Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing

[EN] Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplastreplicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses.This work was supported by the European Research Council (erc.europa.eu; ERC-2011-StG-281191-VIRMUT to RS), the Spanish Ministerio de Economia y Competitividad (www.mineco.gob.es; BFU2013-41329 grant to RS, BFU2014-56812-P grant to RF, and a predoctoral fellowship to ALC), and the Spanish Junta de Comunidades de Castilla-La Mancha (www.castillalamancha.es;postdoctoral fellowship to CB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.López-Carrasco, MA.; Ballesteros Martínez, C.; Sentandreu, V.; Delgado Villar, SG.; Gago Zachert, SP.; Flores Pedauye, R.; Sanjuan Verdeguer, R. (2017). Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing. PLoS Pathogens. 13(9):1-17. https://doi.org/10.1371/journal.ppat.1006547S117139Ganai, R. A., & Johansson, E. (2016). DNA Replication—A Matter of Fidelity. Molecular Cell, 62(5), 745-755. doi:10.1016/j.molcel.2016.05.003Lynch, M. (2010). Evolution of the mutation rate. Trends in Genetics, 26(8), 345-352. doi:10.1016/j.tig.2010.05.003Sanjuán, R., & Domingo-Calap, P. (2016). Mechanisms of viral mutation. Cellular and Molecular Life Sciences, 73(23), 4433-4448. doi:10.1007/s00018-016-2299-6Gago, S., Elena, S. F., Flores, R., & Sanjuan, R. (2009). Extremely High Mutation Rate of a Hammerhead Viroid. Science, 323(5919), 1308-1308. doi:10.1126/science.1169202Flores, 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-091313-103416Flores, 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.2015.02.027Steger, 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. (1989). Circular RNAs: relics of precellular evolution? Proceedings of the National Academy of Sciences, 86(23), 9370-9374. doi:10.1073/pnas.86.23.9370Ambrós, S., Hernández, C., & Flores, R. (1999). Rapid generation of genetic heterogeneity in progenies from individual cDNA clones of peach latent mosaic viroid in its natural host The data reported in this paper are in the EMBL nucleotide sequence database and assigned the accession nos AJ241818–AJ241850. Journal of General Virology, 80(8), 2239-2252. doi:10.1099/0022-1317-80-8-2239Navarro, J.-A., Vera, A., & Flores, R. (2000). A Chloroplastic RNA Polymerase Resistant to Tagetitoxin Is Involved in Replication of Avocado Sunblotch Viroid. Virology, 268(1), 218-225. doi:10.1006/viro.1999.0161Rodio, M.-E., Delgado, S., De Stradis, A., Gómez, M.-D., Flores, R., & Di Serio, F. (2007). A Viroid RNA with a Specific Structural Motif Inhibits Chloroplast Development. The Plant Cell, 19(11), 3610-3626. doi:10.1105/tpc.106.049775Carbonell, A., De la Peña, M., Flores, R., & Gago, S. (2006). Effects of the trinucleotide preceding the self-cleavage site on eggplant latent viroid hammerheads: differences in co- and post-transcriptional self-cleavage may explain the lack of trinucleotide AUC in most natural hammerheads. Nucleic Acids Research, 34(19), 5613-5622. doi:10.1093/nar/gkl717Hutchins, C. J., Rathjen, P. D., Forster, A. C., & Symons, R. H. (1986). Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Research, 14(9), 3627-3640. doi:10.1093/nar/14.9.3627PRODY, G. A., BAKOS, J. T., BUZAYAN, J. M., SCHNEIDER, I. R., & BRUENING, G. (1986). Autolytic Processing of Dimeric Plant Virus Satellite RNA. Science, 231(4745), 1577-1580. doi:10.1126/science.231.4745.1577Nohales, M.-A., Molina-Serrano, D., Flores, R., & Daros, J.-A. (2012). Involvement of the Chloroplastic Isoform of tRNA Ligase in the Replication of Viroids Belonging to the Family Avsunviroidae. Journal of Virology, 86(15), 8269-8276. doi:10.1128/jvi.00629-12Branch, 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.85.23.9128Daros, 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.0401090101Feldstein, 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.95.11.6560Gas, M.-E., Hernández, C., Flores, R., & Daròs, J.-A. (2007). Processing of Nuclear Viroids In Vivo: An Interplay between RNA Conformations. PLoS Pathogens, 3(11), e182. doi:10.1371/journal.ppat.0030182Nohales, M.-A., Flores, R., & Daros, J.-A. (2012). Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase. Proceedings of the National Academy of Sciences, 109(34), 13805-13810. doi:10.1073/pnas.1206187109Brass, J. R. J., Owens, R. A., Matoušek, J., & Steger, G. (2017). Viroid quasispecies revealed by deep sequencing. RNA Biology, 14(3), 317-325. doi:10.1080/15476286.2016.1272745Bull, J. J., Sanjuán, R., & Wilke, C. O. (2007). Theory of Lethal Mutagenesis for Viruses. Journal of Virology, 81(6), 2930-2939. doi:10.1128/jvi.01624-06Cuevas, J. M., González-Candelas, F., Moya, A., & Sanjuán, R. (2009). Effect of Ribavirin on the Mutation Rate and Spectrum of Hepatitis C Virus In Vivo. Journal of Virology, 83(11), 5760-5764. doi:10.1128/jvi.00201-09Ribeiro, R. M., Li, H., Wang, S., Stoddard, M. B., Learn, G. H., Korber, B. T., … Perelson, A. S. (2012). Quantifying the Diversification of Hepatitis C Virus (HCV) during Primary Infection: Estimates of the In Vivo Mutation Rate. PLoS Pathogens, 8(8), e1002881. doi:10.1371/journal.ppat.1002881Acevedo, A., Brodsky, L., & Andino, R. (2013). Mutational and fitness landscapes of an RNA virus revealed through population sequencing. Nature, 505(7485), 686-690. doi:10.1038/nature12861Cuevas, J. M., Geller, R., Garijo, R., López-Aldeguer, J., & Sanjuán, R. (2015). Extremely High Mutation Rate of HIV-1 In Vivo. PLOS Biology, 13(9), e1002251. doi:10.1371/journal.pbio.1002251Acevedo, A., & Andino, R. (2014). Library preparation for highly accurate population sequencing of RNA viruses. Nature Protocols, 9(7), 1760-1769. doi:10.1038/nprot.2014.118Kennedy, S. R., Schmitt, M. W., Fox, E. J., Kohrn, B. F., Salk, J. J., Ahn, E. H., … Loeb, L. A. (2014). Detecting ultralow-frequency mutations by Duplex Sequencing. Nature Protocols, 9(11), 2586-2606. doi:10.1038/nprot.2014.170Franklin, R. M. (1966). Purification and properties of the replicative intermediate of the RNA bacteriophage R17. Proceedings of the National Academy of Sciences, 55(6), 1504-1511. doi:10.1073/pnas.55.6.1504López-Carrasco, A., Gago-Zachert, S., Mileti, G., Minoia, S., Flores, R., & Delgado, S. (2015). The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in theirin vivoRNA conformations. RNA Biology, 13(1), 83-97. doi:10.1080/15476286.2015.1119365Keese, P., & Symons, R. H. (1985). Domains in viroids: evidence of intermolecular RNA rearrangements and their contribution to viroid evolution. Proceedings of the National Academy of Sciences, 82(14), 4582-4586. doi:10.1073/pnas.82.14.4582López-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:10.1080/15476286.2016.1223005Flores, R., Hernandez, C., de la Peña, M., Vera, A., & Daros, J.-A. (2001). Hammerhead Ribozyme Structure and Function in Plant RNA Replication. Ribonucleases - Part A, 540-552. doi:10.1016/s0076-6879(01)41175-xMartick, M., & Scott, W. G. (2006). Tertiary Contacts Distant from the Active Site Prime a Ribozyme for Catalysis. Cell, 126(2), 309-320. doi:10.1016/j.cell.2006.06.036Ruffner, D. E., Stormo, G. D., & Uhlenbeck, O. C. (1990). Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry, 29(47), 10695-10702. doi:10.1021/bi00499a018Flores, 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.2012.00217Owens, R. A., Chen, W., Hu, Y., & Hsu, Y.-H. (1995). Suppression of Potato Spindle Tuber Viroid Replication and Symptom Expression by Mutations Which Stabilize the Pathogenicity Domain. Virology, 208(2), 554-564. doi:10.1006/viro.1995.1186Takeda, R., Petrov, A. I., Leontis, N. B., & Ding, B. (2011). A Three-Dimensional RNA Motif in Potato spindle tuber viroid Mediates Trafficking from Palisade Mesophyll to Spongy Mesophyll in Nicotiana benthamiana. The Plant Cell, 23(1), 258-272. doi:10.1105/tpc.110.081414Zhong, 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.107.056606Thomas, M. J., Platas, A. A., & Hawley, D. K. (1998). Transcriptional Fidelity and Proofreading by RNA Polymerase II. Cell, 93(4), 627-637. doi:10.1016/s0092-8674(00)81191-5Gout, J.-F., Thomas, W. K., Smith, Z., Okamoto, K., & Lynch, M. (2013). Large-scale detection of in vivo transcription errors. Proceedings of the National Academy of Sciences, 110(46), 18584-18589. doi:10.1073/pnas.1309843110Hedtke, B. (1997). Mitochondrial and Chloroplast Phage-Type RNA Polymerases in Arabidopsis. Science, 277(5327), 809-811. doi:10.1126/science.277.5327.809Lerbs-Mache, S. (1993). The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single-subunit enzyme or catalytic core of multimeric enzyme complexes? Proceedings of the National Academy of Sciences, 90(12), 5509-5513. doi:10.1073/pnas.90.12.5509Oldenkott, B., Yamaguchi, K., Tsuji-Tsukinoki, S., Knie, N., & Knoop, V. (2014). Chloroplast RNA editing going extreme: more than 3400 events of C-to-U editing in the chloroplast transcriptome of the lycophyteSelaginella uncinata. RNA, 20(10), 1499-1506. doi:10.1261/rna.045575.114Codoñer, F. M., Darós, J.-A., Solé, R. V., & Elena, S. F. (2006). The Fittest versus the Flattest: Experimental Confirmation of the Quasispecies Effect with Subviral Pathogens. PLoS Pathogens, 2(12), e136. doi:10.1371/journal.ppat.0020136Eigen, M. (1971). Selforganization of matter and the evolution of biological macromolecules. Die Naturwissenschaften, 58(10), 465-523. doi:10.1007/bf00623322Lynch, M. (2011). The Lower Bound to the Evolution of Mutation Rates. Genome Biology and Evolution, 3, 1107-1118. doi:10.1093/gbe/evr066Bradwell, K., Combe, M., Domingo-Calap, P., & Sanjuán, R. (2013). Correlation Between Mutation Rate and Genome Size in Riboviruses: Mutation Rate of Bacteriophage Qβ. Genetics, 195(1), 243-251. doi:10.1534/genetics.113.154963Drake, J. W. (1991). A constant rate of spontaneous mutation in DNA-based microbes. Proceedings of the National Academy of Sciences, 88(16), 7160-7164. doi:10.1073/pnas.88.16.7160Schmitt, M. W., Kennedy, S. R., Salk, J. J., Fox, E. J., Hiatt, J. B., & Loeb, L. A. (2012). Detection of ultra-rare mutations by next-generation sequencing. Proceedings of the National Academy of Sciences, 109(36), 14508-14513. doi:10.1073/pnas.120871510

Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.

PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Elective cancer surgery in COVID-19-free surgical pathways during the SARS-CoV-2 pandemic: An international, multicenter, comparative cohort study

PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Global wealth disparities drive adherence to COVID-safe pathways in head and neck cancer surgery

Peer reviewe

Outcomes from elective colorectal cancer surgery during the SARS-CoV-2 pandemic

This study aimed to describe the change in surgical practice and the impact of SARS-CoV-2 on mortality after surgical resection of colorectal cancer during the initial phases of the SARS-CoV-2 pandemic