Viruses employ a range of strategies to counteract the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR–Cas), including mutational escape and physical blocking of enzymatic function using anti-CRISPR proteins (Acrs). Acrs have been found in many bacteriophages but so far not in archaeal viruses, despite the near ubiquity of CRISPR–Cas systems in archaea. Here, we report the functional and structural characterization of two archaeal Acrs from the lytic rudiviruses, SIRV2 and SIRV3. We show that a 4 kb deletion in the SIRV2 genome dramatically reduces infectivity in Sulfolobus islandicus LAL14/1 that carries functional CRISPR–Cas subtypes I-A, I-D and III-B. Subsequent insertion of a single gene from SIRV3, gp02 (AcrID1), which is conserved in the deleted fragment, successfully restored infectivity. We demonstrate that AcrID1 protein inhibits the CRISPR–Cas subtype I-D system by interacting directly with Cas10d protein, which is required for the interference stage. Sequence and structural analysis of AcrID1 show that it belongs to a conserved family of compact, dimeric αβ-sandwich proteins characterized by extreme pH and temperature stability and a tendency to form protein fibres. We identify about 50 homologues of AcrID1 in four archaeal viral families demonstrating the broad distribution of this group of anti-CRISPR proteins.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
KPT330 improves Cas9 precision genome- and base-editing by selectively regulating mRNA nuclear export
Communications Biology Open Access 17 March 2022
Structural basis for inhibition of an archaeal CRISPR–Cas type I-D large subunit by an anti-CRISPR protein
Nature Communications Open Access 25 November 2020
Machine-learning approach expands the repertoire of anti-CRISPR protein families
Nature Communications Open Access 29 July 2020
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
Koonin, E. V., Makarova, K. S. & Wolf, Y. I. Evolutionary genomics of defense systems in archaea and bacteria. Annu. Rev. Microbiol. 71, 233–261 (2017).
Mohanraju, P. et al. Diverse evolutionary roots and mechanistic variations of the CRISPR–Cas systems. Science 353, aad5147 (2016).
Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).
Koonin, E. V., Makarova, K. S. & Zhang, F. Diversity, classification and evolution of CRISPR–Cas systems. Curr. Opin. Microbiol. 37, 67–78 (2017).
Brouns, S. J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008).
Charpentier, E., Richter, H., van der Oost, J. & White, M. F. Biogenesis pathways of RNA guides in archaeal and bacterial CRISPR–Cas adaptive immunity. FEMS Microbiol. Rev. 39, 428–441 (2015).
Sternberg, S. H., Richter, H., Charpentier, E. & Qimron, U. Adaptation in CRISPR–Cas systems. Mol. Cell 61, 797–808 (2016).
Jackson, S. A. et al. CRISPR–Cas: adapting to change. Science 356, eaal5056 (2017).
Plagens, A., Richter, H., Charpentier, E. & Randau, L. DNA and RNA interference mechanisms by CRISPR–Cas surveillance complexes. FEMS Microbiol. Rev. 39, 442–463 (2015).
Maxwell, K. L. et al. The solution structure of an anti-CRISPR protein. Nat. Commun. 7, 13134 (2016).
Rauch, B. J. et al. Inhibition of CRISPR-Cas9 with bacteriophage proteins. Cell 168, 150–158 (2017).
Borges, A. L., Davidson, A. R. & Bondy-Denomy, J. The discovery, mechanisms, and evolutionary impact of anti-CRISPRs. Annu Rev. Virol. 29, 37–59 (2017).
Pawluk, A., Davidson, A. R. & Maxwell, K. L. Anti-CRISPR: discovery, mechanism and function. Nat. Rev. Microbiol 16, 12–17 (2018).
Bondy-Denomy, J., Pawluk, A., Maxwell, K. L. & Davidson, A. R. Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature 493, 429–432 (2013).
Pawluk, A., Bondy-Denomy, J., Cheung, V. H., Maxwell, K. L. & Davidson, A. R. A new group of phage anti-CRISPR genes inhibits the type I-E CRISPR–Cas system of Pseudomonas aeruginosa. mBio 5, e00896 (2014).
Pawluk, A. et al. Naturally occurring off-switches for CRISPR–Cas9. Cell 167, 1829–1838 (2016).
Pawluk, A. et al. Inactivation of CRISPR–Cas systems by anti-CRISPR proteins in diverse bacterial species. Nat. Microbiol. 1, 16085 (2016).
Hynes, A. P. et al. An anti-CRISPR from a virulent streptococcal phage inhibits Streptococcus pyogenes Cas9. Nat. Microbiol. 2, 1374–1380 (2017).
Prangishvili, D. et al. The enigmatic archaeal virosphere. Nat. Rev. Microbiol. 15, 724–739 (2017).
Jaubert, C. et al. Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon. Open Biol. 3, 130010 (2013).
Wiedenheft, B. et al. RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions. Proc. Natl Acad. Sci. USA 108, 10092–10097 (2011).
Semenova, E. et al. Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed by a seed sequence. Proc. Natl Acad. Sci. USA 108, 10098–10103 (2011).
Manica, A., Zebec, Z., Steinkellner, J. & Schleper, C. Unexpectedly broad target recognition of the CRISPR-mediated virus defence system in the archaeon Sulfolobus solfataricus. Nucleic Acids Res. 41, 10509–10517 (2013).
Mousaei, M., Deng, L., She, Q. & Garrett, R. A. Major and minor crRNA annealing sites facilitate low stringency DNA protospacer binding prior to Type I-A CRISPR–Cas interference in Sulfolobus. RNA Biol. 13, 1166–1173 (2016).
Bize, A. et al. A unique virus release mechanism in the Archaea. Proc. Natl Acad. Sci. USA 106, 11306–11311 (2009).
Okutan, E. et al. Novel insights into gene regulation of the rudivirus SIRV2 infecting Sulfolobus cells. RNA Biol. 10, 875–885 (2013).
Deng, L. et al. Unveiling cell surface and type IV secretion proteins responsible for archaeal rudivirus entry. J. Virol. 88, 10264–10268 (2014).
He, F., Chen, L. & Peng, X. First experimental evidence for the presence of a CRISPR toxin in sulfolobus. J. Mol. Biol. 426, 3683–3688 (2014).
Guo, Y., Kragelund, B. B., White, M. F. & Peng, X. Functional characterization of a conserved archaeal viral operon revealing single-stranded DNA binding, annealing and nuclease activities. J. Mol. Biol. 427, 2179–2191 (2015).
Martinez-Alvarez, L., Bell, S. D. & Peng, X. Multiple consecutive initiation of replication producing novel brush-like intermediates at the termini of linear viral dsDNA genomes with hairpin ends. Nucleic Acids Res. 44, 8799–8809 (2016).
Martinez-Alvarez, L., Deng, L. & Peng, X. Formation of a viral replication focus in Sulfolobus cells infected by the rudivirus Sulfolobus islandicus rod-shaped virus 2. J. Virol. 91, e00486-17 (2017).
Erdmann, S., Le Moine Bauer, S. & Garrett, R. A. Inter-viral conflicts that exploit host CRISPR immune systems of Sulfolobus. Mol. Microbiol. 91, 900–917 (2014).
Shah, S. A., Erdmann, S., Mojica, F. J. & Garrett, R. A. Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol. 10, 891–899 (2013).
Makarova, K. S. et al. Evolution and classification of the CRISPR–Cas systems. Nat. Rev. Microbiol 9, 467–477 (2011).
Niewoehner, O. et al. Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers. Nature 548, 543–548 (2017).
Kazlauskiene, M., Kostiuk, G., Venclovas, C., Tamulaitis, G. & Siksnys, V. A cyclic oligonucleotide signaling pathway in type III CRISPR–Cas systems. Science 357, 605–609 (2017).
Peeters, E. et al. DNA-interacting characteristics of the archaeal rudiviral protein SIRV2_Gp1. Viruses 9, 190 (2017).
Bautista, M. A., Black, J. A., Youngblut, N. D. & Whitaker, R. J. Differentiation and structure in Sulfolobus islandicus rod-shaped virus populations. Viruses 9, 120 (2017).
Quax, T. E. et al. Massive activation of archaeal defense genes during viral infection. J. Virol. 87, 8419–8428 (2013).
Goulet, A. et al. Crystallization and preliminary X-ray diffraction analysis of protein 14 from Sulfolobus islandicus filamentous virus (SIFV). Acta Crystallogr. F 62, 884–886 (2006).
Goulet, A. et al. The crystal structure of ORF14 from Sulfolobus islandicus filamentous virus. Proteins 76, 1020–1022 (2009).
Goulet, A. et al. The thermo- and acido-stable ORF-99 from the archaeal virus AFV1. Protein Sci. 18, 1316–1320 (2009).
Shin, J. et al. Disabling Cas9 by an anti-CRISPR DNA mimic. Sci. Adv. 3, e1701620 (2017).
He, F., Vestergaard, G., Peng, W., She, Q. & Peng, X. CRISPR–Cas type I-A Cascade complex couples viral infection surveillance to host transcriptional regulation in the dependence of Csa3b. Nucleic Acids Res. 45, 1902–1913 (2017).
Zillig, W. et al. Screening for Sulfolobales, their plasmids and their viruses in Icelandic Solfataras. Syst. Appl. Microbiol. 16, 609–628 (1993).
Zhang, C. et al. Revealing the essentiality of multiple archaeal pcna genes using a mutant propagation assay based on an improved knockout method. Microbiology 156, 3386–3397 (2010).
Peng, W. et al. Genetic determinants of PAM-dependent DNA targeting and pre-crRNA processing in Sulfolobus islandicus. RNA Biol. 10, 738–748 (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).
Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006).
Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).
Pape, T. & Schneider, T. R. HKL2MAP: a graphical user interface for macromolecular phasing with SHELX programs. J. Appl. Crystallogr. 37, 843–844 (2004).
Cowtan, K. Fitting molecular fragments into electron density. Acta Crystallogr. D 64, 83–89 (2008).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004).
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).
Soding, J., Biegert, A. & Lupas, A. N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, 244–248 (2005).
Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).
Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2—approximately maximum-likelihood trees for large alignments. PloS ONE 5, e9490 (2010).
Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).
The authors thank M. Krupovic, N. Grishin, D. Prangishvili, Q.X. She and R.A. Garrett for useful discussions, R. Bertelsen for help with protein purification and crystallization, and G.R. Andersen and beamline staff at the P13 beamline at PETRA, Hamburg, for help with data collection. This work was supported by EU FP7 project HotZyme  and Danish Council for Independent Research/Technology and Production (grant number DFF–7017-00060) to XP and the Danish National Research Foundation’s Centre for Bacterial Stress Response and Persistence (BASP, grant no. DNRF120) to D.E.B., K.S.M. and E.V.K. are supported by intramural funds of the US Department of Health and Human Resources (to the National Library of Medicine).
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Tables 1 and 2 and Supplementary Figures 1–9.
Rights and permissions
About this article
Cite this article
He, F., Bhoobalan-Chitty, Y., Van, L.B. et al. Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity. Nat Microbiol 3, 461–469 (2018). https://doi.org/10.1038/s41564-018-0120-z
This article is cited by
Phylogenetic Analysis of Anti-CRISPR and Member Addition in the Families
Molecular Biotechnology (2023)
KPT330 improves Cas9 precision genome- and base-editing by selectively regulating mRNA nuclear export
Communications Biology (2022)
Structure-based functional mechanisms and biotechnology applications of anti-CRISPR proteins
Nature Reviews Molecular Cell Biology (2021)
CRISPR-Cas adaptive immune systems in Sulfolobales: genetic studies and molecular mechanisms
Science China Life Sciences (2021)
Allosteric inhibition of CRISPR-Cas9 by bacteriophage-derived peptides
Genome Biology (2020)