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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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.
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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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