Structural and Functional Characterization of the SARS-CoV-2 Endoribonuclease Nsp15

National Institute Of Environmental Health Sciences

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Abstract

SARS-CoV-2 is the virus responsible for the current Covid-19 global pandemic which has infected millions worldwide. Despite amazing advancements in vaccine and anti-viral development over the past three years, new strategies to treat coronaviruses are still needed. Nsp15 is an endoribonuclease found in all coronaviruses and most nidoviruses, that processes viral RNA to prevent detection by the host immune system. Inactivation of Nsp15 has been shown to lead to an increase in viral double stranded (ds) RNA intermediates, activation of host dsRNA sensors, and stimulation of an immune response. Nsp15 is a promising anti-viral target however, how Nsp15 recognizes and processes viral RNA is poorly understood. Through the combination of cryo-EM, mass-spectrometry, biochemistry, and molecular dynamics simulations we are beginning to establish how Nsp15 recognizes and cleaves both ss- and ds-RNA substrates. Recent research accomplishments are summarized below: To investigate how Nsp15 recognizes dsRNA we recently determined a cryo-EM structure of Nsp15 bound to a 52-nucleotide dsRNA substrate. This structure revealed that oligomerization of Nsp15 is critical for dsRNA binding as we observed that three of the six Nsp15 protomers form a platform for binding dsRNA. Next, we used site-directed mutagenesis coupled with in vitro RNA cleavage assays to determine the significance of Nsp15 residues involved in dsRNA recognition and processing. Nsp15 promotes RNA cleavage by activating the 2'hyroxyl on the ribose of uridine residues for attack on the phosphodiester backbone. Based on our previous structures of Nsp15 bound to ssRNA we hypothesized that dsRNA would have to undergo some type of conformational change for it to be accommodated within the Nsp15 active site. The dsRNA structure revealed that Nsp15 utilizes a base-flipping mechanism to properly orient uridines for cleavage. A critical tryptophan residue from Nsp15 stabilizes the flipped out dsRNA state by forming pi-stacking interactions with the dsRNA. While this work has established that base flipping is critical for dsRNA cleavage it remains unclear if Nsp15 plays an active or passive role in base flipping. To investigate this mechanism further we have determined additional dsRNA bound cryo-EM structures of Nsp15, and we are currently using NMR to study the dynamics of dsRNA base flipping. Another critical question about Nsp15 that we have been trying to address is to establish if any variants of Nsp15 have emerged during the pandemic. Global sequencing efforts during the COVID-19 pandemic have provided unapparelled insight into the evolution of the SARS-CoV-2 genome. We carried out a bioinformatic analysis of millions of publicly deposited SARS-CoV-2 sequences and identified single nucleotide variants across all three domains of Nsp15. We selected a subset of these variants for biochemical analysis and determined what impact these mutations have on Nsp15 activity. Some mutations resulted in decreased cleavage activity while others were shown to influence the ratio of hexamer/monomer during purification of the recombinant protein.