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

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.

Similarity between mutation spectra in hypermutated genomes of rubella virus and in SARS-CoV-2 genomes accumulated during the COVID-19 pandemic.

Genomes of tens of thousands of SARS-CoV2 isolates have been sequenced across the world and the total number of changes (predominantly single base substitutions) in these isolates exceeds ten thousand. We compared the mutational spectrum in the new SARS-CoV-2 mutation dataset with the previously published mutation spectrum in hypermutated genomes of rubella - another positive single stranded (ss) RNA virus. Each of the rubella isolates arose by accumulation of hundreds of mutations during propagation in a single subject, while SARS-CoV-2 mutation spectrum represents a collection events in multiple virus isolates from individuals across the world. We found a clear similarity between the spectra of single base substitutions in rubella and in SARS-CoV-2, with C to U as well as A to G and U to C being the most prominent in plus strand genomic RNA of each virus. Of those, U to C changes universally showed preference for loops versus stems in predicted RNA secondary structure. Similarly, to what was previously reported for rubella, C to U changes showed enrichment in the uCn motif, which suggested a subclass of APOBEC cytidine deaminase being a source of these substitutions. We also found enrichment of several other trinucleotide-centered mutation motifs only in SARS-CoV-2 - likely indicative of a mutation process characteristic to this virus. Altogether, the results of this analysis suggest that the mutation mechanisms that lead to hypermutation of the rubella vaccine virus in a rare pathological condition may also operate in the background of the SARS-CoV-2 viruses currently propagating in the human population.