Flipped over U: structural basis for dsRNA cleavage by the SARS-CoV-2 endoribonuclease

Area of Science:

• Virology
• Structural Biology
• Immunology

Background:

• Coronaviruses produce double-stranded (ds) RNA during replication, which can trigger host immune responses via pattern recognition receptors like MDA5.
• The viral enzyme Nsp15, a uridine-specific endoribonuclease, is crucial for coronaviruses to evade MDA5 activation by cleaving dsRNA intermediates.
• The precise mechanism of Nsp15 dsRNA recognition and processing remained unclear due to limitations in previous structural studies.

Purpose of the Study:

• To elucidate the structural basis of SARS-CoV-2 Nsp15's recognition and cleavage of double-stranded RNA (dsRNA).
• To understand how Nsp15 evades host immune detection by processing viral dsRNA intermediates.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was used to determine the structures of SARS-CoV-2 Nsp15 bound to a 52-nucleotide dsRNA molecule.
• Site-directed mutagenesis and RNA cleavage assays were performed to validate the functional implications of the structural findings.

Main Results:

• The Nsp15 hexamer forms a platform that engages dsRNA across multiple subunits, facilitating substrate binding.
• Structural analysis revealed a base-flipping mechanism employed by Nsp15 to position uridine residues for efficient cleavage.
• Nsp15 demonstrates the capability to effectively cleave both single-stranded (ssRNA) and double-stranded (dsRNA) substrates.

Conclusions:

• SARS-CoV-2 Nsp15 utilizes a unique hexameric platform and a base-flipping mechanism for effective dsRNA processing.
• These findings provide critical insights into how coronaviruses manage viral RNA to evade host innate immunity.
• Nsp15 is confirmed as a versatile endoribonuclease with significant implications for understanding coronavirus replication and immune evasion strategies.