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BREVIA Extremely High Mutation Rate of a Hammerhead Viroid Selma Gago,1 Santiago F. Elena,1 Ricardo Flores,1 Rafael Sanjuán1,2*

merhead viroids are replicated by a proofreadingdeficient chloroplastic DNA-dependent RNA polymerase that is redirected to use RNA instead of its native DNA template (4). This, together with the presence of mutagenic free radicals or unbalanced nucleotide pools, would lead to extremely error-prone replication. Viroids can tolerate such elevated per-site mutation rates owing to their minimal genomes, whereas more complex genomes would accumulate an excessive mutational load (8). Given their genomic simplicity and autocatalytic activity, hammerhead viroids are reminiscent of the postulated RNA world replicons (9). These primitive replicons would also resemble hammerhead viroids in their extremely error-prone replication. Thus, our results support the notion that the emergence of replication fidelity mechanisms was central to the evolution of complexity in the early history of life.

utation rates vary by orders of magni- head mutations sampled in vivo, we recreated the tude across species (1, 2), with the high- mutations by site-directed mutagenesis and asest rates measured so far corresponding sayed for infectivity. Northern-blot hybridizations to RNA viruses (3), but little is known about other indicated that plants inoculated with these mutants RNA replicons. Viroids are plant pathogens with minimal nonprotein-coding RNA genomes replicated by host RNA polymerases (4). We estimated the mutation rate of Chrysanthemum chlorotic mottle viroid (CChMVd), a 399-nucleotide chloroplastic viroid with hammerhead ribozymes. Hammerheads are RNA References and Notes motifs formed by three double1. J. W. Drake, B. Charlesworth, helix regions flanking a core of D. Charlesworth, J. F. Crow, 15 highly conserved nucleotides Genetics 148, 1667 (1998). critical for catalytic activity (5), 2. P. D. Sniegowski, P. J. Gerrish, T. Johnson, A. Shaver, Bioessays which mediate self-cleavage of 22, 1057 (2000). replicative intermediates and, 3. S. Duffy, L. A. Shackelton, hence, are essential for viroid E. C. Holmes, Nat. Rev. Genet. 9, replication. Hammerhead viroids 267 (2008). 4. R. Flores, C. Hernández, show elevated genetic variabilA. E. Martínez de Alba, J. A. Daròs, ity (6), but this variability results Fig. 1. Per-site mutation rate versus genome size for CChMVd and other biological F. Di Serio, Annu. Rev. Phytopathol. from the combined action of entities [reviewed in (2) and updated with more recent data from (3)]. RNA viruses (left to 43, 117 (2005). right) are tobacco mosaic virus, human rhinovirus, poliovirus, vesicular stomatitis virus, mutation and selection and there5. M. Martick, W. G. Scott, Cell 126, bacteriophage F6, and measles virus. Single-stranded DNA viruses are bacteriophage fore cannot be used to directly 309 (2006). FX174 and bacteriophage m13. Double-stranded DNA viruses are bacteriophage l, 6. N. Duran-Vila, S. F. Elena, estimate mutation rates. J. A. Daròs, R. Flores, in Origin and To achieve this goal, we in- herpes simplex virus, bacteriophage T2, and bacteriophage T4. Bacteria is Escherichia Evolution of Viruses, E. Domingo, coli. Lower eukaryotes are Saccharomyces cerevisiae and Neurospora crassa. Higher oculated plants with an in vitro C. R. Parrish, J. J. Holland, eukaryotes are Caenorhabditis elegans, Drosophila melanogaster, Mus musculus, and transcript of CChMVd (7), and Eds. (Elsevier, London, 2008), Homo sapiens. When several estimations were available, the mean value is shown. pp. 43–64. at the onset of symptoms we 7. Materials and methods are available as supporting screened for mutations at the 15 core nucleotides had no detectable viroid RNA (fig. S2A). Further, material on Science Online. plus the nucleotide preceding the self-cleavage site self-cleavage analyses confirmed that all except 8. M. Eigen, Naturwissenschaften 58, 465 (1971). in each of the two hammerheads (32 sites). one of the mutant hammerheads showed severely 9. T. O. Diener, Proc. Natl. Acad. Sci. U.S.A. 86, 9370 (1989). Considering that these mutations are lethal for reduced or null catalytic activity (fig. S2B). 10. This work was supported by grants from the Spanish To determine the strength of selection against the viroid, their population frequency must equal Ministerio de Ciencia e Innovación: BFU2006-14819the mutation rate because, despite multiple mutations elsewhere in the viroid genome, we C02-01/BMC to S.F.E., BFU2008-03154/BMC to R.F., and replication rounds downstream from inoculation, competed 24 random-point mutants against the BFU2008-03978/BMC to R.S. GenBank sequence accession numbers are FJ647228 to FJ647553. they have necessarily been generated during the wild type. Sequencing of 138 RT-PCR clones last one. In three independent experiments, we revealed that 20/24 mutations had been purged Supporting Online Material found three, seven, and five mutations in 63, 64, by selection at the onset of symptoms. In con- www.sciencemag.org/cgi/content/full/323/5919/1308/DC1 and 61 reverse transcription polymerase chain trast, 51 new polymorphisms appeared in this Materials and Methods reaction (RT-PCR) clones, respectively (188 × 32 time interval, showing that genetic variability is Figs. S1 to S3 = 6016 total target sites), yielding a mutation rate rapidly regenerated because of highly error-prone References of 0.0025 T 0.0006 (SEM) per site and replication replication (fig. S3). We also inferred that ham- 1 December 2008; accepted 2 February 2009 cycle, that is, one mutation per 400 nucleotides merheads are unlikely to constitute mutational 10.1126/science.1169202 hotspots because polymorphisms did not map (fig. S1). In a control experiment, we sequenced RT- more frequently in hammerheads than in the rest 1 Instituto de Biología Molecular y Celular de Plantas, Consejo PCR clones from the in vitro transcript used for of the genome (Fisher exact test, P = 0.963) Superior de Investigaciones Científicas–Universidad Politécnica inoculations and found a single substitution in whereas the fraction of point mutations that were de València, 46022 València, Spain. 2Institut Cavanilles de 6525 sites. This result gives an error rate 17-fold selected against was also similar for these two Biodiversitat i Biologia Evolutiva, Universitat de València, lower than the estimated CChMVd mutation rate regions (7/8 and 13/16, respectively). 46980 València, Spain. The CChMVd mutation rate is the highest *To whom correspondence should be addressed. E-mail: and discards any significant effect of RT-PCR artifacts. To confirm the lethality of the hammer- reported for any biological entity (Fig. 1). Ham- rafael.sanjuan@uv.es

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Extremely High Mutation Rate of a Hammerhead Viroid  

Mutation rates vary by orders of magnitude across species (1, 2), with the highest rates measured so far corresponding to RNAviruses (3), bu...

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