Direct visualization of the native structure of viroid RNAs at single-molecule resolution by atomic force microscopy

[EN] Viroids are small infectious, non-protein-coding circular RNAs that replicate independently and, in some cases, incite diseases in plants. They are classified into two families: Pospiviroidae, composed of species that have a central conserved region (CCR) and replicate in the cell nucleus, and Avsunviroidae, containing species that lack a CCR and whose multimeric replicative intermediates of either polarity generated in plastids self-cleave through hammerhead ribozymes. The compact, rod-like or branched, secondary structures of viroid RNAs have been predicted by RNA folding algorithms and further examined using different in vitro and in vivo experimental techniques. However, direct data about their native tertiary structure remain scarce. Here we have applied atomic force microscopy (AFM) to image at single-molecule resolution different variant RNAs of three representative viroids: potato spindle tuber viroid (PSTVd, family Pospiviroidae), peach latent mosaic viroid and eggplant latent viroid (PLMVd and ELVd, family Avsunviroidae). Our results provide a direct visualization of their native, three-dimensional conformations at 0 and 4 mM Mg2+ and highlight the role that some elements of tertiary structure play in their stabilization. The AFM images show that addition of 4 mM Mg2+ to the folding buffer results in a size contraction in PSTVd and ELVd, as well as in PLMVd when the kissing-loop interaction that stabilizes its 3D structure is preserved.This work was supported by the Spanish Ministerio de Economia y Competitividad (MINECO) grants BIO2016-79618-R (funded by EU under the FEDER programme) to C.B. and BFU2104-56812-P to R.F., as well as by the Comunidad de Madrid grant S2018/NMT-4349 to L.V. CIBERehd is funded by the Instituto de Salud Carlos III (ISCIII).Moreno, M.; Vázquez, L.; López Carrasco, A.; Martín-Gago, JA.; Flores Pedauye, R.; Briones, C. (2019). Direct visualization of the native structure of viroid RNAs at single-molecule resolution by atomic force microscopy. RNA Biology. 16(3):295-308. https://doi.org/10.1080/15476286.2019.1572436S295308163Diener, T. O. (2003). Discovering viroids — a personal perspective. Nature Reviews Microbiology, 1(1), 75-80. doi:10.1038/nrmicro736Flores, R., Hernández, C., Alba, A. E. M. de, Daròs, J.-A., & Serio, F. D. (2005). Viroids and Viroid-Host Interactions. Annual Review of Phytopathology, 43(1), 117-139. doi:10.1146/annurev.phyto.43.040204.140243Ding, B. (2009). The Biology of Viroid-Host Interactions. Annual Review of Phytopathology, 47(1), 105-131. doi:10.1146/annurev-phyto-080508-081927Zhang, Z., Qi, S., Tang, N., Zhang, X., Chen, S., Zhu, P., … Wu, Q. (2014). Discovery of Replicating Circular RNAs by RNA-Seq and Computational Algorithms. PLoS Pathogens, 10(12), e1004553. doi:10.1371/journal.ppat.1004553Serra, P., Messmer, A., Sanderson, D., James, D., & Flores, R. (2018). Apple hammerhead viroid-like RNA is a bona fide viroid: Autonomous replication and structural features support its inclusion as a new member in the genus Pelamoviroid. Virus Research, 249, 8-15. doi:10.1016/j.virusres.2018.03.001Hadidi, A., Barba, M., Hong, N., & Hallan, V. (2017). Apple Scar Skin Viroid. Viroids and Satellites, 217-228. doi:10.1016/b978-0-12-801498-1.00021-8Flores, R., Minoia, S., Carbonell, A., Gisel, A., Delgado, S., López-Carrasco, A., … Di Serio, F. (2015). Viroids, the simplest RNA replicons: How they manipulate their hosts for being propagated and how their hosts react for containing the infection. Virus Research, 209, 136-145. doi:10.1016/j.virusres.2015.02.027Hammann, C., & Steger, G. (2012). Viroid-specific small RNA in plant disease. RNA Biology, 9(6), 809-819. doi:10.4161/rna.19810Kovalskaya, N., & Hammond, R. W. (2014). Molecular biology of viroid–host interactions and disease control strategies. Plant Science, 228, 48-60. doi:10.1016/j.plantsci.2014.05.006Tsagris, E. M., Martínez de Alba, Á. E., Gozmanova, M., & Kalantidis, K. (2008). Viroids. Cellular Microbiology, 10(11), 2168-2179. doi:10.1111/j.1462-5822.2008.01231.xGrill, L. K., & Semancik, J. S. (1978). RNA sequences complementary to citrus exocortis viroid in nucleic acid preparations from infected Gynura aurantiaca. Proceedings of the National Academy of Sciences, 75(2), 896-900. doi:10.1073/pnas.75.2.896Branch, A. D., Benenfeld, B. J., & Robertson, H. D. (1988). Evidence for a single rolling circle in the replication of potato spindle tuber viroid. Proceedings of the National Academy of Sciences, 85(23), 9128-9132. doi:10.1073/pnas.85.23.9128Branch, A. D., & Robertson, H. D. (1984). A Replication Cycle for Viroids and Other Small Infectious RNA’s. Science, 223(4635), 450-455. doi:10.1126/science.6197756Daros, J. A., Marcos, J. F., Hernandez, C., & Flores, R. (1994). Replication of avocado sunblotch viroid: evidence for a symmetric pathway with two rolling circles and hammerhead ribozyme processing. Proceedings of the National Academy of Sciences, 91(26), 12813-12817. doi:10.1073/pnas.91.26.12813Feldstein, P. A., Hu, Y., & Owens, R. A. (1998). Precisely full length, circularizable, complementary RNA: An infectious form of potato spindle tuber viroid. Proceedings of the National Academy of Sciences, 95(11), 6560-6565. doi:10.1073/pnas.95.11.6560Daros, J.-A., & Flores, R. (2004). Arabidopsis thaliana has the enzymatic machinery for replicating representative viroid species of the family Pospiviroidae. Proceedings of the National Academy of Sciences, 101(17), 6792-6797. doi:10.1073/pnas.0401090101Flores, R., Gago-Zachert, S., Serra, P., Sanjuán, R., & Elena, S. F. (2014). Viroids: Survivors from the RNA World? Annual Review of Microbiology, 68(1), 395-414. doi:10.1146/annurev-micro-091313-103416Diener, T. O. (1989). Circular RNAs: relics of precellular evolution? Proceedings of the National Academy of Sciences, 86(23), 9370-9374. doi:10.1073/pnas.86.23.9370Ruiz-Mirazo, K., Briones, C., & de la Escosura, A. (2013). Prebiotic Systems Chemistry: New Perspectives for the Origins of Life. Chemical Reviews, 114(1), 285-366. doi:10.1021/cr2004844Flores, R., Serra, P., Minoia, S., Di Serio, F., & Navarro, B. (2012). Viroids: From Genotype to Phenotype Just Relying on RNA Sequence and Structural Motifs. Frontiers in Microbiology, 3. doi:10.3389/fmicb.2012.00217Steger, G., & Perreault, J.-P. (2016). Structure and Associated Biological Functions of Viroids. Advances in Virus Research, 141-172. doi:10.1016/bs.aivir.2015.11.002Diener, T. O. (1972). Potato spindle tuber viroid. Virology, 50(2), 606-609. doi:10.1016/0042-6822(72)90412-6Gross, H. J., Domdey, H., Lossow, C., Jank, P., Raba, M., Alberty, H., & Sänger, H. L. (1978). Nucleotide sequence and secondary structure of potato spindle tuber viroid. Nature, 273(5659), 203-208. doi:10.1038/273203a0Gast, F.-U., Kempe, D., Spieker, R. L., & Sänger, H. L. (1996). Secondary Structure Probing of Potato Spindle Tuber Viroid (PSTVd) and Sequence Comparison with Other Small Pathogenic RNA Replicons Provides Evidence for Central Non-canonical Base-pairs, Large A-rich Loops, and a Terminal Branch. Journal of Molecular Biology, 262(5), 652-670. doi:10.1006/jmbi.1996.0543Giguère, T., Raj Adkar-Purushothama, C., & Perreault, J.-P. (2014). Comprehensive Secondary Structure Elucidation of Four Genera of the Family Pospiviroidae. PLoS ONE, 9(6), e98655. doi:10.1371/journal.pone.0098655López-Carrasco, A., & Flores, R. (2016). Dissecting the secondary structure of the circular RNA of a nuclear viroid in vivo: A «naked» rod-like conformation similar but not identical to that observed in vitro. RNA Biology, 14(8), 1046-1054. doi:10.1080/15476286.2016.1223005Wang, Y., Zirbel, C. L., Leontis, N. B., & Ding, B. (2018). RNA 3-dimensional structural motifs as a critical constraint of viroid RNA evolution. PLOS Pathogens, 14(2), e1006801. doi:10.1371/journal.ppat.1006801Zhong, X., Leontis, N., Qian, S., Itaya, A., Qi, Y., Boris-Lawrie, K., & Ding, B. (2006). Tertiary Structural and Functional Analyses of a Viroid RNA Motif by Isostericity Matrix and Mutagenesis Reveal Its Essential Role in Replication. Journal of Virology, 80(17), 8566-8581. doi:10.1128/jvi.00837-06Zhong, X., Tao, X., Stombaugh, J., Leontis, N., & Ding, B. (2007). Tertiary structure and function of an RNA motif required for plant vascular entry to initiate systemic trafficking. The EMBO Journal, 26(16), 3836-3846. doi:10.1038/sj.emboj.7601812Zhong, X., Archual, A. J., Amin, A. A., & Ding, B. (2008). A Genomic Map of Viroid RNA Motifs Critical for Replication and Systemic Trafficking. The Plant Cell, 20(1), 35-47. doi:10.1105/tpc.107.056606Hernandez, C., & Flores, R. (1992). Plus and minus RNAs of peach latent mosaic viroid self-cleave in vitro via hammerhead structures. Proceedings of the National Academy of Sciences, 89(9), 3711-3715. doi:10.1073/pnas.89.9.3711Fadda, Z., Daròs, J. A., Fagoaga, C., Flores, R., & Duran-Vila, N. (2003). Eggplant Latent Viroid , the Candidate Type Species for a New Genus within the Family Avsunviroidae (Hammerhead Viroids). Journal of Virology, 77(11), 6528-6532. doi:10.1128/jvi.77.11.6528-6532.2003Navarro, B., & Flores, R. (1997). Chrysanthemum chlorotic mottle viroid: Unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes. Proceedings of the National Academy of Sciences, 94(21), 11262-11267. doi:10.1073/pnas.94.21.11262Bussière, F., Ouellet, J., Côté, F., Lévesque, D., & Perreault, J. P. (2000). Mapping in Solution Shows the Peach Latent Mosaic Viroid To Possess a New Pseudoknot in a Complex, Branched Secondary Structure. Journal of Virology, 74(6), 2647-2654. doi:10.1128/jvi.74.6.2647-2654.2000GAGO, S. (2005). A kissing-loop interaction in a hammerhead viroid RNA critical for its in vitro folding and in vivo viability. RNA, 11(7), 1073-1083. doi:10.1261/rna.2230605Dube, A., Baumstark, T., Bisaillon, M., & Perreault, J.-P. (2010). The RNA strands of the plus and minus polarities of peach latent mosaic viroid fold into different structures. RNA, 16(3), 463-473. doi:10.1261/rna.1826710Sogo, J. M., Koller, T., & Diener, T. O. (1973). Potato spindle tuber viroid. Virology, 55(1), 70-80. doi:10.1016/s0042-6822(73)81009-8Goodman, T. C., Nagel, L., Rappold, W., Klotz, G., & Riesner, D. (1984). Viroid replication: equilibrium association constant and comparative activity measurements for the viroid-polymerase interaction. Nucleic Acids Research, 12(15), 6231-6246. doi:10.1093/nar/12.15.6231Sanger, H. L., Klotz, G., Riesner, D., Gross, H. J., & Kleinschmidt, A. K. (1976). Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures. Proceedings of the National Academy of Sciences, 73(11), 3852-3856. doi:10.1073/pnas.73.11.3852McClements, W. L., & Kaesberg, P. (1977). Size and secondary structure of potato spindle tuber viroid. Virology, 76(2), 477-484. doi:10.1016/0042-6822(77)90230-6Bustamante, C., & Keller, D. (1995). Scanning Force Microscopy in Biology. Physics Today, 48(12), 32-38. doi:10.1063/1.881478Hansma, H. G., Kasuya, K., & Oroudjev, E. (2004). Atomic force microscopy imaging and pulling of nucleic acids. Current Opinion in Structural Biology, 14(3), 380-385. doi:10.1016/j.sbi.2004.05.005Kuznetsov, Y. G., Daijogo, S., Zhou, J., Semler, B. L., & McPherson, A. (2005). Atomic Force Microscopy Analysis of Icosahedral Virus RNA. Journal of Molecular Biology, 347(1), 41-52. doi:10.1016/j.jmb.2005.01.006Alvarez, D. E., Lodeiro, M. F., Ludueña, S. J., Pietrasanta, L. I., & Gamarnik, A. V. (2005). Long-Range RNA-RNA Interactions Circularize the Dengue Virus Genome. Journal of Virology, 79(11), 6631-6643. doi:10.1128/jvi.79.11.6631-6643.200

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