Gaughan, C. | bioRxiv Science

bioRxiv2023

Initiation of a ZAKa-dependent Ribotoxic Stress Response by the Innate Immunity Endoribonuclease RNase L

RNase L is a regulated endoribonuclease in higher vertebrates that functions in antiviral innate immunity. Interferons induce OAS enzymes that sense double-stranded RNA of viral origin leading to synthesis of 2,5-oligoadenylate (2-5A) activators of RNase L. However, it is unknown precisely how RNase L inhibits viral infections. To isolate effects of RNase L from other effects of double-stranded RNA or virus, 2-5A was directly introduced into cells. Here we report that RNase L activation by 2-5A causes a ribotoxic stress response that requires the ribosome-associated MAP3K, ZAK. Subsequently, the stress-activated protein kinases (SAPK) JNK and p38 are phosphorylated. RNase L activation profoundly altered the transcriptome by widespread depletion of mRNAs associated with different cellular functions, but also by SAPK-dependent induction of inflammatory genes. Our findings show that 2-5A is a ribotoxic stressor that causes RNA damage through RNase L triggering a ZAK kinase cascade leading to proinflammatory signaling and apoptosis. HighlightsO_LIRNase L signals antiviral innate immunity from the ribosome C_LIO_LIThe ribotoxic stress response requires the MAP3K ZAK C_LIO_LIRNA cleavage leads to transcription of proinflammatory genes C_LIO_LIZAK contributes to apoptosis downstream of RNase L activity C_LI

immunology↗

bioRxiv2021

Specificity and Mechanism of Coronavirus, Rotavirus and Mammalian Two-Histidine-Phosphoesterases That Antagonize Antiviral Innate Immunity

2,5-oligoadenylate(2-5A)-dependent endoribonuclease, RNase L, is a principal mediator of the interferon (IFN) antiviral response. Therefore, regulation of cellular levels of 2-5A is a key point of control in antiviral innate immunity. Cellular 2-5A levels are determined by IFN-inducible 2,5-oligoadenylate synthetases (OASs) and by enzymes that degrade 2-5A. Importantly, many coronaviruses and rotaviruses encode 2-5A degrading enzymes thereby antagonizing RNase L and its antiviral effects. A-kinase anchoring protein 7 (AKAP7), a mammalian counterpart, could possibly limit tissue damage from excessive or prolonged RNase L activation during viral infections or from self double-stranded-RNAs that activate OAS. We show these enzymes, members of the two-histidine-phosphoesterase (2H-PE) superfamily, constitute a sub-family referred here as 2,5-PEs. 2,5-PEs from mouse coronavirus (CoV) MHV (NS2), MERS-CoV (NS4b), group A rotavirus (VP3), and mouse (AKAP7) were investigated for their evolutionary relationships and activities. While there was no activity against 3,5-oligoribonucleotides, all cleaved 2,5-oligoadenylates efficiently, but with variable activity against other 2,5-oligonucleotides. The 2,5-PEs are shown to be metal ion-independent enzymes that cleave trimer 2-5A (2,5-p3A3) producing mono- or di- adenylates with 2,3-cyclic phosphate termini. Our results suggest that elimination of 2-5A might be the sole function of viral 2,5-PEs, thereby promoting viral escape from innate immunity by preventing or limiting the activation of RNase L. IMPORTANCEViruses often encode accessory proteins that antagonize the host antiviral immune response. Here we probed the evolutionary relationships and biochemical activities of two-histidine-phosphoesterases (2H-PEs) that allow some coronaviruses and rotaviruses to counteract antiviral innate immunity. In addition, we investigated the mammalian enzyme, AKAP7, which has homology and shared activities with the viral enzymes and might reduce self-injury. These viral and host enzymes, that we refer to as 2,5-PEs, specifically degrade 2,5-oligoadenylate activators of the antiviral enzyme RNase L. We show that the host and viral enzymes are metal ion independent and exclusively cleave 2,5- and not 3,5-phosphodiester bonds, producing cleavage products with cyclic 2,3-phosphate termini. Our study defines 2,5-PEs as enzymes that share characteristic conserved features with the 2H-PE superfamily but which have specific and distinct biochemical cleavage activities. These findings may eventually lead to pharmacologic strategies for developing antiviral drugs against coronaviruses, rotaviruses, and other viruses.

microbiology↗