SARS‐CoV‐2 3CLpro displays faster self‐maturation in vitro than SARS‐CoV 3CLpro due to faster C‐terminal cleavage

The coronavirus (CoV) disease 2019 (COVID‐19) caused by the severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2) has become a worldwide pandemic. The 3C‐like protease (3CLpro), which cleaves 11 sites including its own N‐ and C‐termini on the viral polyproteins, is essential for SARS‐CoV‐2 replication. In this study, we constructed the full‐length inactive 3CLpro with N‐ and C‐terminal extensions as substrates for monitoring self‐cleavage by wild‐type 3CLpro. We found that the rate‐limiting C‐terminal self‐cleavage rate of SARS‐CoV‐2 3CLpro was 35‐fold faster than that of SARS‐CoV 3CLpro using the Trx/GST‐tagged C145A 3CLpro substrates. Since self‐cleavage of 3CLpro is the initial step for maturation of other viral proteins, our study suggests more facile SARS‐CoV‐2 replication than that of SARS‐CoV. The 3C‐like protease (3CLpro), which cleaves 11 sites including its own N‐ and C‐termini on the viral polyproteins, is essential for SARS‐CoV‐2 replication. We found that the rate‐limiting C‐terminal self‐cleavage rate of SARS‐CoV‐2 3CLpro was 35‐fold faster than that of SARS‐CoV 3CLpro using the Trx/GST‐tagged C145A 3CLpro substrates, suggesting more facile SARS‐CoV‐2 replication than SARS‐CoV replication.