World lineages. This suggests that the common ancestor of both groups likely assembled cores using interaction networks similar to those seen in present-day Old-World viruses. We propose that early divergence in these interactions altered assembly efficiency and assembly pathways, potentially contributing to differences in replication dynamics and pathogenicity among New-World alphaviruses. This attenuation may reflect an adaptive trade-off, in which reduced assembly efficiency lowers viral replication and virulence, supporting long-term persistence in enzootic cycles. By revealing how specific residues control capsid architecture and tracing their evolutionary history, this study provides new insights into alphavirus assembly mechanisms, opening new avenues for antiviral strategies and rational vaccine design. These findings establish a framework for investigating the relationship between capsid architecture, assembly pathways, and viral evolution.