A key process in the regulation of protein activities and thus cellular signaling pathways is the modification of proteins by post-translational mechanisms. Knowledge about the enzymes (writers and erasers) that attach and remove post-translational modifications, the targets that are modified and the functional consequences elicited by specific modifications, is crucial for understanding cell biological processes. Moreover detailed knowledge about these mechanisms and pathways helps to elucidate the molecular causes of various diseases and in defining potential targets for therapeutic approaches. Intracellular adenosine diphosphate (ADP)-ribosylation refers to the nicotinamide adenine dinucleotide (NAD⁺)-dependent modification of proteins with ADP-ribose and is catalyzed by enzymes of the ARTD (ADP-ribosyltransferase diphtheria toxin like, also known as PARP) family as well as some members of the Sirtuin family. Poly-ADP-ribosylation is relatively well understood with inhibitors being used as anti-cancer agents. However, the majority of ARTD enzymes and the ADP-ribosylating Sirtuins are restricted to catalyzing mono-ADP-ribosylation. Although writers, readers and erasers of intracellular mono-ADP-ribosylation have been identified only recently, it is becoming more and more evident that this reversible post-translational modification is capable of modulating key intracellular processes and signaling pathways. These include signal transduction mechanisms, stress pathways associated with the endoplasmic reticulum and stress granules, and chromatin-associated processes such as transcription and DNA repair. We hypothesize that mono-ADP-ribosylation controls, through these different pathways, the development of cancer and infectious diseases.

A subset of intracellular mono-ADP-ribosyltransferases diphtheria toxin-like (ARTDs, aka mono-PARPs) is induced by type I interferons. Some of these mono-ARTDs feature antiviral activity while certain RNA viruses, including Chikungunya virus (CHIKV), encode mono-ADP-ribosylhydrolases, suggesting a role for mono-ADP-ribosylation (MARylation) in host-virus conflicts. CHIKV expresses four non-structural proteins (nsP1-nsP4), with nsP3 containing a macrodomain that hydrolyzes and thereby reverses protein MARylation in vitro and in cells. This de-MARylation activity is essential as hydrolase inactivating mutations result in replication defective virus. However, the substrates of MARylation during CHIKV infection are unknown and thus it is unclear how the macrodomain contributes to virus replication and how mono-ARTD-dependent MARylation confers antiviral immunity. We identified ARTD10 and ARTD12 as restriction factors for CHIKV replication in a catalytic activity-dependent manner. CHIKV replication requires processing of the non-structural polyprotein nsP1-4 by the nsP2-encoded protease and the assembly of the four individual nsPs into a functional replication complex. Expression of ARTD10 and ARTD12 resulted in a reduction of processed nsPs. Similarly, MAR hydrolase inactive CHIKV replicon mutants revealed a decrease in processed nsPs, comparable to an nsP2 protease defective mutant. This suggested that the macrodomain contributes to nsP2 protease activity. In support, a hydrolase-deficient virus was complemented by a protease-deficient virus. We hypothesized that MARylation regulates the proteolytic function of nsP2. Indeed, we found that nsP2 is MARylated by ARTD10. This inhibited nsP2 protease activity, thereby preventing polyprotein processing and consequently virus replication. This inhibition was antagonized by the MAR hydrolase activity of nsP3. Together, our findings provide a mechanistic explanation for the need of the viral MAR hydrolase for efficient replication of CHIKV. Author Summary: Infectious diseases still pose major health threats. Especially fast evolving viruses find ever new strategies to manipulate the immune response. With climate warming and increased human mobility vector-borne pathogens like Chikungunya virus (CHIKV) spread and cause world-wide epidemics. Beyond the acute phase, CHIKV patients regularly suffer from chronic rheumatism. This entails a decline in life quality and an economic burden. To date no drugs are approved and the mode of pathogenesis remains elusive. Here we describe a mechanistic function of the CHIKV nsP3 macrodomain. We found that the viral nsP2 is mono-ADP-ribosylated interfering with its auto-proteolytic function. The nsP3 macrodomain removes this modification and restores the protease activity that is essential for replication. Because macrodomains are highly conserved they might represent broad antiviral targets.

Lethal Toxin from Clostridium sordellii (TcsL), which is casually involved in the toxic shock syndrome and in gas gangrene, enters its target cells by receptor-mediated endocytosis. Inside the cell, TcsL mono-O-glucosylates and thereby inactivates Rac/Cdc42 and Ras subtype GTPases, resulting in actin reorganization and an activation of p38 MAP kinase. While a role of p38 MAP kinase in TcsL-induced cell death is well established, data on a role of p38 MAP kinase in TcsL-induced actin reorganization are not available. In this study, TcsL-induced Rac/Cdc42 glucosylation and actin reorganization are differentially analyzed in p38_alpha^−/− MSCV empty vector MEFs and the corresponding cell line with reconstituted p38_alpha expression (p38_alpha^−/− MSCV p38_alpha MEFs). Genetic deletion of p38_alpha results in reduced susceptibility of cells to TcsL-induced Rac/Cdc42 glucosylation and actin reorganization. Furthermore, SB203580, a pyridinyl imidazole inhibitor of p38_alpha/beta MAP kinase, also protects cells from TcsL-induced effects in both p38^−/− MSCV empty vector MEFs and in p38_alpha^−/− MSCV p38_alpha MEFs, suggesting that inhibition of p38_beta contributes to the protective effect of SB203580. In contrast, the effects of the related C. difficile Toxin B are responsive neither to SB203580 treatment nor to p38_alpha deletion. In conclusion, the protective effects of SB203580 and of p38_alpha deletion are likely not based on inhibition of the toxins’ glucosyltransferase activity rather than on inhibited endocytic uptake of specifically TcsL into target cells.