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

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