The Palm Subdomain-based Active Site is Internally Permuted in Viral RNA-dependent RNA Polymerases of an Ancient Lineage

Template-dependent polynucleotide synthesis is catalyzed by enzymes whose core component includes a ubiquitous αβ palm subdomain comprising A, B and C sequence motifs crucial for catalysis. Due to its unique, universal conservation in all RNA viruses, the palm subdomain of RNA-dependent RNA polymerases (RdRps) is widely used for evolutionary and taxonomic inferences. We report here the results of elaborated computer-assisted analysis of newly sequenced replicases from Thosea asigna virus (TaV) and the closely related Euprosterna elaeasa virus (EeV), insect-specific ssRNA+ viruses, which revise a capsid-based classification of these viruses with tetraviruses, an Alphavirus-like family. The replicases of TaV and EeV do not have characteristic methyltransferase and helicase domains, and include a putative RdRp with a unique C–A–B motif arrangement in the palm subdomain that is also found in two dsRNA birnaviruses. This circular motif rearrangement is a result of migration of ∼22 amino acid (aa) residues encompassing motif C between two internal positions, separated by ∼110 aa, in a conserved region of ∼550 aa. Protein modeling shows that the canonical palm subdomain architecture of poliovirus (ssRNA+) RdRp could accommodate the identified sequence permutation through changes in backbone connectivity of the major structural elements in three loop regions underlying the active site. This permutation transforms the ferredoxin-like β1αAβ2β3αBβ4 fold of the palm subdomain into the β2β3β1αAαBβ4 structure and brings β-strands carrying two principal catalytic Asp residues into sequential proximity such that unique structural properties and, ultimately, unique functionality of the permuted RdRps may result. The permuted enzymes show unprecedented interclass sequence conservation between RdRps of true ssRNA+ and dsRNA viruses and form a minor, deeply separated cluster in the RdRp tree, implying that other, as yet unidentified, viruses may employ this type of RdRp. The structural diversification of the palm subdomain might be a major event in the evolution of template-dependent polynucleotide polymerases in the RNA–protein world.