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

Figures

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  Figure 1.. Genomic architecture and proteome composition of arterivirus genomes. (A) Genomic structure and protein domain organization of representative mammalian arteriviruses. ORFs are depicted as colored arrows, with their encoded domains labeled and highlighted. The color scheme is consistently applied across the figure for homologous regions and domains. Newly identified functional domains—the C-terminal winged helix-turn-helix (wHTH) domain of NSP3, the small β-barrel (SBB) domain of NSP7, and the N7-MTase-like domain of NSP12—are outlined with black borders. (B) Genomic structure and protein domain organization of arterivirus genomes from non-mammalian hosts. In cases where ORF1a and ORF1b regions are merged into a single, larger ORF [as observed in NGSA, Guangdong greater green snake arterivirus (GGGSA), HOFA, HHPA, and Chinese broad-headed pond turtle arterivirus (CBPTA) genomes] or split into multiple shorter ORFs (as in TSHSA genome), the corresponding regions of ORF1a and ORF1b are highlighted using the same color scheme as their mammalian counterparts (light brown background for ORF1a and light blue for ORF1b) to aid easier comparison. Highly divergent genomic regions predicted…

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  Figure 2.. Comparative analysis and structural characteristics of the arterivirus NSP3C wHTH and NSP7 SBB domains. (A) The first two 3D structures in panel (A) illustrate the AF3-predicted C-terminal wHTH domain of NSP3 (NSP3C) from arteriviruses, alongside the experimentally resolved and corresponding wHTH domain in the C-terminal region of NSP4 (NSP4C) from coronaviruses (PDB ID: 3VC8). The structural superpositions on the right display the alignment of NSP3C with NSP4C, as well as NSP3C with the canonical wHTH domain in GnTRs (PDB ID: 3IHU). Panel (B) displays a linear topological representation and comparison of the arterivirus NSP7 SBB domain with closely related SBB domains that, while representing distinct folds, share a broader structural framework. These domains are collectively classified within the SBB assemblage, with NSP7 being identified as a new member in this study. (C) 3D structure and topological comparisons of the arterivirus NSP7 SBB (AF3-predicted), HerA/FtsK ATPase-associated (HAS) β-barrel (PDB ID: 4D2I), and ribosomal SBB L21 (PDB ID: 8CVM). As shown in panel (B), these three domains are closely related and share two β-strands that are common between the nearly…

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  Figure 3.. Conserved structural and sequence features of ExoN and 2′-O-MTase domains in non-mammalian arteriviruses. (A) The first two images in the panel display the representative AF3-predicted 3D structure and the corresponding schematic topology of the ExoN domain from non-mammalian arteriviruses. The structural superpositions on the right show the alignment of the arterivirus ExoN domain with the DnaQ exonuclease (PDB ID: 8H18), with a zoomed-in view (far right) highlighting the alignment of the DEDDH catalytic residues. (B) The first two illustrations in the panel depict the representative AF3-predicted 3D structure and the corresponding schematic topology of the 2′-O-MTase domain from non-mammalian arteriviruses. The structural superpositions on the right illustrate the alignment of the 2′-O-MTase domains from arteriviruses and coronaviruses (NSP16 2′-O-MTase), with a zoomed-in inset (far right) emphasizing the catalytic K-D-K-E tetrad. (C) Representative 2'-O-MTase (PDB: ID 2XYQ) from coronavirus, and AF3-predicted 3D structure of 2′-O-MTases from other Nidovirales. (D) Representative MSA of 2′-O-MTases across all Nidovirales, highlighting key conserved motifs for S-adenosyl…

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  Figure 4.. Structural features of the arterivirus NSP12 N7-MTase-like domain and its comparison with Nidovirale N7-MTases. Panels (A) and (B) display the 3D structure and corresponding topology of the arterivirus NSP12 N7-MTase-like domain (AF3 predicted) and the coronavirus NSP14 N7-MTases (PDB ID: 7QGI). (C) The illustration on the top shows the structural superposition of the arterivirus NSP12 N7-MTase-like domain with the coronavirus NSP14 N7-MTase (front view), while the middle unit presents a top view, focusing primarily on the superposition of the core sheet and the anterior core αH1. The illustration at the bottom of the panel shows the topological comparison of the core β-sheet of the arterivirus NSP12 N7-MTase-like domain with the coronavirus NSP14 N7-MTase. The shared core sheet and the αH1 are colored. (D-F) 3D structure and corresponding topology diagrams of N7-MTases from mesnidovirus, ronidovirus, and tornidovirus. (G) Structural superposition of coronavirus NSP14 N7-MTase with N7-MTases from (i) tornidovirus, (ii) mesnidovirus, and (iii) ronidovirus.

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  Figure 5.. MSA of the arterivirus NSP12 N7-MTase-like domain with N7-MTases across Nidovirales. Panel (A) presents a linear topological comparison of the arterivirus NSP12 N7-MTase-like domain with N7-MTases across Nidovirales. The core α-helices and β-sheet forming the Rossmann fold are shown in blue and red, respectively, with the three-stranded insert in yellow and the C-terminal insert in teal. Secondary structural elements positioned outside of the αβα sandwich is colored gray. (B) Representative MSAs of N7-MTase domains from all five distinct Nidovirales families, with secondary structural elements colored as described. In the coronavirus N7-MTase, a short, inconsistently predicted βS3, which occasionally causes the structure to resemble and portray a seven-strand sheet, is highlighted with a yellow border. A consensus sequence is indicated at the bottom of each alignment. SAM-binding motifs, substrate-binding motifs, and Zn-chelating residues are highlighted in red, black, and magenta, respectively. Excluding the Zn-chelating residues, the SAM and substrate-binding (guanine-binding) residues exhibit erosion and lack conservation in arteriviruses. Arterivirus-specific conserved…

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  Figure 6.. CLANS-based clustering and phylogenetic analysis of arterivirus structural glycoproteins. (A) CLANS-based clustering patterns of structural glycoproteins from both mammalian and non-mammalian arteriviruses. Using CLANS, all-versus-all pairwise sequence similarity-based clustering was conducted, with each glycoprotein cluster shown in distinct colors. (B) Representative AF3-predicted structures of each glycoprotein, with colors consistent with their respective CLANS clusters. (C) ML trees of each glycoprotein. The ML trees show clear clustering patterns that reflect host-specific divergence, especially between primate and non-primate hosts—primate clusters are highlighted in red and brown, non-primate clusters in blue. Likewise, the non-mammalian GPs are distinct, and their clusters are highlighted in green. Nodes with high confidence bootstrap values are marked in red dots.