overexpressing-and-purifying-a-toxic-nuclease-from-escherichia-coli

Overexpressing and Purifying a Toxic Nuclease from <em>Escherichia coli</em>

Escherichia coli has been widely employed as an expression system to produce recombinant proteins. Yet some types of recombinant proteins, such as ...

College of Charleston

Global sequencing efforts from the ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, continue to provide insight into the evolution of the viral genome. Coronaviruses encode 16 nonstructural proteins, within the first two-thirds of their genome, that facilitate viral replication and transcription as well as evasion of the host immune response. However, many of these viral proteins remain understudied. Nsp15 is a uridine-specific endoribonuclease conserved across all coronaviruses. The nuclease activity of Nsp15 helps the virus evade triggering an innate immune response. Understanding how Nsp15 has changed over the course of the pandemic, and how mutations affect its RNA processing function, will provide insight into the evolution of an oligomerization-dependent endoribonuclease and inform drug design. In combination with previous structural data, bioinformatics analyses of 1.9 + million SARS-CoV-2 sequences revealed mutations across Nsp15's three structured domains (N-terminal, Middle, EndoU). Selected Nsp15 variants were characterized biochemically and compared to wild type Nsp15. We found that mutations to important catalytic residues decreased cleavage activity but increased the hexamer/monomer ratio of the recombinant protein. Many of the highly prevalent variants we analyzed led to decreased nuclease activity as well as an increase in the inactive, monomeric form. Overall, our work establishes how Nsp15 variants seen in patient samples affect nuclease activity and oligomerization, providing insight into the effect of these variants in vivo.

Biochemical Characterization of Emerging SARS-CoV-2 Nsp15 Endoribonuclease Variants.

Positive-strand RNA viruses have been the cause of several recent outbreaks and epidemics, including the Zika virus epidemic in 2015, the SARS outbreak in 2003, and the ongoing SARS-CoV-2 pandemic. On June 18-22, 2022, researchers focusing on positive-strand RNA viruses met for the Keystone Symposium "Positive-Strand RNA Viruses" to share the latest research in molecular and cell biology, virology, immunology, vaccinology, and antiviral drug development. This report presents concise summaries of the scientific discussions at the symposium.

Positive-strand RNA viruses-a Keystone Symposia report.

Coronaviruses evade detection by the host immune system with the help of the endoribonuclease Nsp15, which regulates levels of viral double stranded RNA by cleaving 3' of uridine (U). While prior structural data shows that to cleave double stranded RNA, Nsp15's target U must be flipped out of the helix, it is not yet understood whether Nsp15 initiates flipping or captures spontaneously flipped bases. We address this gap by designing fluorinated double stranded RNA substrates that allow us to directly relate a U's sequence context to both its tendency to spontaneously flip and its susceptibility to cleavage by Nsp15. Through a combination of nuclease assays, F NMR spectroscopy, mass spectrometry, and single particle cryo-EM, we determine that Nsp15 acts most efficiently on unpaired Us, particularly those that are already flipped. Across sequence contexts, we find Nsp15's cleavage efficiency to be directly related to that U's tendency to spontaneously flip. Overall, our findings unify previous characterizations of Nsp15's cleavage preferences, and suggest that activity of Nsp15 during infection is partially driven by bulged or otherwise relatively accessible Us that appear at strategic positions in the viral RNA.

Article	Year
Biochemical Characterization of Emerging SARS-CoV-2 Nsp15 Endoribonuclease Variants. Journal of molecular biology\| PubMed ID: 35995266	2022
Positive-strand RNA viruses-a Keystone Symposia report. Annals of the New York Academy of Sciences\| PubMed ID: 36697369	2023
Spontaneous base flipping helps drive Nsp15's preferences in double stranded RNA substrates. Nature communications\| PubMed ID: 39755678	2025

Meredith N. Frazier

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