Site-Directed Mutagenesis To Characterize Manganese Binding Site of SARS-CoV-2 NSP15 Protein

To carry out successful infection, coronaviruses must avoid detection from their host’s immune system. All vertebrates infecting Coronaviridae, like SARS, MERS, and the novel SARS-CoV-2 (COVID-19), contain homologous versions of Nsp15, a specialized endoribonuclease enzyme that preferentially targets uridine nucleotides (EndoU) which assists coronaviruses in avoidance of the immune system throughout the viral lifecycle. Nsp15’s function and activity are dependent on the divalent metal manganese, thus we investigated the potential location and biochemistry of the metal-binding site (which is currently unknown). In fact, in the absence of Nsp15 coronaviruses are not viable and if the metal manganese is not present, Nsp15 consistently lacks activity (Bhardwaj et al., 2004; Ancar et al., 2020; Deng et al., 2017; Kindler et al., 2017). Four amino acid residues were mutated on NSP15, three of which represent putative metal-binding sites, and the fourth catalytically dead mutant were generated using a specific Polymerase Chain Reaction technique, Site-Directed Mutagenesis. Mutated plasmids were propagated in Escherichia coli, cultured, and induced to produce wild-type and mutant NSP15s. Each protein was purified by Fast-Paced Liquid Chromatography (FPLC) incorporating Nickel Immobilized Metal Affinity (Ni IMAC), glutathione-S-Transferase (GST), and size exclusion chromatography techniques. Future research will focus on biochemically characterizing and determining the precise location of the manganese binding site within NSP15, which may be necessary for developing novel treatments for inhibiting SARS-CoV-2 replication among other related viruses containing NSP15.