Realtime 2-5A kinetics suggests interferons ß and ? evade global arrest of translation by RNase L

Alisha Chitrakar; Sneha Rath; Jesse Donovan; Kaitlin Demarest; Yize Li; Raghavendra Rao Sridhar; Susan R. Weiss; Sergei V. Kotenko; Ned S. Wingreen; Alexei Korennykh

These observations suggest that 2-5A production may precede the IFN response and that 2-5A is supplied by basal rather than IFN-induced OASs. Therefore, 2-5A/OAS activation does not require IFN stimulation, as reported 39 . Similarly, basal OASs are solely responsible for protection of mouse myeloid cells from murine coronavirus 40 . We also found that the OAS/RNase L activation was not inhibited by pre-treatment with a To determine whether cellular 2-5A dynamics corresponds to a rapid arrest of translation by RNase L, we measured nascent protein synthesis by puromycin pulse labeling in WT and RNase L -/-A549 cells 5 . Treatment of WT, but not RNase L -/cells with poly-IC halted global translation before ISG induction ( Fig. 4F and 4B ). RNase L -/cells exhibited a delayed and incomplete translational attenuation, presumably due to PKR. Translational arrest ahead of ISG induction was also present in HeLa cells (Fig. S4 , B and C). Disengagement of basal protein synthesis before the IFN response was further confirmed using metabolic labeling of nascent proteome with 35 S (Fig. 4G) . The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/476341 doi: bioRxiv preprint IFNs β and  escape the translational shutoff caused by 2-5A 2-5A rapidly stops cell-wide protein synthesis. To examine IFN protein production under these conditions, we treated WT and RNase L -/cells with poly-IC and assayed the media for IFN activity (Fig. 5A) . These tests revealed a time-dependent increase of media antiviral activity and media ability to induce ISGs, which developed after RNase L-mediated translational arrest (Fig. 5B, and fig. S8 ). At time points well beyond translational arrest, we observed an increase in antiviral activity (Fig. 5C ) and ISG induction by two orders of magnitude from media of poly I:C-treated WT and RNase L -/cells (Fig. 5D ). In agreement with the ISG induction readout, media from WT and RNase L -/cells exhibited comparable antiviral activity and similar time-dependent increase of IFN potency (Fig. S9) . These data suggest that shutdown of bulk translation by RNase L thus does not inhibit IFN production.