A viral ring nuclease anti-CRISPR subverts type III CRISPR immunity

Abstract

The CRISPR system provides adaptive immunity against mobile genetic elements in bacteria and archaea. On detection of viral RNA, type III CRISPR systems generate a cyclic oligoadenylate (cOA) second messenger 1-3 , activating defence enzymes and sculpting a powerful antiviral response that can drive viruses to extinction 4,5 . Cyclic nucleotides are increasingly implicated as playing an important role in host-pathogen interactions 6,7 . Here, we identify a widespread new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease (AcrIII-1) is the first Acr described for type III CRISPR systems and is widely distributed in archaeal and bacterial viruses, and proviruses. The enzyme uses a novel fold to bind cA4 specifically and utilizes a conserved active site to rapidly cleave the signalling molecule, allowing viruses to neutralise 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. This study highlights the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.Type III CRISPR-Cas systems synthesise the signalling molecule cyclic oligoadenylate (cOA) from ATP 1,2 when they detect viral RNA. cOA molecules are synthesised with a range of ring sizes with 3-6 AMP subunits (denoted cA3, cA4 etc.) by the cyclase domain of the Cas10 protein [1][2][3]9,10 . cOA binds to a specific protein domain, known as a CARF (CRISPR Associated Rossman Fold) domain. CARF domains are found fused to a variety of effector domains that are known or predicted to cleave RNA, DNA, or function as transcription factors 11 . The best characterised CARF protein family is the Csx1/Csm6 family of HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) ribonucleases, which are activated by cOA binding and cleave RNA with minimal sequence dependence 1-3 . A number of studies have demonstrated that the cOA signalling component of type III systems is crucial for effective immunity against viruses 4,12-15 , highlighting the importance of this facet of CRISPR immunity.Recently, we identified a cellular enzyme in Sulfolobus solfataricus, hereafter referred to as the Crn1 family (for "CRISPR associated ring nuclease 1"), that degrades cA4 molecules and thus deactivates the Csx1 ribonuclease in vitro 16 . These enzymes exhibit very slow kinetics, and are thought to act by mopping up cA4 molecules in the cell without compromising the immunity provided by the type III CRISPR system. Unsurprisingly, viruses have responded to the threat of the CRISPR system by evolving a range of anti-CRISPR (Acr) proteins, which are used to inhibit and overcome the cell's CRISPR defences (reviewed in 17 ). Acr's have been identified for the type I-D 18 , I-F , II-A and V-A effector complexes (reviewed in 17,19,20 ), numbering over 40 families 21 , but importantly not for type III systems. We focussed on one of the protein families, DUF1874, conserved and widespread ...

Tetramerisation of the CRISPR ring nuclease Csx3 facilitates cyclic oligoadenylate cleavage

“…386 387 Crn3 has a highly unusual cooperative catalytic mechanism 388 A. fulgidus Crn3/Csx3 is a much faster enzyme than Crn1. The single turnover reaction rate for cA 4 389 cleavage of 3.6 min -1 is closer to that of the AcrIII-1 family, which utilise a distinct active site 390 architecture, employing a conserved histidine residue as a general acid to stabilise the oxyanion 391 leaving group (Athukoralage et al, 2020b). This raises an important question: why does Crn3/Csx3 392 degrade cA 4 so quickly when it plays a role in cellular defence rather than viral offense?…”

“…Each datapoint is the average of three technical replicates and error bars show the standard deviation of the mean. nuclease 2 (Crn2) enzymes (Athukoralage et al, 2020b, Samolygo et al, 2020 picture is becoming clearer for the specific ring nucleases as they are identified in different 382 archaeal and bacterial genomes (Figure 7). It appears to be an emerging paradigm that cells with a 383 type III CRISPR defence require a mechanism to remove the cOA signal, either once viral infection 384…”

“…and return the cell to a basal state. Some archaea encode a dedicated ring nuclease 55 , recently named as the Crn1 family (CRISPR-associated ring nuclease 56 1) (Athukoralage et al, 2020b). Crn1 is a specialised CARF domain protein that splits the cA 4 ring 57 into two linear A 2 products to switch off the antiviral response .…”

“…an anti-CRISPR (Acr). This enzyme, known as AcrIII-1, binds cA 4 by means of a distinct protein 62 fold (DUF1874) unrelated to the CARF domain, and degrades cA 4 rapidly using conserved active 63 site residues (Athukoralage et al, 2020b). These viral ring nucleases can abrogate type III 64 CRISPR immunity by rapidly destroying the cA 4 infection signal.…”

“…nuclease AcrIII-1 appears in many archaeal virus and some bacteriophage genomes 375 (Athukoralage et al, 2020b). Homologues of AcrIII-1 are also found associated with some bacterial 376 type III CRISPR systems, and in this context have been named as CRISPR associated ring 377…”