Cryo-EM reveals a C15 annular stacking assembly of the VEEV nsP3 Alphavirus Unique Domain.

Alphaviruses are arthropod-borne pathogens that cause significant human disease, including severe arthritis and encephalitis. Among the four nonstructural proteins (nsP1-nsP4) that form the viral replication complex (RC), nsP3 is the least functionally characterized. Recent studies have shown that CHIKV-nsP3 forms a right-handed helical scaffold, while Venezuelan equine encephalitis virus (VEEV) nsP3 forms gel-like biomolecular condensates mediated by its Alphavirus Unique Domain (AUD). In this study, we used cryo-electron microscopy to reveal a closed annular stacking assembly of VEEV-nsP3, composed of C15-symmetric rings that stack axially without helical offset-fundamentally differing from the helical arrangement of CHIKV. Structural comparisons demonstrate that this divergence arises from the loss of three key hydrogen bond networks, particularly an axial hydrogen bond that drives helical propagation in CHIKV but is uniquely absent in VEEV. Furthermore, we propose that the proteolytic cleavage at the nsP2/nsP3 junction regulates the annular to tubular transition. Our work provides insights into the structural diversity and dynamic regulation of alphavirus RC crown formation, with implications for understanding replication complex assembly across this viral lineage.