Physiologic RNA targets and refined sequence specificity of coronavirus EndoU

Area of Science:

• Virology
• Molecular Biology
• Immunology

Background:

• Coronaviruses possess an Endonuclease U (EndoU) enzyme that suppresses host antiviral responses.
• The specific physiological RNA substrates targeted by viral EndoU remain unidentified.

Purpose of the Study:

• To identify the physiological RNA targets of Coronavirus EndoU.
• To elucidate the mechanism by which EndoU evades host antiviral responses.

Main Methods:

• Infection of bone marrow-derived macrophages (BMM) with mouse hepatitis virus (MHV).
• Cyclic phosphate cDNA sequencing to identify EndoU RNA targets.
• Analysis of MHV mutants with EndoU (EndoU^mut) or 2'-5' phosphodiesterase (PDE^mut) mutations.

Main Results:

• EndoU targets viral RNA, preferentially cleaving at UA and CA sequences (endoYA).
• Cleavage occurs throughout the MHV genome, including the 5' and 3' untranslated regions (NTR) and transcriptional regulatory sequences (TRS).
• MHV mutants lacking functional EndoU or PDE activated RNase L, indicating increased dsRNA recognition.

Conclusions:

• Coronavirus EndoU's physiological targets are viral RNA templates essential for negative-strand RNA synthesis and dsRNA accumulation.
• By cleaving viral RNA at specific dinucleotides, EndoU suppresses the activation of host antiviral pathways, particularly those involving dsRNA detection.