The dynamic interplay of host and viral enzymes in type III CRISPR-mediated cyclic nucleotide signalling

Abstract

Cyclic nucleotide second messengers are increasingly implicated in prokaryotic anti-viral defence systems. Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) upon detecting foreign RNA, activating ancillary nucleases that can be toxic to cells, necessitating mechanisms to remove cOA in systems that operate via immunity rather than abortive infection. Previously, we demonstrated that the Sulfolobus solfataricus type III-D CRISPR complex generates cyclic tetra-adenylate (cA4), activating the ribonuclease Csx1, and showed that subsequent RNA cleavage and dissociation acts as an ‘off-switch’ for the cyclase activity. Subsequently, we identified the cellular ring nuclease Crn1, which slowly degrades cA4 to reset the system (Rouillon et al., 2018), and demonstrated that viruses can subvert type III CRISPR immunity by means of a potent anti-CRISPR ring nuclease variant AcrIII-1. Here, we present a comprehensive analysis of the dynamic interplay between these enzymes, governing cyclic nucleotide levels and infection outcomes in virus-host conflict.

Fuse to defuse: a self-limiting ribonuclease-ring nuclease fusion for type III CRISPR defence

“…We first investigated cA4 binding by the H495A variant, which can bind but not cleave cA4 in the Crn2 site, by electrophoretic mobility shift assay (EMSA), using ~10 nM radioactivelylabelled cA4 ( Figure 5B). A clear gel-shift was observed yielding an apparent KD of around 20 nM, which is similar to that previously observed for stand-alone ring nucleases (13). As there are two cA4 binding sites in the protein, we next designed variant enzymes to abolish cA4 binding in either the CARF (H129W) or Crn2 (S452Y) binding sites, guided by the structural model shown in figure 1.…”

“…Examples include cA4 and cA6 for type III CRISPR (5,6), cA3 for the HORMA-DncV-NucC system (24,25) and a variety of cyclic dinucleotides synthesised by diverse CBASS enzymes (26,27). Viruses have a clear imperative to degrade these molecules and circumvent immunity, but it is increasing apparent that cellular immune systems also need a mechanism to turnover these second messengers if they are to prevent runaway toxicity and cell death (13). For type III CRISPR systems at least, immunity rather than abortive infection may be the modus operandi, as mechanisms exist to "kill the messenger".…”

“…This suggests that the RNase activity of Csx1 will only be licensed once cA4 concentrations reach high µM levels in the cell. This is not necessarily a limitation, as the large degree of signal amplification generated by type III CRISPR systems can result in a 6 µM concentration of cA4 in cells detecting only 1 viral RNA (13). It should also be noted that a second cA4-activated defence enzyme is encoded by the CRISPR locus of M. piezophila -a predicted RelE enzyme that may be licensed by cA4 binding to cleave mRNA undergoing translation.…”

“…immunity) or lead to cell dormancy or even death (akin to Abortive Infection) (12). Detection of 1 viral RNA by the Sulfolobus solfataricus type III-D CRISPR system can lead to the generation of 1000 molecules of cyclic tetra-adenylate (cA4), equivalent to an intracellular concentration of 6 µM cA4, which is sufficient to fully activate the Csx1 ribonuclease (13).…”

Heavily Armed Ancestors: CRISPR Immunity and Applications in Archaea with a Comparative Analysis of CRISPR Types in Sulfolobales

“…As described above, cOAs produced by type III effector complexes serve as activators for Csx1/Csm6, and in turn those signaling molecules are degraded by ring nucleases Crn allowing the CRISPR immune response to return to ground state (see above). Thus, the type III effector complex, Csx1/Csm6 and Crn should co-occur in order to govern the controlled catalytic cycle of the type III immune response [259]. Indeed, we generally find all three components in one genome, however, there are exceptions to this rule (Figure 4).…”