Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (M(pro)), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. M(pro) is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show M(pro) is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated M(pro) could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated M(pro) primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated M(pro) exists as a monomer. Tryptic and chymotryptic digestions of M(pro) as well as experiments using a C300S M(pro) mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon M(pro) glutathionylation. These findings indicate that M(pro) dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for M(pro) activity, and provides a novel target for the development of agents to block M(pro) dimerization and activity. This feature of M(pro) may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses.