Positive single stranded RNA ((+)ssRNA) viruses pose a major health threat due to their high mutation rates and adaptability. To counteract arising resistances against existing treatments and vaccines, novel therapeutic targets are urgently needed. A subset of (+)ssRNA viruses encodes for so-called macrodomains (macros). These comprise a conserved protein fold that is highly associated with ADP-ribosylation. ADP-ribosylation is a post-translational modification (PTM) of proteins. Recently, it

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... s also found as a modification of DNA and RNA. Intracellularly ADP-ribosylation is mainly mediated by the ADP-ribosyltransferase Diphtheria toxin-like (ARTD aka PARP) family. These enzymes mediate the transfer of ADP-ribose (ADPr) from their co-factor NAD+ to the substrate. ADP-ribosylation comes in two variants: mono-ADP-ribosylation (MARylation) which is the transfer of a single ADPr moiety and poly-ADP-ribosylation (PARylation) which constitutes the iterative addition of ADPr units resulting in polymers. The majority of ARTDs mediate MARylation and a subset, including ARTD10, is interferon(IFN)-inducible proposing a role in innate immunity. In this work, the isolated, viral macros of four alphaviruses, two ortho-hepeviruses and one alphacoronavirus were characterized as efficient MAR-hydrolases on several protein substrates in vitro. Meanwhile, their activity towards PARylation was shown to be inefficient. A more detailed characterization focused on the macro of Chikungunya virus (CHIKV), a (re)emerging alphavirus that has already infected millions of people worldwide. CHIKV causes an acute flu-like disease that progresses to a chronic arthralgia in about 30% of the patients. The MAR-hydrolase activity was also confirmed for the macro within the full-length non-structural (ns) protein 3 (nsP3) and catalytically important amino acids were identified. Further the MAR-hydrolase activity was demonstrated in cells. In the following, the mechanistical relevance for the MAR-hydrolase activity of the CHIKV macro and for MARylat [...]