RNA three-dimensional structure drives the sequence organization of potato spindle tuber viroid quasispecies

RNA viruses and viroids exist and evolve as quasispecies due to error-prone replication. Quasispecies consist of a few dominant master sequences alongside numerous variants that contribute to genetic diversity. Upon environmental changes, certain variants within quasispecies have the potential to become the dominant sequences, leading to the emergence of novel infectious strains. However, the emergence of new infectious variants remains unpredictable. Using mutant pools prepared by saturation mutagenesis of selected stem and loop regions, our study of potato spindle tuber viroid (PSTVd) demonstrates that mutants forming local three-dimensional (3D) structures similar to the wild type (WT) are more likely to accumulate in PSTVd quasispecies. The selection mechanisms underlying this biased accumulation are likely associated with cell-to-cell movement and long-distance trafficking. Moreover, certain trafficking-defective PSTVd mutants can be spread by functional sister genomes in the quasispecies. Our study reveals that the RNA 3D structure of stems and loops constrains the evolution of viroid quasispecies. Mutants with a structure similar to WT have a higher likelihood of being maintained within the quasispecies and can potentially give rise to novel infectious variants. These findings emphasize the potential of targeting RNA 3D structure as a more robust approach to defend against viroid infections. Our study investigates the world of RNA viruses and viroids, which are constantly evolving due to error-prone replication. These pathogens exist as quasispecies, featuring dominant master sequences and numerous variants. Understanding how new infectious strains emerge from this genetic diversity remains challenging. Focusing on potato spindle tuber viroid (PSTVd), targeted mutagenesis of specific stem and loop regions was performed to generate mutant pools. Plants were infected with mutant pools, and progeny were analyzed following cell-to-cell movement and long-distance trafficking. Our results reveal that mutants with 3D structures similar to the wild type viroid are more likely to accumulate within quasispecies. This accumulation is associated with the ability to move between cells and travel long distances within the plant host. Even mutants with impaired mobility can be spread by functional counterparts within the quasispecies. This study highlights the role of RNA 3D structures in shaping viroid quasispecies evolution. Mutants resembling the wild type viroid have a greater chance of persisting within quasispecies and may give rise to new infectious variants. These findings offer insight into viroid quasispecies dynamics and identify RNA 3D structure as a promising avenue for resistance strategies.