RNase L amplifies Interferon signaling by inducing PKR-mediated antiviral stress granules

Virus infection leads to activation of the interferon-induced endoribonuclease, RNase L, which results in degradation of viral and cellular RNAs. Both cellular and viral RNA cleavage products of RNase L bind pattern recognition receptors (PRR) like Retinoic acid-inducible I (Rig-I) and or melanoma differentiation-associated protein 5 (MDA5) to further amplify interferon (IFN) production and antiviral response. Although much is known about the mechanics of ligand binding and PRR activation, how the cells coordinate RNA sensing to signaling response and interferon production remains unclear. We show that RNA cleavage products of RNase L activity induce formation of antiviral stress granule (avSG) by regulating activation of double-stranded RNA (dsRNA)-dependent protein kinase R (PKR), and recruit antiviral proteins Rig-I, PKR, OAS and RNase L to avSG. Biochemical analysis of purified avSG showed interaction of key stress granule protein, G3BP1, with only PKR and Rig-I and not with OAS or RNase L. AvSG assembly during RNase L activation is required for IRF3-mediated IFN production and not IFN signaling or proinflammatory cytokine induction. Consequently, cells lacking avSG formation or RNase L signaling produced less IFN and showed higher susceptibility during Sendai virus infection demonstrating the importance of avSG in RNase L-mediated host defense. During viral infection, we propose a role for RNase L-cleaved RNAs in inducing avSG containing antiviral proteins to provide a platform for efficient interaction of RNA ligands with pattern recognition receptors to enhance IFN production to effectively mount antiviral response. IMPORTANCE Double-stranded RNAs produced during viral infections serve as pathogen associated molecular patterns (PAMPs) and bind pattern recognition receptors to stimulate IFN production. RNase L is an IFN-regulated endoribonuclease that is activated in virus-infected cells and cleaves single-stranded viral and cellular RNAs. The RNase L-cleaved dsRNAs signal to Rig-like helicases to amplify IFN production. This study identifies a novel role of antiviral stress granules induced by RNase L as an antiviral signaling hub to coordinate the RNA ligands with cognate receptors to mount effective host response during viral infections.

RNase L with G3BP1. We observed significant co-localization of these antiviral proteins 156 on RNase L activation in avSGs (Fig. 1B). Following 2-5A transfection, compared to mock 157 treated cells, 32% cells formed stress granules (Fig. 1C). To demonstrate that avSGs 158 were formed in response to RNase L activation, we generated CRISPR-mediated 159 knockout of RNase L in HT1080 cells (49) and observed no avSG formation with 2-5A 160 transfection (Fig.1D). AvSG formation was restored in these cells only by expression of    Interaction of PKR and Rig-I with G3BP1 in the avSG core was seen only after 2-5A 223 11 transfection, however, OAS and RNase L were present in the avSG core but did not 224 interact with G3BP1. MAVS (IPS-1), a mitochondrial adaptor protein required for IFN 225 production and RNase L-mediated IFN induction was not present in avSG core (Fig. 3B).

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As expected, cells lacking G3BP1 or RNase L did not assemble avSG core in response 227 to 2-5A. These results are consistent with avSG co-localization and interaction of G3BP1  (Fig. 5C, D). Taken together, these results indicate that avSG assembly that requires 301 G3BP1 protein, is required for IFN production in response to RNase L activation.

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However, following IFN production, G3BP1 is dispensable for activation of JAK-STAT 303 signaling pathway to transcriptionally induce ISGs. and OAS and RNase L localized to avSG but did not interact with G3BP1 (Fig. 7B). We RNase L as well as avSG assembly in SeV replication (Fig. 7F, G).

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A recent report showed formation of unique RNase L-dependent bodies (RLB) distinct 407 from SG in cells treated with polyI:C (61). The RLB they identify is distinct from avSGs 408 we observe in that RLBs were formed with polyI:C treatment in cells lacking G3BP1 and 409 was independent of SG, and did not require PKR or phosphorylation of eIF2α which were 410 essential in our study for avSG formation. Also, the study did not explore if antiviral 411 proteins localized with RLBs they observed. These differences may be attributed to the 412 use of polyI:C that can bind and activate other dsRNA-binding proteins as described following purification and recruitment of PKR, Rig-I, OAS and RNase L (Fig. 7A, B).

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Cells were harvested at indicated time points in luciferase lysis buffer and luciferase 599 activity was determined using luciferase reagents (Goldbio,USA) and normalized to β-600 galactosidase levels (75).

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Stress granules were isolated as described before (51). Briefly, cells were grown on six  Table 1 638 Table 2 639 ACKNOWLEDGEMENTS 640 We thank Robert Silverman, Ganes Sen and George Stark (Cleveland Clinic) for