An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity.

The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA)1,2 and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP)3-5. Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain6, sculpting a powerful antiviral response7-10 that can drive viruses to extinction7,8. Cyclic nucleotides are increasingly implicated in host-pathogen interactions11-13. Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize the type III CRISPR defence system. The AcrIII-1 family has a broad host range, as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

A seed motif for target RNA capture enables efficient immune defence by a type III-B CRISPR-Cas system.

CRISPR-Cas systems provide an adaptive defence against foreign nucleic acids guided by small RNAs (crRNAs) in archaea and bacteria. The Type III CRISPR systems are reported to carry RNase, RNA-activated DNase and cyclic oligoadenylate (cOA) synthetase activity, and are significantly different from other CRISPR systems. However, detailed features of target recognition, which are essential for enhancing target specificity remain unknown in Type III CRISPR systems. Here, we show that the Type III-B Cmr-α system in S. islandicus generates two constant lengths of crRNA independent of the length of the spacer. Either mutation at the 3'-end of crRNA or target truncation greatly influences the target capture and cleavage by the Cmr-α effector complex. Furthermore, we found that cleavage at the tag-proximal site on the target RNA by the Cmr-α RNP complex is delayed relative to the other sites, which probably provides Cas10 more time to function as a guard against invaders. Using a mutagenesis assay in vivo, we discovered that a seed motif located at the tag-distal region of the crRNA is required by Cmr1α for target RNA capture by the Cmr-α system thereby enhancing target specificity and efficiency. These findings further refine the model for immune defence of Type III-B CRISPR-Cas system, commencing on capture, cleavage and regulation.

Stable maintenance of the rudivirus SIRV3 in a carrier state in Sulfolobus islandicus despite activation of the CRISPR-Cas immune response by a second virus SMV1.

Carrier state viral infection constitutes an equilibrium state in which a limited fraction of a cellular population is infected while the remaining cells are transiently resistant to infection. This type of infection has been characterized for several bacteriophages but not, to date, for archaeal viruses. Here we demonstrate that the rudivirus SIRV3 can produce a host-dependent carrier state infection in the model crenarchaeon Sulfolobus. SIRV3 only infected a fraction of a Sulfolobus islandicus REY15A culture over several days during which host growth was unimpaired and no chromosomal DNA degradation was observed. CRISPR spacer acquisition from SIRV3 DNA was induced by coinfecting with the monocaudavirus SMV1 and it was coincident with increased transcript levels from subtype I-A adaptation and interference cas genes. However, this response did not significantly affect the carrier state infection of SIRV3 and both viruses were maintained in the culture over 12 days during which SIRV3 anti-CRISPR genes were shown to be expressed. Transcriptome and proteome analyses demonstrated that most SIRV3 genes were expressed at varying levels over time whereas SMV1 gene expression was generally low. The study yields insights into the basis for the stable infection of SIRV3 and the resistance to the different host CRISPR-Cas interference mechanisms. It also provides a rationale for the commonly observed coinfection of archaeal cells by different viruses in natural environments.

Tolerance of Sulfolobus SMV1 virus to the immunity of I-A and III-B CRISPR-Cas systems in Sulfolobus islandicus.

Sulfolobus islandicus Rey15A encodes one Type I-A and two Type III-B systems, all of which are active in mediating nucleic acids interference. However, the effectiveness of each CRISPR system against virus infection was not tested in this archaeon. Here we constructed S. islandicus strains that constitutively express the antiviral immunity from either I-A, or III-B, or I-A plus III-B systems against SMV1 and tested the response of each host to SMV1 infection. We found that, although both CRISPR immunities showed a strong inhibition to viral DNA replication at an early stage of incubation, the host I-A CRISPR immunity gradually lost the control on virus proliferation, allowing accumulation of cellular viral DNA and release of a large number of viral particles. In contrast, the III-B CRISPR immunity showed a tight control on both viral DNA replication and virus particle formation. Furthermore, the SMV1 tolerance to the I-A CRISPR immunity did not result from the occurrence of escape mutations, suggesting the virus probably encodes an anti-CRISPR protein (Acr) to compromise the host I-A CRISPR immunity. Together, this suggests that the interplay between viral Acrs and CRISPR-Cas systems in thermophilic archaea could have shaped the stable virus-host relationship that is observed for many archaeal viruses.

Coupling transcriptional activation of CRISPR-Cas system and DNA repair genes by Csa3a in Sulfolobus islandicus.

CRISPR-Cas system provides the adaptive immunity against invading genetic elements in prokaryotes. Recently, we demonstrated that Csa3a regulator mediates spacer acquisition in Sulfolobus islandicus by activating the expression of Type I-A adaptation cas genes. However, links between the activation of spacer adaptation and CRISPR transcription/processing, and the requirement for DNA repair genes during spacer acquisition remained poorly understood. Here, we demonstrated that de novo spacer acquisition required Csa1, Cas1, Cas2 and Cas4 proteins of the Sulfolobus Type I-A system. Disruption of genes implicated in crRNA maturation or DNA interference led to a significant accumulation of acquired spacers, mainly derived from host genomic DNA. Transcriptome and proteome analyses showed that Csa3a activated expression of adaptation cas genes, CRISPR RNAs, and DNA repair genes, including herA helicase, nurA nuclease and DNA polymerase II genes. Importantly, Csa3a specifically bound the promoters of the above DNA repair genes, suggesting that they were directly activated by Csa3a for adaptation. The Csa3a regulator also specifically bound to the leader sequence to activate CRISPR transcription in vivo. Our data indicated that the Csa3a regulator couples transcriptional activation of the CRISPR-Cas system and DNA repair genes for spacer adaptation and efficient interference of invading genetic elements.

The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease.

Prokaryotes are frequently exposed to potentially harmful invasive nucleic acids from phages, plasmids, and transposons. One method of defense is the CRISPR-Cas adaptive immune system. Diverse CRISPR-Cas systems form distinct ribonucleoprotein effector complexes that target and cleave invasive nucleic acids to provide immunity. The Type III-B Cmr effector complex has been found to target the RNA and DNA of the invader in the various bacterial and archaeal organisms where it has been characterized. Interestingly, the gene encoding the Csx1 protein is frequently located in close proximity to the Cmr1-6 genes in many genomes, implicating a role for Csx1 in Cmr function. However, evidence suggests that Csx1 is not a stably associated component of the Cmr effector complex, but is necessary for DNA silencing by the Cmr system in Sulfolobus islandicus. To investigate the function of the Csx1 protein, we characterized the activity of recombinant Pyrococcus furiosus Csx1 against various nucleic acid substrates. We show that Csx1 is a metal-independent, endoribonuclease that acts selectively on single-stranded RNA and cleaves specifically after adenosines. The RNA cleavage activity of Csx1 is dependent upon a conserved HEPN motif located within the C-terminal domain of the protein. This motif is also key for activity in other known ribonucleases. Collectively, the findings indicate that invader silencing by Type III-B CRISPR-Cas systems relies both on RNA and DNA nuclease activities from the Cmr effector complex as well as on the affiliated, trans-acting Csx1 endoribonuclease.

Inter-viral conflicts that exploit host CRISPR immune systems of Sulfolobus.

Infection of Sulfolobus islandicus REY15A with mixtures of different Sulfolobus viruses, including STSV2, did not induce spacer acquisition by the host CRISPR immune system. However, coinfection with the tailed fusiform viruses SMV1 and STSV2 generated hyperactive spacer acquisition in both CRISPR loci, exclusively from STSV2, with the resultant loss of STSV2 but not SMV1. SMV1 was shown to activate adaptation while itself being resistant to CRISPR-mediated adaptation and DNA interference. Exceptionally, a single clone S-1 isolated from an SMV1 + STSV2-infected culture, that carried STSV2-specific spacers and had lost STSV2 but not SMV1, acquired spacers from SMV1. This effect was also reproducible on reinfecting wild-type host cells with a variant SMV1 isolated from the S-1 culture. The SMV1 variant lacked a virion protein ORF114 that was shown to bind DNA. This study also provided evidence for: (i) limits on the maximum sizes of CRISPR loci; (ii) spacer uptake strongly retarding growth of infected cultures; (iii) protospacer selection being essentially random and non-directional, and (iv) the reversible uptake of spacers from STSV2 and SMV1. A hypothesis is presented to explain the interactive conflicts between SMV1 and the host CRISPR immune system.

CRISPR-mediated defense mechanisms in the hyperthermophilic archaeal genus Sulfolobus.

CRISPR (clustered regularly interspaced short palindromic repeats)-mediated virus defense based on small RNAs is a hallmark of archaea and also found in many bacteria. Archaeal genomes and, in particular, organisms of the extremely thermoacidophilic genus Sulfolobus, carry extensive CRISPR loci each with dozens of sequence signatures (spacers) able to mediate targeting and degradation of complementary invading nucleic acids. The diversity of CRISPR systems and their associated protein complexes indicates an extensive functional breadth and versatility of this adaptive immune system. Sulfolobus solfataricus and S. islandicus represent two of the best characterized genetic model organisms in the archaea not only with respect to the CRISPR system. Here we address and discuss in a broader context particularly recent progress made in understanding spacer recruitment from foreign DNA, production of small RNAs, in vitro activity of CRISPR-associated protein complexes and attack of viruses and plasmids in in vivo test systems.

An archaeal immune system can detect multiple protospacer adjacent motifs (PAMs) to target invader DNA.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system provides adaptive and heritable immunity against foreign genetic elements in most archaea and many bacteria. Although this system is widespread and diverse with many subtypes, only a few species have been investigated to elucidate the precise mechanisms for the defense of viruses or plasmids. Approximately 90% of all sequenced archaea encode CRISPR/Cas systems, but their molecular details have so far only been examined in three archaeal species: Sulfolobus solfataricus, Sulfolobus islandicus, and Pyrococcus furiosus. Here, we analyzed the CRISPR/Cas system of Haloferax volcanii using a plasmid-based invader assay. Haloferax encodes a type I-B CRISPR/Cas system with eight Cas proteins and three CRISPR loci for which the identity of protospacer adjacent motifs (PAMs) was unknown until now. We identified six different PAM sequences that are required upstream of the protospacer to permit target DNA recognition. This is only the second archaeon for which PAM sequences have been determined, and the first CRISPR group with such a high number of PAM sequences. Cells could survive the plasmid challenge if their CRISPR/Cas system was altered or defective, e.g. by deletion of the cas gene cassette. Experimental PAM data were supplemented with bioinformatics data on Haloferax and Haloquadratum.

CRISPR associated diversity within a population of Sulfolobus islandicus.

BACKGROUND: Predator-prey models for virus-host interactions predict that viruses will cause oscillations of microbial host densities due to an arms race between resistance and virulence. A new form of microbial resistance, CRISPRs (clustered regularly interspaced short palindromic repeats) are a rapidly evolving, sequence-specific immunity mechanism in which a short piece of invading viral DNA is inserted into the host's chromosome, thereby rendering the host resistant to further infection. Few studies have linked this form of resistance to population dynamics in natural microbial populations. METHODOLOGY/PRINCIPAL FINDINGS: We examined sequence diversity in 39 strains of the archeaon Sulfolobus islandicus from a single, isolated hot spring from Kamchatka, Russia to determine the effects of CRISPR immunity on microbial population dynamics. First, multiple housekeeping genetic markers identify a large clonal group of identical genotypes coexisting with a diverse set of rare genotypes. Second, the sequence-specific CRISPR spacer arrays split the large group of isolates into two very different groups and reveal extensive diversity and no evidence for dominance of a single clone within the population. CONCLUSIONS/SIGNIFICANCE: The evenness of resistance genotypes found within this population of S. islandicus is indicative of a lack of strain dominance, in contrast to the prediction for a resistant strain in a simple predator-prey interaction. Based on evidence for the independent acquisition of resistant sequences, we hypothesize that CRISPR mediated clonal interference between resistant strains promotes and maintains diversity in this natural population.