The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA)1,2 and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP)3,4,5. Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain6, sculpting a powerful antiviral response7,8,9,10 that can drive viruses to extinction7,8. Cyclic nucleotides are increasingly implicated in host–pathogen interactions11,12,13. Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize the type III CRISPR defence system. The AcrIII-1 family has a broad host range, as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.
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The structural coordinates and data have been deposited in the Protein Data Bank (PDB) with deposition code 6SCF. The genome sequence of the SSeV virus has been submitted to GenBank with accession code MN53972. Raw data are available in the Supplementary Information for the plasmid immunity analysis presented in Fig. 1 and Extended Data Fig. 3, and the kinetic analysis presented in Fig. 2 and Extended Data Figs. 5, 6.
Samai, P. et al. Co-transcriptional DNA and RNA cleavage during type III CRISPR-Cas immunity. Cell 161, 1164–1174 (2015).
Tamulaitis, G. et al. Programmable RNA shredding by the type III-A CRISPR-Cas system of Streptococcus thermophilus. Mol. Cell 56, 506–517 (2014).
Kazlauskiene, M., Kostiuk, G., Venclovas, Č., Tamulaitis, G. & Siksnys, V. A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science 357, 605–609 (2017).
Niewoehner, O. et al. Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers. Nature 548, 543–548 (2017).
Rouillon, C., Athukoralage, J. S., Graham, S., Grüschow, S. & White, M. F. Control of cyclic oligoadenylate synthesis in a type III CRISPR system. eLife 7, e36734 (2018).
Makarova, K. S., Anantharaman, V., Grishin, N. V., Koonin, E. V. & Aravind, L. CARF and WYL domains: ligand-binding regulators of prokaryotic defense systems. Front. Genet. 5, 102 (2014).
Rostøl, J. T. & Marraffini, L. A. Non-specific degradation of transcripts promotes plasmid clearance during type III-A CRISPR-Cas immunity. Nat. Microbiol. 4, 656–662 (2019).
Pyenson, N. C., Gayvert, K., Varble, A., Elemento, O. & Marraffini, L. A. Broad targeting specificity during bacterial type III CRISPR-Cas immunity constrains viral escape. Cell Host Microbe 22, 343–353 (2017).
Deng, L., Garrett, R. A., Shah, S. A., Peng, X. & She, Q. A novel interference mechanism by a type IIIB CRISPR-Cmr module in Sulfolobus. Mol. Microbiol. 87, 1088–1099 (2013).
Jiang, W., Samai, P. & Marraffini, L. A. Degradation of phage transcripts by CRISPR-associated RNases enables type III CRISPR-Cas immunity. Cell 164, 710–721 (2016).
Whiteley, A. T. et al. Bacterial cGAS-like enzymes synthesize diverse nucleotide signals. Nature 567, 194–199 (2019).
Maelfait, J. & Rehwinkel, J. RECONsidering sensing of cyclic dinucleotides. Immunity 46, 337–339 (2017).
Cohen, D. et al. Cyclic GMP-AMP signalling protects bacteria against viral infection. Nature 574, 691–695 (2019).
Athukoralage, J. S., Rouillon, C., Graham, S., Grüschow, S. & White, M. F. Ring nucleases deactivate type III CRISPR ribonucleases by degrading cyclic oligoadenylate. Nature 562, 277–280 (2018).
Borges, A. L., Davidson, A. R. & Bondy-Denomy, J. The discovery, mechanisms, and evolutionary impact of anti-CRISPRs. Annu. Rev. Virol. 4, 37–59 (2017).
Hwang, S. & Maxwell, K. L. Meet the anti-CRISPRs: widespread protein inhibitors of CRISPR-Cas systems. CRISPR J. 2, 23–30 (2019).
Oke, M. et al. The Scottish structural proteomics facility: targets, methods and outputs. J. Struct. Funct. Genomics 11, 167–180 (2010).
Larson, E. T. et al. A new DNA binding protein highly conserved in diverse crenarchaeal viruses. Virology 363, 387–396 (2007).
Wirth, J. F. et al. Development of a genetic system for the archaeal virus Sulfolobus turreted icosahedral virus (STIV). Virology 415, 6–11 (2011).
Bautista, M. A., Zhang, C. & Whitaker, R. J. Virus-induced dormancy in the archaeon Sulfolobus islandicus. MBio 6, e02565-14 (2015).
Grüschow, S., Athukoralage, J. S., Graham, S., Hoogeboom, T. & White, M. F. Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence. Nucleic Acids Res. 47, 9259–9270 (2019).
Bondy-Denomy, J. et al. A unified resource for tracking anti-CRISPR names. CRISPR J. 1, 304–305 (2018).
Auchtung, J. M., Aleksanyan, N., Bulku, A. & Berkmen, M. B. Biology of ICEBs1, an integrative and conjugative element in Bacillus subtilis. Plasmid 86, 14–25 (2016).
Yang, W. Nucleases: diversity of structure, function and mechanism. Q. Rev. Biophys. 44, 1–93 (2011).
Broo, K. S., Brive, L., Sott, R. S. & Baltzer, L. Site-selective control of the reactivity of surface-exposed histidine residues in designed four-helix-bundle catalysts. Fold. Des. 3, 303–312 (1998).
Bhoobalan-Chitty, Y., Johansen, T. B., Di Cianni, N. & Peng, X. Inhibition of type III CRISPR-Cas immunity by an archaeal virus-encoded anti-CRISPR protein. Cell 179, 448–458 (2019).
Knott, G. J. et al. Broad-spectrum enzymatic inhibition of CRISPR-Cas12a. Nat. Struct. Mol. Biol. 26, 315–321 (2019).
Dong, L. et al. An anti-CRISPR protein disables type V Cas12a by acetylation. Nat. Struct. Mol. Biol. 26, 308–314 (2019).
Keller, J. et al. Crystal structure of AFV3-109, a highly conserved protein from crenarchaeal viruses. Virol. J. 4, 12 (2007).
Anderson, R. E., Kouris, A., Seward, C. H., Campbell, K. M. & Whitaker, R. J. Structured populations of Sulfolobus acidocaldarius with susceptibility to mobile genetic elements. Genome Biol. Evol. 9, 1699–1710 (2017).
Held, N. L., Herrera, A. & Whitaker, R. J. Reassortment of CRISPR repeat-spacer loci in Sulfolobus islandicus. Environ. Microbiol. 15, 3065–3076 (2013).
Zhang, C. & Whitaker, R. J. Microhomology-mediated high-throughput gene inactivation strategy for the hyperthermophilic crenarchaeon Sulfolobus islandicus. Appl. Environ. Microbiol. 84, e02167-17 (2017).
Zhang, C., Cooper, T. E., Krause, D. J. & Whitaker, R. J. Augmenting the genetic toolbox for Sulfolobus islandicus with a stringent positive selectable marker for agmatine prototrophy. Appl. Environ. Microbiol. 79, 5539–5549 (2013).
Deng, L., Zhu, H., Chen, Z., Liang, Y. X. & She, Q. Unmarked gene deletion and host-vector system for the hyperthermophilic crenarchaeon Sulfolobus islandicus. Extremophiles 13, 735–746 (2009).
Rouillon, C., Athukoralage, J. S., Graham, S., Grüschow, S. & White, M. F. Investigation of the cyclic oligoadenylate signaling pathway of type III CRISPR systems. Methods Enzymol. 616, 191–218 (2019).
Linkert, M. et al. Metadata matters: access to image data in the real world. J. Cell Biol. 189, 777–782 (2010).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Sternberg, S. H., Haurwitz, R. E. & Doudna, J. A. Mechanism of substrate selection by a highly specific CRISPR endoribonuclease. RNA 18, 661–672 (2012).
Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).
Pei, J. & Grishin, N. V. PROMALS3D: multiple protein sequence alignment enhanced with evolutionary and three-dimensional structural information. Methods Mol. Biol. 1079, 263–271 (2014).
Capella-Gutiérrez, S., Silla-Martínez, J. M. & Gabaldón, T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972–1973 (2009).
Guindon, S. et al. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321 (2010).
Letunic, I. & Bork, P. Interactive tree of life (iTOL) v4: recent updates and new developments. Nucleic Acids Res. 47 (W1), W256–W259 (2019).
Winter, G. xia2: an expert system for macromolecular crystallography data reduction. J. Appl. Crystallogr. 43, 186–190 (2010).
Kabsch, W. Xds. Acta Crystallogr. D 66, 125–132 (2010).
Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D 67, 282–292 (2011).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Long, F. et al. AceDRG: a stereochemical description generator for ligands. Acta Crystallogr. D 73, 112–122 (2017).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).
Gerlt, J. A. Genomic enzymology: web tools for leveraging protein family sequence-function space and genome context to discover novel functions. Biochemistry 56, 4293–4308 (2017).
This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/S000313/1 to M.F.W. and BB/R008035/1 to T.M.G.) and by a NASA Exobiology and Evolutionary Biology grant (NNX14AK23G to R.J.W.). We thank J. Black and M. Alejandra-Bautista for isolating and characterizing the SSeV virus, and R. Wipfler and W. Zhu for technical assistance.
The University of St Andrews has filed a patent application (UK Patent Application 1902256.5, “Novel enzyme for phage therapy”; filed 19 February 2019), on which J.S.A. and M.F.W. are inventors. The other authors declare no competing interests.
Peer review information Nature thanks Joseph Bondy-Denomy and John van der Oost for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Multiple sequence alignment of DUF1874-family members, and purity of DUF1874 and CRISPR ancillary enzymes used in biochemical assays.
a, This multiple sequence alignment includes the AcrIII-1 proteins from the archaeal viruses SIRV1, STIV, AFV3, ARV1, SIFV, SMV4 and ATV, the ICEBs1 protein YddF from B. subtilis, the bacteriophage proteins from Thermoanaerobacterium phage THSA-485A, Synechococcus phage S-CBWM1, Fusobacterium phage Fnu1 and Hydrogenobaculum phage 1, and the Crn2 protein from Crenothrix polyspora. Conserved residues H47, R66, R85 and E88 are indicated by asterisks. Light and dark grey shading indicate regions of partial and strong sequence conservation, respectively. b, SDS–PAGE of SIRV1 gp29 (wild-type, H47A and E88A variants), YddF, the Crn1 enzyme Sso2081, and the Csx1 enzyme Sso1389. The gel is representative of two or more biological replicates.
a, Genomic context of the CRISPR system in the genetic host (S. islandicus RJW007) and in mutant strains. A1 and A2 denote two different CRISPR arrays, the orientations of which are indicated with arrows. b, PCR verification of Δtype I-A mutants. A representative Sulfolobus transformant with integrated type I-A knockout plasmid was grown in dextrin-tryptone liquid medium, and the cell cultures were plated on dextrin-tryptone plates containing 5-fluoroorotic acid (5-FOA, 50 µg mg−1), uracil (20 µg ml−1), and agamatine (1 mg ml−1). Seven randomly selected 5-FOA-resistant (5-FOAR) colonies were screened using the primers that bind outside of the flanking homologous regions to confirm the type I-A deletion. A representative Δtype I-A mutant was further colony purified for subsequent experiments. The expected sizes of the PCR products amplified from the genomic DNA of the parental strain (referred to wild type, wt) and the Δtype I-A mutant are 8,830 base pairs (bp) and 3,001 bp, respectively. The minus symbol denotes a negative control (using water as the template for PCR). L, log-2 DNA ladder (NEB). Seven biological replicates were screened. c, PCR analysis of the RJW007 Δtype I-AΔcsx1 mutant and its parental strain RJW007 Δtype I-A using primers that anneal to the outside of the flanking homologous regions of csx1, generating amplicons of 2,312 bp and 3,650 bp, respectively. Minus symbol, negative control (using water as the template for PCR). L, Gene Ruler Express DNA ladder (Thermo Scientific). The experiment carried out once. d, Plaque counts for the three strains tested (n = 3 biological replicates).
Extended Data Fig. 3 Effect of DUF1874 on plasmid immunity provided by a heterologously expressed M. tuberculosis type III-A CRISPR system, providing cA4- or cA6-mediated immunity.
Unprocessed images of sample plates are shown for all replicates (two biological replicates with four technical replicates each; n = 8). Cell-culture dilutions are indicated above the plates.
Extended Data Fig. 4 Substrate preference of the AcrIII-1 proteins SIRV1 gp29 and YddF, and effective range of cA4 degradation.
a–d, TLC images visualizing (under 254 nm UV light) cA4 and cA6 (450 μM) degradation by SIRV1 gp29 (a, b) and YddF (c, d) over time (in minutes). Both AcrIII-1 enzymes display a clear preference for cA4 over cA6. All TLC images are representative of three technical replicates. e, Denaturing PAGE showing activation of Csx1 (0.5 μM dimer) by the indicated amounts (500–0.5 μM) of HPLC-purified cA4, and its subsequent deactivation when either AcrIII-1 or Crn1 (2 μM dimer) was present to degrade cA4. The AcrIII-1 enzyme degraded 100-fold more cA4 than did Crn1. The control reaction (C) shows RNA incubated with Csx1 in the absence of cA4 (n = 3 technical replicates). For gel source data, see Supplementary Fig. 1.
a, b, Orthogonal views of SIRV1 gp29 dimer in complex with cA4. The protein monomers are coloured purple and gold, with catalytic residue H47 from the apo structure shown in salmon. cA4 is shown as a spacefill model, with green, blue, red and orange representing carbon, nitrogen, oxygen and phosphorus atoms, respectively. Conserved residues (Extended Data Fig. 1) in the AcrIII-1 family are indicated and discussed in the text. c, Interactions between each monomer of the SIRV1 dimer (orange and blue), with cA4 shown in green. d, Diagram showing the interaction between SIRV1 gp29 and cA4. Dotted lines represent hydrogen bonds, with distances annotated. Spheres represent water molecules.
Extended Data Fig. 6 Single-turnover cA4 cleavage by SIRV1 gp29 and variants, and chemical rescue with imidazole.
a, Phosphorimage of TLC visualizing cA4 cleavage by SIRV1 gp29 H47A (4 μM dimer, 50 °C) in the presence or absence of 500 mM imidazole, over time. The rate of cA4 cleavage to generate A2>P and A2-P was calculated by quantifying densiometric signals from the phosphorimage (n = 3 technical replicates). b, Plot comparing the single-turnover rates of cA4 by SIRV1 gp29, its E88A variant and its H47A variant, in the presence or absence of imidazole. Cleavage of cA4 by the H47A variant can be partially restored when the reaction is supplemented with 500 mM imidazole. Data are mean and s.d. (n = 3 technical replicates). For gel source data, see Supplementary Fig. 1.
The maximum likelihood phylogenetic tree was constructed with automatic selection of the best-fit substitution model for a given alignment (LG + G + I). Red circles indicate 95–100% branch support, as assessed using aBayes implemented in PhyML. The scale bar represents the number of substitutions per site. Branches and labels are colour coded: red, archaea; black, bacteria; blue, bacteria and archaea in which AcrIII-1 homologues are associated with CRISPR loci; green, archaeal viruses and plasmids; orange, bacteriophages.
Type III CRISPR loci in the bacterial species Crenothrix polyspora, Methylovulum psychrotolerans, Methylomagnum ishizawai, Thioalkalivibrio sufidiphilus and Marinitoga piezophilia are shown, with genes labelled and colour coded. The crn2 gene is shown in pale yellow with a bold outline; CRISPRs are indicated by small black arrowheads; and unrelated/hypothetical genes are shown as small white arrows. The sizes and orientations of genes are not reflected. Ago, Argonaute; CARF, CRISPR-associated Rossman fold; CARF-RelE, CARF domain fused to the RelE toxin; DUF1887, predicted CARF nuclease; RT, reverse transcriptase.
Genomic neighbourhoods were analysed using the enzyme function initiative–genome neighbourhood tool (EFI-GNT) against the Pfam profile database53. Gene annotations are colour coded according to the key at the right.
Supplementary information Fig. 1: uncropped phosphorimages of gels and TLC plates.
Dataset S1: Gene distribution of the crn2/AcrIII-1 family.
Dataset S2: Raw data for kinetic analyses.
Dataset S3: Raw data for colony counts.
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Athukoralage, J.S., McMahon, S.A., Zhang, C. et al. An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity. Nature 577, 572–575 (2020). https://doi.org/10.1038/s41586-019-1909-5
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