Review 3: "Viral Proteins Activate PARIS-Mediated tRNA Degradation and Viral tRNAs Rescue Infection"

RR:C19 Evidence Scale rating by reviewer:

• Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review: Burman, Belukhina, Depardieu, et al describe a model for the mechanism of phage defense provided by the PARIS system along with a structure of the AriA-AriB complex. The authors show that six AriA ATPase and three AriB TOPRIM nuclease subunits form a complex, with each AriA dimer binding an AriB monomer. The authors convincingly show that the presence of the T7 phage protein Ocr, a known trigger of PARIS, induces the release of AriB from AriA, and the free AriB is an active nuclease that cleaves tRNA^Lys and leads to translational arrest. The authors also describe two new trigger proteins for the PARIS system encoded by T5, that are, like Ocr, negatively charged proteins. T5 encodes these triggers along with a tRNA^Lys that is resistant to cleavage by AriB, which allows T5 to overcome PARIS-mediated restriction despite encoding activating proteins. Overall, the model being proposed is well-support by the data that is presented. There are a few areas that, while currently supported by the available data, could be more explicitly tested using techniques already established within this work:

1. Some of the conclusions drawn from the mutagenesis could be better supported by additional controls. For example, can the AriA mutants that are proposed to be auto-active (e.g R168E, R168A, R172E, R172A) be used to pull down catalytically dead AriB? This could begin to separate the potential roles of the central and radial pores of AriA.

2. Is AriB a dimer when expressed alone? The original experiments describing PARIS suggest AriB would not be toxic expressed individually, and knowing multimerization state of inactive AriB could further test the model that dimerization induces activation. Do the AriB mutants at the proposed dimerization interface, E285R and F137A, maintain protein stability and disrupt dimerization? Can these AriB mutants be loaded on to AriA? These additional experiments could provide further support for oligomerization-induced activation model.

3. Is there a way to quantify the changes in chromosomal compactness that occur upon PARIS activation? The differences shown here (Fig. S8) are challenging to interpret; though the main conclusions of how the system functions do not rely on these conclusions.

4. Is AriB specifc for tRNA^Lys? Are other tRNAs degraded? The evidence that the phage-encoded tRNA^Lysis sufficient to rescue T5 infectivity suggests this is the main target, but degradation of other tRNAs would not be seen here without a dedicated Northern blot.