- Taha, Taha Y;
- Suryawanshi, Rahul K;
- Chen, Irene P;
- Correy, Galen J;
- McCavitt-Malvido, Maria;
- O’Leary, Patrick C;
- Jogalekar, Manasi P;
- Diolaiti, Morgan E;
- Kimmerly, Gabriella R;
- Tsou, Chia-Lin;
- Gascon, Ronnie;
- Montano, Mauricio;
- Martinez-Sobrido, Luis;
- Krogan, Nevan J;
- Ashworth, Alan;
- Fraser, James S;
- Ott, Melanie
- Editor(s): Yount, Jacob S
Despite unprecedented efforts, our therapeutic arsenal against SARS-CoV-2 remains limited. The conserved macrodomain 1 (Mac1) in NSP3 is an enzyme exhibiting ADP-ribosylhydrolase activity and a possible drug target. To determine the role of Mac1 catalytic activity in viral replication, we generated recombinant viruses and replicons encoding a catalytically inactive NSP3 Mac1 domain by mutating a critical asparagine in the active site. While substitution to alanine (N40A) reduced catalytic activity by ~10-fold, mutations to aspartic acid (N40D) reduced activity by ~100-fold relative to wild-type. Importantly, the N40A mutation rendered Mac1 unstable in vitro and lowered expression levels in bacterial and mammalian cells. When incorporated into SARS-CoV-2 molecular clones, the N40D mutant only modestly affected viral fitness in immortalized cell lines, but reduced viral replication in human airway organoids by 10-fold. In mice, the N40D mutant replicated at >1000-fold lower levels compared to the wild-type virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection. Our data validate the critical role of SARS-CoV-2 NSP3 Mac1 catalytic activity in viral replication and as a promising therapeutic target to develop antivirals.