Real-time 2-5A kinetics suggest that interferons β and λ evade global arrest of translation by RNase L

Abstract

Cells of all mammals recognize double-stranded RNA (dsRNA) as a foreign material. In response, they release interferons (IFNs) and activate a ubiquitously expressed pseudokinase/endoribonuclease RNase L. RNase L executes regulated RNA decay and halts global translation. Here, we developed a biosensor for 2′,5′-oligoadenylate (2-5A), the natural activator of RNase L. Using this biosensor, we found that 2-5A was acutely synthesized by cells in response to dsRNA sensing, which immediately triggered cellular RNA cleavage by RNase L and arrested host protein synthesis. However, translation-arrested cells still transcribed IFN-stimulated genes and secreted IFNs of types I and III (IFN-β and IFN-λ). Our data suggest that IFNs escape from the action of RNase L on translation. We propose that the 2-5A/RNase L pathway serves to rapidly and accurately suppress basal protein synthesis, preserving privileged production of defense proteins of the innate immune system. interferon | translation reprogramming | 2-5A

Zika Virus Production Is Resistant to RNase L Antiviral Activity

“…5B), correlating with the aforementioned data indicating ZIKV evasion of type I IFN responses. These findings are also in agreement with a recently published study indicating that production of type I, as well as type III, IFNs are spared from RNase L-mediated translation inhibition, providing additional explanation for the lack of difference in ZIKV titers between infected RNase L KO and MAVS/RNase L DKO cells (47).…”

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

“…We found dORF fLuc activity increased relative to that of the main-ORF rLuc in a 2-5A concentration dependent manner (0.1-10 µM) ( Figure 3D). Notably, this concentration dependence of 3'UTR translation was similar to that observed for the dimerization of an RNase L based 2-5A sensor, consistent with the idea that dORF translation is correlated with RNase L dimerization and activation (Chitrakar et al, 2019). This trend therefore provides added evidence that activation of RNase L increases translation of 3'UTR dORFs relative to the corresponding main ORF.…”

“…Activation of RNase L is therefore traditionally assessed by using an rRNA cleavage assay (Wreschner et al, 1981). This activation can be specifically achieved by transfection with purified 2-5A (prepared as described in Methods) or the double stranded RNA mimic, poly I:C, that also activates additional dsRNA sensors, such as PKR, RIG-I and MDA5 (Chitrakar et al, 2019;de Haro et al, 1996;Martinand et al, 1998). We performed most experiments in the A549 lung carcinoma cell line since these cells had demonstrated robust RNase L activation in previous studies (Burke et al, 2019;Chitrakar et al, 2019;Rath et al, 2019).…”

“…This activation can be specifically achieved by transfection with purified 2-5A (prepared as described in Methods) or the double stranded RNA mimic, poly I:C, that also activates additional dsRNA sensors, such as PKR, RIG-I and MDA5 (Chitrakar et al, 2019;de Haro et al, 1996;Martinand et al, 1998). We performed most experiments in the A549 lung carcinoma cell line since these cells had demonstrated robust RNase L activation in previous studies (Burke et al, 2019;Chitrakar et al, 2019;Rath et al, 2019). We found that 4.5 hours treatment of wild type (WT) A549 cells with 1-10 µM 2-5A was sufficient to generate readily detectable rRNA cleavage products by using electrophoretic analysis (BioAnalyzer) of purified total RNA ( Figure 1B).…”

A ribosomal RNA fragment with 2′,3′-cyclic phosphate and GTP-binding activity acts as RIG-I ligand

“…2′-5′-oligoadenylate (2–5A) containing solution was generated according to Chitrakar A et al ( 32 ). Briefly, HeLa cells were plated in 10 cm plates at 70% confluency, incubated for 4 h with 100 U/ml universal type I interferon alpha (PBL, Piscataway, USA, #11200-2) and transfected with 1 μg/ml poly(I:C) complexed to Lipofectamine 2000.…”