A single inactivating amino acid change in the SARS-CoV-2 NSP3 Mac1 domain attenuates viral replication and pathogenesis in vivo.

Taha, Taha Y, Rahul K Suryawanshi, Irene P Chen, Galen J Correy, Patrick C O’Leary, Manasi P Jogalekar, Maria McCavitt-Malvido, et al. 2023. “A Single Inactivating Amino Acid Change in the SARS-CoV-2 NSP3 Mac1 Domain Attenuates Viral Replication and Pathogenesis in Vivo.”. BioRxiv : The Preprint Server for Biology.

Abstract

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 therapeutic potential of Mac1 inhibition, 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 wildtype. 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, N40D replicated at >1000-fold lower levels compared to the wildtype virus while inducing a robust interferon response; all animals infected with the mutant virus survived infection and showed no signs of lung pathology. Our data validate the SARS-CoV-2 NSP3 Mac1 domain as a critical viral pathogenesis factor and a promising target to develop antivirals.

Last updated on 06/12/2023
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