Macrodomain Mac1 of SARS-CoV-2 Nonstructural Protein 3 Hydrolyzes Diverse ADP-ribosylated Substrates

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that resulted in more than 6-million deaths worldwide. The virus encodes several non-structural proteins (Nsps) that contain elements capable of disrupting cellular processes. Among these Nsp proteins, Nsp3 contains macrodomains, e.g., Mac1, Mac2, Mac3, with potential effects on host cells. Mac1 has been shown to increase SARS-CoV-2 virulence and disrupt ADP-ribosylation pathways in mammalian cells. ADP-ribosylation results from the transfer of the ADP-ribose moiety of NAD+ to various acceptors, e.g., proteins, DNA, RNA, contributing on a cells biological processes. ADP-ribosylation is the mechanism of action of bacterial toxins, e.g., Pseudomonas toxins, diphtheria toxin that disrupt protein biosynthetic and signaling pathways. On the other hand, some viral macrodomains cleavage ADP-ribose-acceptor bond, generating free ADP-ribose. By this reaction, the macrodomain-containing proteins interfere ADP-ribose homeostasis in host cells. Here, we examined potential hydrolytic activities of SARS-CoV-2 Mac1, 2, and 3 on substrates containing ADP-ribose. Mac1 cleaved -NAD+, but not {beta}-NAD+, consistent with stereospecificity at the C-1" bond. In contrast to ARH1 and ARH3, Mac1 did not require Mg2+ for optimal activity. Mac1 also hydrolyzed O-acetyl-ADP-ribose and ADP-ribose-1"-phosphat, but not Mac2 and Mac3. However, Mac1 did not cleave -ADP-ribose-(arginine) and ADP-ribose-(serine)-histone H3 peptide, suggesting that Mac1 hydrolyzes ADP-ribose attached to O- and N-linked functional groups, with specificity at the catalytic site in the ADP-ribose moiety. We conclude that SARS-CoV-2 Mac1 may exert anti-viral activity by reversing host-mediated ADP-ribosylation. New insights on Nsp3 activities may shed light on potential SARS-CoV-2 therapeutic targets. IMPORTANCESARS-CoV-2, the virus responsible for COVID-19, encodes 3 macrodomain-containing proteins, e.g., Mac1, Mac2, Mac3, within non-structural proteins 3 (Nsp3). Mac1 was shown previously to hydrolyze ADP-ribose-phosphate. Inactivation of Mac1 reduced viral proliferation. Here we report that Mac1, but not Mac2 and Mac3, has multiple activities, i.e., Mac1 hydrolyzed. -NAD+ and O-acetyl-ADP-ribose. However, Mac1 did not hydrolyze {beta}-NAD+, ADP-ribose-serine on a histone 3 peptide (aa1-21), and ADP-ribose-arginine, exhibiting substrate selectivity. These data suggest that Mac1 may have multi-function as a -NAD+ consumer for viral replication and a disruptor of host-mediated ADP-ribosylation pathways. Understanding Mac1s mechanisms of action is important to provide possible therapeutic targets for COVID-19.