Uncovering the Structural and Functional Implications of Uncharacterized NSPs and Variations in the Molecular Toolkit Across Mammalian and Non-Mammalian Arteriviruses

Despite considerable scrutiny of mammalian arterivirus genomes, their genomic architecture remains incomplete, with several unannotated non-structural proteins (NSPs) and the puzzling 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 identify 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, showing structural alignment with N7-MTases in Nidovirales, yet it 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 2ʹ O-MTase and an exonuclease domain, which are typically absent in mammalian arteriviruses, highlighting contrasting evolutionary trends and variations in their molecular armory. Similar lineage-specific paterns are observed in the diversification of papain-like proteases and structural proteins. Overall, the study extends our knowledge of arterivirus genomic diversity and evolution.