Ribonuclease L Targets Discrete Sites in Influenza Virus RNA

We used 2', 3'-cyclic phosphate cDNA synthesis and Illumina sequencing to identify and quantify ribonuclease L cleavage sites in host and viral RNAs. To make cDNA libraries, we exploited the 2’, 3’-cyclic phosphate at the end of RNA fragments produced by RNase L and other metal-ion-independent endoribonucleases. Using Arabidopsis thaliana tRNA ligase, RNA fragments with 2’, 3’-cyclic phosphates were covalently attached to defined RNA linkers containing an 8-base long unique molecular identifier (UMI) sequence. Libraries prepared in this manner contain cDNA derived exclusively from RNA fragments with 2', 3'-cyclic phosphates. The UMI sequence allows for detailed quantitation. We applied these methods to total RNA from A549 cells infected by influenza A virus unable to express NS1. NS1 inhibits the activation of RNase L.