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Anti-CRISPR proteins encoded by archaeal lytic viruses inhibit subtype I-D immunity

A Publisher Correction to this article was published on 21 June 2018

This article has been updated

Abstract

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.

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Fig. 1: Isolation of a SIRV2 mutant, SIRV2M, that is non-infectious in S. islandicus LAL14/1.
Fig. 2: SIRV3 gp02 is essential and sufficient for restoring SIRV2M infectivity in LAL14/1.
Fig. 3: SIRV3 gp02 facilitates SIRV2M propagation in LAL14/1.
Fig. 4: SIRV3 gp02 (AcrID1) protein interacts with Cas10d protein thereby inhibiting CRISPR–Cas subtype I-D immunity.
Fig. 5: Phylogeny of AcrID1 homologues.
Fig. 6: Crystal structure of AcrID1.

Change history

  • 21 June 2018

    In the original version of this Article, molecular weight markers in Figs 1c, 2c,d and 4d were displaced during the production process, so that they were not correctly aligned with the corresponding bands. In addition, in Fig. 4c, molecular masses given for three different elution volumes were displaced so that they appeared to the left of the correct positions. These errors have now been corrected.

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Acknowledgements

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 [265933] 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).

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F.H. and X.P. conceived the experiments. F.H., Y.B.C., L.B.V., A.L.K. and M.D. performed the experiments. D.E.B. analysed the crystal structure of AcrID1. K.S.M. and E.V.K. performed phylogenetic analysis of the AcrID homologues. F.H., Y.B.C., A.L.K., E.V.K., D.E.B. and X.P. wrote the paper which was read and approved by all authors.

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Correspondence to Xu Peng.

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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

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