Understanding evolutionary and functional relationships of RNA polymerases in plant and fungal viruses through structural modeling and divergence date estimations

Abstract

RNA-dependent RNA polymerases (RdRps) are crucial for RNA virus replication and serve as key marker genes for defining deep taxonomic ranks and for understanding viral evolutionary history. Despite their conserved functions and motifs, the high genetic diversity of RdRps complicates precise sequence comparisons across viral families, hindering accurate taxonomic classification of new species. Three-dimensional (3D) RdRp structures can help overcome these challenges through structure-guided alignments. However, such data are rare for myco- and phytoviruses, limiting investigation of their ecological and evolutionary links. In this study, we focused on the highly divergent order Sobelivirales, which includes sobemoviruses infecting plants -- a taxon known for its ancient origin -- and barnaviruses infecting fungi, which remain relatively unknown. Using deep-learning structural modeling, we generated reliable 3D models for 44 sobemoviral and sobelivirad species. Structure-guided alignments, together with new barnaviral and relevant outgroup sequences, enabled robust phylogenetic reconstruction, allowing us to propose revisions of existing viral families and suggest new evolutionary scenarios. Divergence dates were estimated for the first time at this taxonomical rank using the Prisoner of War model, which revealed a divergence of plant and fungal sobelivirads 27.0 {+/-} 10 million years ago -- much more recent than the separation of their respective hosts. This result suggests that cross-kingdom host shifts have contributed more likely to the evolutionary history of Sobelivirales than strict virus-host codivergence. Based on an extended dataset of 127 species, structure and sequence conservation analyses identified molecular signatures of sobeliviral families. These conserved and extented RdRp motifs will facilitate future taxonomic assignments and the development of diagnostic tools. Our interdisciplinary approach, integrating structure modeling and divergence dating, offers new insights into the evolutionary divergence of plant and fungal viruses, with potential applications to other viral orders and families. Author summaryRNA-dependent RNA polymerases (RdRps) are essential for RNA virus replication and serve as important markers for classifying viruses and understanding their evolution. However, with metagenomic studies that rapidly expand the known diversity of RNA viruses, it is increasingly difficult to compare highly divergent RdRps and accurately classify new species. When available, 3D structures of RdRps can help overcome this challenge through structure-guided alignments. Here, we focused on the highly divergent order Sobelivirales, which groups phytoviruses and mycoviruses. Deep-learning models were used to generate reliable 3D structures for 44 representative viral species. Structure-guided alignments combined with the identification of new barnaviruses and outgroups allowed us to build a more accurate viral phylogeny. Based on this finding, we proposed updates to existing viral families and genera within the order Sobelivirales. We estimated divergence dates using a model that previously uncovered the ancient origins of sobemovirus. Notably, we provided the first estimate of when these plant and fungal viruses diverged [~]27.0 {+/-} 10 million years ago, suggesting cross-kingdom host shifts rather than strict virus-host codivergence. We also identified molecular signatures that are useful for future virus classification and diagnosis, with potential applications to other viral groups.