Review 1: "Viral Proteins Activate PARIS-Mediated tRNA Degradation and Viral tRNAs Rescue Infection"

RR:C19 Evidence Scale rating by reviewer:

• Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

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Review:

In this manuscript, Burman, Belukhina, Depardieu et al. characterized the recently discovered PARIS abortive defense system using complementary structural, genetic, and biochemical approaches.

The authors previously reported that PARIS detects anti-restriction proteins expressed by viruses in bacteria and kills the host cell, preventing the infection from spreading through an act of altruistic suicide. In this study, the authors present an exquisitely detailed mechanism of how PARIS affects cell death, tease apart active and inactive conformations of PARIS, and uncover new triggers for PARIS activation in phage genomes. The novel triggers are especially interesting as they present a new insight into the story of how bacterial immune systems sense the presence of phage factors – suggesting that phage proteins with potentially diverse functions (but similar features) can be detected by the same host defense system. This leads to the intriguing possibility that there might be a finite set of phage strategies that are detected by myriad host defenses, possibly simplifying the dizzying repertoire of PhAMPs into sets of phage factors with convergent characteristics. As more mechanisms of bacterial phage-exclusion mechanisms are elucidated, this picture will perhaps become more finely resolved. Moreover, this study adds to examples of other known tRNA-cleaving defense systems (it is particularly interesting that another such example, PrrC, also cleaves the Lys tRNA – this should perhaps be mentioned explicitly), suggesting an alternate explanation for the proliferation of tRNA genes in phage genomes. It is still a stretch to imagine that antiviral defense systems explain the majority of this variation (as the complexities of translational takeover of the cell likely contribute a significant evolutionary imperative here), however they certainly play a more prominent role than previously thought. Finally, the authors conduct a phylogenetic analysis that broadens the classifications of ABC ATPase/TOPRIM defense systems in bacteria, and suggests that the ABC ATPase might generally be a sensor of diverged phage factors that modularly co-evolves with several effector strategies to carry out antiviral defense.

Overall, the study is conducted carefully and thoroughly, the data presented are convincing and the conclusions drawn are well supported by the experiments performed. The paper is fit to be published in its current form in any suitable journal. The following is intended as a point of discussion, and not a critique. This reviewer was particularly struck by the mechanism of Ocr sensing by AriA. A naïve expectation might be that Ocr binds AriA, thereby triggering its ATPase activity, and post-translationally modifying and releasing AriB in an active state. Instead, it appears that Ocr blocks the ATPase activity of AriA, suggesting that in the absence of Ocr, AriA is a constitutively active ATPase. It is admittedly difficult to imagine why maintaining the inactive state (of AriB associated with AriA) would demand ATP hydrolysis. It is perhaps wise to temper the conclusions drawn here, as it is technically possible that the dampening of AriA ATPase activity is simply due to the much higher concentration of Ocr used in these in vitro assays (60x relative to PARIS). Could the K39A mutant fail to bind Ocr simply for stearic reasons in addition to the ATPase defect that this mutation confers? Thus, Ocr sensing might not be dependent on the ATPase activity of AriA, even though it most likely modulates this activity somehow, and affects the release of active AriB.