Physiologic RNA targets and refined sequence specificity of coronavirus EndoU

Endoribonuclease cleavage of cellular RNAs and changes in host gene expression. (A,B) Gene ontology (GO) analysis of host gene expression during MHV infection. Categories of biological processes enriched with significantly up-regulated genes (P < 0.01, log₂FC > 2) from (A) MHV^(s)-infected or (B) EndoU^mut-infected wt BMM. The top five significantly enriched categories (weightFisher < 0.01) are shown. (C) Expression of host genes in GO category “response to exogenous dsRNA.” Expression (log₁₀-normalized counts) of genes in the GO category “response to exogenous dsRNA” for wt BMM at 12 hpi. (D,E) Volcano plots of changes in host gene expression during MHV infection. (C) Plot comparing MHV^(s)-infected and mock-infected wt BMM and (D) comparing EndoU^mut-infected and MHV^(s)-infected wt BMM. Host genes were considered significantly differentially expressed at FDR < 0.05 and logFC > 2 (up-regulated) or logFC < −2 (down-regulated). (F,G) Relationship between cyclic phosphate and RNA-seq enrichment scores. An enrichment ratio was calculated for all mRNAs using the total sum of cyclic phosphate or RNA-seq normalized counts in MHV^(S) infected samples/total sum of cyclic phosphate or RNA-seq normalized counts in EndoU^mut-infected samples at 9 and 12 hpi in wt and RNase L^−/− BMM (enrichment score = [MHV^(S)/[EndoU^mut]). Genes were assigned to bins as follows: bin 1 = cyclic phosphate and RNA-seq enrichment ratio <1, bin 2 = cyclic phosphate and RNA-seq enrichment ratio ≥1, bin 3 = cyclic phosphate ratio <1 and RNA-seq ratio ≥1, bin 4 = cyclic phosphate ratio ≥1 and RNA-seq ratio <1. Each bin includes genes assigned from 9 and 12 hpi and highlighted genes represent those identified in the dsRNA response GO category (Fig. 7C). (H,I) Dinucleotide specificity analysis for cleavage of transcripts involved in the dsRNA response (Fig. 7C) during infection with MHV^(s) and PDE^mut for positions −2:−1 (H) or MHV^(s) and EndoU^mut for positions −1:+1 (I). Percent of cleavage at each 3′-dinucleotide calculated relative to the total cDNA reads aligned to the mm10 transcriptome per library in wt and RNase L^−/− BMM at 9 and 12 hpi.