Coronaviruses are large RNA viruses, with genomes of over 30 kb in some species. They cause a range of diseases in birds, mammals, and humans. While most human coronaviruses lead to mild respiratory infections, in the last two decades, we saw two major epidemics and a global pandemic, mainly caused by the virus SARS-CoV-2, causing severe respiratory illnesses and fatalities. Within the coronavirus genome, there is a gene encoding a non-structural protein 14 (nsp14), which possesses 3’-5’exonuclease and methyltransferase enzymatic activities. The exonuclease nsp14 participates in the repair of misincorporated nucleotides during viral genome replication, and its presence is exceptionally rare among RNA viruses. The exonuclease activity of nsp14 is significantly enhanced by the binding of another viral protein, nsp10, which lacks enzymatic activity, but acts as a critical cofactor for several enzymatically active coronavirus nsps. Both nsp14 and nsp10 are highly conserved and have similar sequences among the various coronavirus species.
To characterize the exonuclease activity of nsp14 and its interactions with nsp10, mutant versions of the nsp14 and nsp10 proteins were created using PCR mutagenesis. Both the wild-type and mutant versions of nsp14 and nsp10, as well as a truncated version of nsp10, were produced in a bacterial expression system utilizing E. coli bacteria. The exonuclease activity of each nsp14 variant was observed in vitro using activity assays with ssRNA, dsRNA, and dsRNA, with mismatched base pairs as the substrates in the presence or absence of nsp10 variants. The results of this study provide insights into the functioning of nsp14 exonuclease and its interactions with nsp10, which may serve as a basis for more detailed characterization in future research.
This research was funded by the project the National Institute Virology and Bacteriology (Programme EXCELES; Project No. LX22NPO5103) and by the European Union, Next Generation EU. RVO: 61388963 is also acknowledged.