ABSTRACT 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.
【저자키워드】 COVID-19, SARS-CoV-2, main protease, oxidative stress, drug targets, dimerization, glutaredoxin, glutathionylation, thioltransferase, 【초록키워드】 coronavirus disease, coronavirus, protease, activity, Replication, Protein, pathophysiology, viral replication, cleavage, mutant, experiment, pp1a, bat coronaviruses, cysteine, acute respiratory syndrome, gel filtration, cysteines, M pro, therapeutic interventions, monomer, Modification, ultracentrifugation, ENCODE, polyprotein, polyproteins, pp1ab, susceptible, Cell, reactive, responsible, inhibit, required, undergo, provide, reducing, demonstrated, regulated, restored, chymotryptic digestion, 【제목키워드】 activity, Reversible,