Identification and functional characterization of binding partners and substrates of viral macrodomains with a focus on Chikungunya virus

Krieg, Sarah; Lüscher, Bernhard (Thesis advisor); Huber, Michael (Thesis advisor)

Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2022


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 was 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 MARylation mediated by the IFN-responsive ARTDs was investigated in more detail. An RNA replicon-based system was established where Gaussia luciferase was used as a measure for viral replication. Using this setup, ARTD10 was identified as a restriction factor for CHIKV dependent on its catalytic activity and the MAR-interaction of the viral macro was discovered to be crucial for replication. These effects of MARylation on replication were at least in part attributed to a defect in processing of the viral ns polyprotein, which is mediated by the viral protease within nsP2. NsP2 was further identified as a substrate for ARTD10 in vitro and in cells. Its MARylation inhibited protease activity in vitro, while reversal of the modification by the CHIKV macro rescued this effect. Lastly, complementary approaches were used to identify potential host factors that are regulated by (de-)MARylation. Therefore, the BioID system and classical pulldown experiments were performed with nsP3, nsP3-macro and ARTD10 to identify potential common interactors and substrates by mass spectrometry analysis. Additionally, the influence of IFN and catalytic activity was assessed for the interactome of ARTD10. Initial verification could confirm novel and known interactors of nsP3 and Ras GTPase-activating protein-binding protein 1 (G3BP1) was identified as a new, substrate of (de-)MARylation in vitro with potential relevance in CHIKV infection.