Unveiling the structural and functional implications of uncharacterized NSPs and variations in the molecular toolkit across arteriviruses.

Despite considerable scrutiny of mammalian arterivirus genomes, their genomic architecture remains incomplete, with several unannotated non-structural proteins (NSPs) and the enigmatic absence of methyltransferase (MTase) domains. Additionally, the host range of arteriviruses has expanded to include seven newly sequenced genomes from non-mammalian hosts, which remain largely unannotated and await detailed comparisons alongside mammalian isolates. Utilizing comparative genomics approaches and comprehensive sequence-structure analysis, we provide enhanced genomic architecture and annotations for arterivirus genomes. We identified the previously unannotated C-terminal domain of NSP3 as a winged helix-turn-helix domain and classified NSP7 as a new small β-barrel domain, both likely involved in interactions with viral RNA. NSP12 is identified as a derived variant of the N7-MTase-like Rossmann fold domain that retains core structural alignment with N7-MTases in Nidovirales but likely lacks enzymatic functionality due to the erosion of catalytic residues, indicating a unique role specific to mammalian arteriviruses. In contrast, non-mammalian arteriviruses sporadically retain a 2'-O-MTase and an exonuclease (ExoN) domain, which are typically absent in mammalian arteriviruses, highlighting contrasting evolutionary trends and variations in their molecular toolkit. Similar lineage-specific patterns are observed in the diversification of papain-like proteases and structural proteins. Overall, the study extends our knowledge of arterivirus genomic diversity and evolution.