RNase L Amplifies Interferon Signaling by Inducing Protein Kinase R-Mediated Antiviral Stress Granules

Abstract

Virus infection leads to activation of the interferon (IFN)-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 (PRRs), like retinoic acid-inducible I (Rig-I) and melanoma differentiation-associated protein 5 (MDA5), to further amplify IFN production and antiviral response. Although much is known about the mechanics of ligand binding and PRR activation, how cells coordinate RNA sensing with signaling response and interferon production remains unclear. We show that RNA cleavage products of RNase L activity induce the formation of antiviral stress granules (avSGs) by regulating activation of double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) and recruit the antiviral proteins Rig-I, PKR, OAS, and RNase L to avSGs. Biochemical analysis of purified avSGs showed interaction of a 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, but 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 avSGs in RNase L-mediated host defense. We propose a role during viral infection for RNase L-cleaved RNAs in inducing avSGs containing antiviral proteins to provide a platform for efficient interaction of RNA ligands with pattern recognition receptors to enhance IFN production to mount an effective 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 an effective host response during viral infections.

Dance with the Devil: Stress Granules and Signaling in Antiviral Responses

“…However, it may be important to note that viral dsRNA could not be detected within SGs in this infection model, which may indicate that SGs adopt an antiviral function as platforms for RIG-I or MDA5 activation only when they accumulate viral RNA [95]. Another study showed that disruption of SGs in IAV∆NS1and SeV-infected cells did not reduce but actually increased RIG-I-mediated IFN-β production [202], which is in direct contradiction with previous observations [242,263]. It is therefore still unclear whether the changes observed in the IFN response ensue from the disruption of SGs as a coordinating platform or are due to the immunomodulatory function of single SG components.…”

“…Recently, RNase L was reported to localize in SGs upon transfection of 2-5A. The resulting RNase L cleavage products were found to activate PKR, hence triggering SG formation and subsequent induction of IRF3-mediated IFN response [263]. Contradictory to these findings, earlier reports indicate that RNase L activity reduces PKR protein levels and eIF2α phosphorylation via destabilizing PKR mRNA [349].…”

“…At later stages of EMCV infection, cleavage of G3BP1 by the EMCV protease 3C resulted in SG dissolution concomitant with a reduced IFN-β and cytokine response, which could be rescued expressing a cleavage-resistant G3BP1 mutant [245]. The characterization of avSGs formed upon infection with SeV, again revealed the presence of many antiviral components and SG formation was shown to be important for IFN-β production and viral restriction [263]. Furthermore, late during NDV infection, uncapped viral RNA(+) derived from read-through transcription was found to accumulate together with RIG-I in SGs and trigger RIG-I activation.…”

Activation of the antiviral factor RNase L triggers translation of non-coding mRNA sequences

“…For example, they could sequester ribosomes and thereby prevent translation of viral transcripts. On the other hand, translation of fragments could be detrimental to the host since ribosomes melt mRNA secondary structure (Mizrahi et al, 2018) and this structure in the fragments was suggested to be important for activating other dsRNA sensors that trigger interferon production and assembly of stress granules (Burke et al, 2019;Malathi et al, 2007;Manivannan et al, 2020).…”

“…RNase L could therefore be important for generally reshaping the transcriptome and translatome to enhance the response to viral infection. Intriguingly, the RNase L cleavage fragments themselves have been proposed to be important since they can activate dsRNA sensors, such as RIG-I, MDA5, and PKR, that lead to IFN production or shutdown of translation initiation (Malathi et al, 2007;Manivannan et al, 2020). It remains unknown whether the fragments themselves have additional roles, such as the ability to be translated into short peptides.…”