Assessing the interplay between the mono-ADP-ribosyltransferase ARTD10 and the Chikungunya viral macrodomain in modulating the antiviral immune response.

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Viruses entering host cells are recognized by the innate immune system. For efficient replication viruses evolved strategies to antagonize the immune system and to hijack the cellular processes. Recent findings suggest that mono-ADP-ribosylation (MARylation), a post-translational modification, is part of the innate immune response. In this proposal we plan to define the role of MARylation in the conflict between mammalian hosts and Chikungunya virus (CHIKV).CHIKV caused large epidemic outbreaks worldwide in recent years with severe health and economic burden. Presently neither a vaccine nor therapeutic treatments are available. CHIKV encodes only 4 non-structural proteins (nsPs), of which nsP3 harbors a macrodomain, a conserved protein fold, closely linked to MARylation. MARylation involves the transfer of ADP-ribose from NAD+ onto substrate proteins and is intracellularly mainly catalyzed by ADP-ribosyltransferases diphtheria toxin-like (ARTDs). We focus on ARTD10, which is induced in response to type I interferons (IFNs). We identified the CHIKV macrodomain as a de-MARylating enzyme, which is required for efficient viral replication. Moreover, preliminary work demonstrates that nsP1-nsP3 are substrates of ARTD10 and thus their activities potentially controlled by MARylation. These findings led us to hypothesize a role for MARylation in innate immunity, which we propose to function in two distinct ways: (I) Host-dependent MARylation of the CHIKV nsPs directly affects their function and thereby impacts on the viral life cycle. (II) MARylation of host-factors enables them to promote an antiviral state. Both hypotheses imply a critical role of the viral macrodomain and its MAR hydrolase function.Therefore, I propose to clarify the biochemical and physiological consequences of ARTD10-catalyzed MARylation of the nsPs. This will involve mapping the modification sites, addressing the impact of MARylation on specific functions of the individual nsPs, and in consequence on the viral life cycle. In addition, we aim at the identification of host factors regulated by MARylation. To determine those, we will identify common substrates of ARTD10 and the CHIKV nsP3 hydrolase. A further selection criterion will be whether the MARylation of a particular substrate is subject to IFN regulation. I assume that these substrates are important host factors that we plan to investigate regarding their relevance for viral replication. This will involve proteomic studies, the analysis of MARylation of individual cellular proteins, enzymatic MARylation and de-MARylation assays, the knock-down and knock-out of cellular host factors and the consequences on the viral life cycle. Together these studies will define how MARylation is used in antiviral defense, and how CHIKV antagonizes this innate immune strategy. The findings of these studies will be relevant to define novel entry points for antiviral therapies.