Human pathogenic positive single strand RNA ((+)ssRNA) viruses, including Chikungunya virus, pose severe health problems as for many neither efficient vaccines nor therapeutic strategies exist. To interfere with propagation, viral enzymatic activities are considered potential targets. Here we addressed the function of the viral macrodomains, conserved folds of non-structural proteins of many (+)ssRNA viruses. Macrodomains are closely associated with ADP-ribose function and metabolism. ADP-ribosylation is a post-translational modification controlling various cellular processes, including DNA repair, transcription and stress response. We found that the viral macrodomains possess broad hydrolase activity towards mono-ADP-ribosylated substrates of the mono-ADP-ribosyltransferases ARTD7, ARTD8 and ARTD10 (aka PARP15, PARP14 and PARP10, respectively), reverting this post-translational modification both in vitro and in cells. In contrast, the viral macrodomains possess only weak activity towards poly-ADP-ribose chains synthesized by ARTD1 (aka PARP1). Unlike poly-ADP-ribosylglycohydrolase, which hydrolyzes poly-ADP-ribose chains to individual ADP-ribose units but cannot cleave the amino acid side chain - ADP-ribose bond, the different viral macrodomains release poly-ADP-ribose chains with distinct efficiency. Mutational and structural analyses identified key amino acids for hydrolase activity of the Chikungunya viral macrodomain. Moreover, ARTD8 and ARTD10 are induced by innate immune mechanisms, suggesting that the control of mono-ADP-ribosylation is part of a host-pathogen conflict.

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.

Background: ADP-ribosylation is a posttranslational modification catalyzed in cells by ADP-ribosyltransferases (ARTD or PARP enzymes). The ARTD family consists of 17 members. Some ARTDs modify their substrates by adding ADP-ribose in an iterative process, thereby synthesizing ADP-ribose polymers, the best-studied example being ARTD1/PARP1. Other ARTDs appear to mono-ADP-ribosylate their substrates and are unable to form polymers. The founding member of this latter subclass is ARTD10/PARP10, which we identified as an interaction partner of the nuclear oncoprotein MYC. Biochemically ARTD10 uses substrate-assisted catalysis to modify its substrates. Our previous studies indicated that ARTD10 may shuttle between the nuclear and cytoplasmic compartments. We have now addressed this in more detail. Results: We have characterized the subcellular localization of ARTD10 using live-cell imaging techniques. ARTD10 shuttles between the cytoplasmic and nuclear compartments. When nuclear, ARTD10 can interact with MYC as measured by bimolecular fluorescence complementation. The shuttling is controlled by a Crm1-dependent nuclear export sequence and a central ARTD10 region that promotes nuclear localization. The latter lacks a classical nuclear localization sequence and does not promote full nuclear localization. Rather this non-conventional nuclear localization sequence results in an equal distribution of ARTD10 between the cytoplasmic and the nuclear compartments. ARTD10 forms discrete and dynamic bodies primarily in the cytoplasm but also in the nucleus. These contain poly-ubiquitin and co-localize in part with structures containing the poly-ubiquitin receptor p62/SQSTM1. The co-localization depends on the ubiquitin-associated domain of p62, which mediates interaction with poly-ubiquitin. Conclusions: Our findings demonstrate that ARTD10 is a highly dynamic protein. It shuttles between the nuclear and cytosolic compartments dependent on a classical nuclear export sequence and a domain that mediates nuclear uptake. Moreover ARTD10 forms discrete bodies that exchange subunits rapidly. These bodies associate at least in part with the poly-ubiquitin receptor p62. Because this protein is involved in the uptake of cargo into autophagosomes, our results suggest a link between the formation of ARTD10 bodies and autophagy. Lay Post-translational modifications refer to changes in the chemical appearance of proteins and occur, as the name implies, after proteins have been synthesized. These modifications frequently affect the behavior of proteins, including alterations in their activity or their subcellular localization. One of these modifications is the addition of ADP-ribose to a substrate from the cofactor NAD⁺. The enzymes responsible for this reaction are ADP-ribosyltransferases (ARTDs or previously named PARPs). Presently we know very little about the role of mono-ADP-ribosylation of proteins that occurs in cells. We identified ARTD10, a mono-ADP-ribosyltransferase, as an interaction partner of the oncoprotein MYC. In this study we have analyzed how ARTD10 moves within a cell. By using different live-cell imaging technologies that allow us to follow the position of ARTD10 molecules over time, we found that ARTD10 shuttles constantly in and out of the nucleus. In the cytosol ARTD10 forms distinct structures or bodies that themselves are moving within the cell and that exchange ARTD10 subunits rapidly. We have identified the regions within ARTD10 that are required for these movements. Moreover we defined these bodies as structures that interact with p62. This protein is known to play a role in bringing other proteins to a structure referred to as the autophagosome, which is involved in eliminating debris in cells. Thus our work suggests that ARTD10 might be involved in and is regulated by ADP-riboslyation autophagosomal processes.

ADP-ribosylation describes an ancient and highly conserved posttranslational modification (PTM) of proteins. Many cellular processes have been identified that are regulated by ADP-ribosylation, including DNA repair, gene transcription and signaling processes. Enzymes catalyzing ADP-ribosylation use NAD+ as a cofactor to transfer ADP-ribose to a substrate under release of nicotinamide. In mammals extracellular and intracellular enzymes have been described. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs) and some Sirtuins. Extracellular and intracellular ARTs belong to the cholera toxin-like (ARTC) and the diphtheria toxin-like (ARTD) subclass, respectively. ARTDs can be further subdivided depending on their ability to either generate poly-ADP-ribose chains, or to mono-ADP-ribosylate substrates. Similar to the latter, ARTCs and Sirtuins are restricted to mono-ADP-ribosylation. Recent findings have provided information about the functional consequences of ADP-ribosylation. Analogous to other PTMs, ADP-ribosylation can exert allosteric effects on enzymes, thereby controlling their catalytic activity. Moreover, this PTM can be read by multiple protein motifs and domains mediating protein-protein interactions. Typically these readers can distinguish between mono- and poly-ADP-ribosylation. Furthermore, with the description of proteins that can erase ADP-ribosylation, this posttranslational modification is fully reversible and thus provides an additional mechanism to transiently control protein functions and networks. In this review we will describe the most recent findings on motifs and domains that are related to ADP-ribosylation processes with a particular focus on readers and erasers. These new findings provide evidence for broad functional roles of ADP-ribosylation and a high diversity of mechanisms that contribute to the downstream consequences of this modification.

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.