부산대학교 도서관

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. Author summary: The coronavirus disease 2019 (CoVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and remains a public health issue worldwide. Despite unprecedented efforts to develop vaccines and therapeutics against SARS-CoV-2, our therapeutic arsenal remains limited to only a limited number of therapeutics due to the lack of validated viral drug targets. Here, we identify a potentially new drug target in SARS-CoV-2. The Mac1 domain is an evolutionarily conserved enzyme in coronaviruses that can remove ADP-ribose post-translational modifications from proteins thereby combating host innate immune responses. We generated SARS-CoV-2 with a single point mutation that renders the Mac1 domain catalytically inactive and demonstrated that this mutant virus is less fit in mini lungs in the dish (organoids) and in mice. This work paves the way for the development of novel therapeutics targeting the Mac1 catalytic activity as SARS-CoV-2 antivirals. [ABSTRACT FROM AUTHOR]