Physiologic RNA targets and refined sequence specificity of coronavirus EndoU

Coronavirus EndoU inhibits dsRNA-activated antiviral responses; however, the physiologic RNA substrates of EndoU are unknown. In this study, we used mouse hepatitis virus (MHV)-infected bone marrow–derived macrophage (BMM) and cyclic phosphate cDNA sequencing to identify the RNA targets of EndoU. EndoU targeted viral RNA, cleaving the 3^′′ side of pyrimidines with a strong preference for U^↓A and C^↓A sequences (endoY^↓A). EndoU-dependent cleavage was detected in every region of MHV RNA, from the 5^′′ NTR to the 3^′′ NTR, including transcriptional regulatory sequences (TRS). Cleavage at two CA dinucleotides immediately adjacent to the MHV poly(A) tail suggests a mechanism to suppress negative-strand RNA synthesis and the accumulation of viral dsRNA. MHV with EndoU (EndoU^mut) or 2^′′–5^′′ phosphodiesterase (PDE^mut) mutations provoked the activation of RNase L in BMM, with corresponding cleavage of RNAs by RNase L. The physiologic targets of EndoU are viral RNA templates required for negative-strand RNA synthesis and dsRNA accumulation. Coronavirus EndoU cleaves U^↓A and C^↓A sequences (endoY^↓A) within viral (+) strand RNA to evade dsRNA-activated host responses.