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, their nucleocapsid core forms in the cytoplasm and adopts a T=4 icosahedral symmetry, a hallmark of Alphaviruses. Here, we combine structural and phylogenetic analyses to identify the amino acids most likely to govern capsomer formation (pentamers and hexamers) and core organization. We find that these residues are highly conserved in present-day Old-World alphaviruses but are divergent in New-World lineages. This suggests that the common ancestor of both groups likely assembled cores using the same molecular interactions seen in present-day Old-World viruses. We propose that early divergence in these interactions weakened core assembly efficiency, potentially contributing to the attenuation observed in encephalitic 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. Revealing how specific residues control capsid architecture and tracing their evolutionary history, this study provides key insights into alphavirus assembly mechanisms, opening new avenues for antiviral strategies and rational vaccine design.