Structural basis for inhibition of an archaeal CRISPR-Cas type I-D large subunit by an anti-CRISPR protein.

A hallmark of type I CRISPR-Cas systems is the presence of Cas3, which contains both the nuclease and helicase activities required for DNA cleavage during interference. In subtype I-D systems, however, the histidine-aspartate (HD) nuclease domain is encoded as part of a Cas10-like large effector complex subunit and the helicase activity in a separate Cas3' subunit, but the functional and mechanistic consequences of this organisation are not currently understood. Here we show that the Sulfolobus islandicus type I-D Cas10d large subunit exhibits an unusual domain architecture consisting of a Cas3-like HD nuclease domain fused to a degenerate polymerase fold and a C-terminal domain structurally similar to Cas11. Crystal structures of Cas10d both in isolation and bound to S. islandicus rod-shaped virus 3 AcrID1 reveal that the anti-CRISPR protein sequesters the large subunit in a non-functional state unable to form a cleavage-competent effector complex. The architecture of Cas10d suggests that the type I-D effector complex is similar to those found in type III CRISPR-Cas systems and that this feature is specifically exploited by phages for anti-CRISPR defence.

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 αß-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.

First experimental evidence for the presence of a CRISPR toxin in sulfolobus.

Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (cas) genes constitute the adaptive immune system in bacteria and archaea. Although the CRISPR-Cas systems have been hypothesized to encode potential toxins, no experimental data supporting the hypothesis are available in the literature. In this work, we provide the first experimental evidence for the presence of a toxin gene in the type I-A CRISPR system of hyperthermophilic archaeon Sulfolobus. csa5, under the control of its native promoter in a shuttle vector, could not be transformed into CRISPR-deficient mutant Sulfolobus solfataricus Sens1, demonstrating a strong toxicity in the cells. A single-amino-acid mutation destroying the intersubunit bridge of Csa5 attenuated the toxicity, indicative of the importance of Csa5 oligomerization for its toxicity. In line with the absence of Csa5 toxicity in S. solfataricus InF1 containing functional CRISPR systems, the expression of csa5 is repressed in InF1 cells. Induced from the arabinose promoter in Sens1 cells, Csa5 oligomers resistant to 1% SDS co-occur with chromosome degradation and cell death, reinforcing the connection between Csa5 oligomerization and its toxicity. Importantly, a rudivirus was shown to induce Csa5 expression and the formation of SDS-resistant Csa5 oligomers in Sulfolobus cells. This demonstrates that the derepression of csa5 and the subsequent Csa5 oligomerization take place in native virus-host systems. Thus, csa5 is likely to act as a suicide gene under certain circumstances to inhibit virus spreading.

Transcription of the rod-shaped viruses SIRV1 and SIRV2 of the hyperthermophilic archaeon sulfolobus.

The double-stranded DNA genomes of the crenarchaeal rudiviruses SIRV1 (32 kb) and SIRV2 (35 kb) were previously sequenced. Here we present results of the analysis of gene expression of these viruses at different time points after infection of the host cell, Sulfolobus islandicus, and of the mapping of transcriptional start sites. Transcription of both genomes starts simultaneously at multiple sites spread over the total length of the genome and from both strands. The earliest time point when viral transcripts could be detected in cells was 30 min after infection. At this time point all the viral genes, except one, were transcribed. Many genes were clustered and appeared to be transcribed as polycistronic messengers. Although the coat protein-encoding gene was initially also transcribed as a polycistronic messenger, an abundant monocistronic transcript of this gene was detected 2 to 3 h after infection, just before assembly of viral particles. The expression of a single gene, adjacent to the coat protein gene, was upregulated at the late phase of infection, suggesting that it might be involved in specific processing and activation of the coat protein messenger. Start sites of 13 transcripts from the SIRV1 genome have been mapped by primer extension, and promoter sequences have been identified. Similar to host promoters, these viral promoters all contain potential binding sites for the archaeal transcription factors TATA binding protein and transcription factor B. In addition, most of them contain a virus-specific consensus element, suggesting the involvement of alternative transcription factors.