The SARS-CoV-2 Conserved Macrodomain Is a Mono-ADP-Ribosylhydrolase

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low μM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity. IMPORTANCE SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused greater than 1.2 million deaths worldwide. With, no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode for a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic post-translational process increasingly recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose, and describe its ADP-ribose binding and hydrolysis activities in direct comparison to SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

CD38 in the age of COVID-19: a medical perspective

“…The SARS-CoV-2 genome encodes for nsp (15): among them, an ADP-ribosylhydrolase (ARH), an enzyme required for virulence that removes ADPR from proteins ribosylated by mART (FIGURE 4). (108,109).…”

Production of Proteins of the SARS-CoV-2 Proteome for Drug Discovery

“… 74 , 75 RNA viruses, including coronaviruses, counter this protective step by the host by their ARH (ARH domain) activity. 75 − 77 This function has been shown in vivo to be essential for pathogenesis of coronavirus infection and that of other RNA viruses. 76 , 78 − 80 In light of the presence of the ARH domain in coronaviruses and other viruses, the ARH activity is likely a broad-spectrum target for antiviral compound discovery.…”

Host ADP-ribosylation and the SARS-CoV-2 macrodomain

“…In this context, it is important to note that both mono- or poly-ADP-ribose can be attached to different aminoacid residues, including glutamates, aspartates, lysines, arginines, serines or cysteines [ 80 ] and that the MacroD-type viral macrodomains discussed here are thought to selectively hydrolyze mono-ADP-ribose-modified glutamates and aspartates [ 70 , 81 , 82 ]. While the aminoacid preference of many PARPs is insufficiently defined, particularly for the less-well studied PARPs discussed here, there is evidence for the SARS-CoV-2 macrodomain acting on PARP10, PARP12 and PARP14-catalyzed modifications in vitro [ 58 , 61 , 83 ].…”