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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.

Physiologic RNA targets and refined sequence specificity of coronavirus EndoU