Physiologic RNA targets and refined sequence specificity of coronavirus EndoU

RNase L–dependent and EndoU-dependent cleavage sites in MHV RNA. (A) Schematic outline of analysis to identify EndoU/RNase L–dependent cyclic phosphate reads. (B,C) Fold change values for the top 100 RNase L–dependent or EndoU-dependent cleavage sites. Fold change in cyclic phosphate signal when comparing wt or IFNAR^(−/−) BMM infected with MHV^(S), MHV^(V), PDE^mut, and EndoU^mut virus to RNase L^−/− BMM (B) or MHV^(S), MHV^(V), PDE^mut virus to infection with EndoU^mut virus across all cell types (C) displayed as a violin scatterplot. Log₂-fold change in the absence of RNase L activity (B) or in the absence of EndoU activity (C) was calculated for each position in the MHV RNA. Fold change values for the top 100 RNase L–dependent or EndoU-dependent sites were compared in wt and IFNAR^−/− BMM under conditions of infection with MHV^(S), MHV^(V), PDE^mut, and EndoU^mut virus at 12 hpi (B) or in all cell types across conditions of infection with MHV^(S), MHV^(V), PDE^mut virus at 12 hpi. (D) Frequency and location of RNase L–dependent cleavage sites in MHV RNA. Cyclic phosphate counts at each position in the viral genome were normalized by removing signal that occurred in the absence of RNase L, which emphasizes sites that are RNase L–dependent in wt BMM infected with MHV^(S), and PDE^mut at 9 and 12 hpi. Labeled positions and dinucleotides (−2 base:−1 base) on the graph of PDE^mut represent the top 15 RNase L–dependent cleavage sites (from B) with the greatest fold change in RNase L activity (*site with robust cleavage without canonical RNase L dinucleotide preference and independent of EndoU activity; not identified as top site by RNase L–fold-change analysis). (E) Frequency and location of EndoU-dependent cleavage sites in MHV RNA. Cyclic phosphate counts at each position in the viral genome were normalized by removing signal that occurred in the absence of EndoU, which emphasizes sites that are EndoU-dependent and RNase L–independent in RNase L^−/− BMM infected with wt MHV^(V) at 9 and 12 hpi. Labeled positions and dinucleotides (−1 base:+1 base) represent the top 15 EndoU-dependent cleavage sites with the greatest fold change in EndoU activity (from C). (F) Cumulative distribution of normalized counts by position of MHV genome for every position with ≥10 cyclic phosphate counts across all cell types and infection conditions.