RESEARCH ARTICLE The Structure of the RNA-Dependent RNA Polymerase of a Permutotetravirus Suggests a Link between Primer-Dependent and

Thosea asigna virus (TaV), an insect virus belonging to the Permutatetraviridae family, has a positive-sense single-stranded RNA (ssRNA) genome with two overlapping open reading frames, encoding for the replicase and capsid proteins. The particular TaV replicase includes a structurally unique RNA-dependent RNA polymerase (RdRP) with a sequence permutation in the palm sub-domain, where the active site is anchored. This non-canonical arrangement of the RdRP palm is also found in double-stranded RNA viruses of the Birna-viridae family. Both virus families also share a conserved VPg sequence motif at the poly-merase N-terminus which in birnaviruses appears to be used to covalently link a fraction of the replicase molecules to the 5’-end of the genomic segments. Birnavirus VPgs are pre-sumed to be used as primers for replication initiation. Here we have solved the crystal struc-ture of the TaV RdRP, the first non-canonical RdRP of a ssRNA virus, in its apo- form and bound to different substrates. The enzyme arranges as a stable dimer maintained by mutual interactions between the active site cleft of one molecule and the flexible N-terminal tail of the symmetrically related RdRP. The latter, partially mimicking the RNA template backbone