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bioRxiv · 10.1101/2022.03.02.480688

Flipped Over U: Structural Basis for dsRNA Cleavage by the SARS-CoV-2 Endoribonuclease

Frazier, M. N.·Wilson, I. M.·Krahn, J. M.·Butay, K. J.·Dillard, L. B.·Borgnia, M. J. N.·Stanley, R. E.
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Abstract

Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is uridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively.

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Frazier, M. N., Wilson, I. M., Krahn, J. M., Butay, K. J., Dillard, L. B., Borgnia, M. J. N., Stanley, R. E.. 2022-03-02. Flipped Over U: Structural Basis for dsRNA Cleavage by the SARS-CoV-2 Endoribonuclease. https://doi.org/10.1101/2022.03.02.480688

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