Mono-ADP-ribosylation by ARTD10 restricts Chikungunya virus replication by interfering with the proteolytic activity of nsP2

Sarah Krieg; Fabian Pott; Laura Eckei; Maud Verheirstraeten; Mareike Bütepage; Barbara Lippok; Christine Goffinet; Bernhard Lüscher; Patricia Verheugd

Consequences of protein MARylation are poorly understood. Our previous studies indicated 262 that ARTD10-dependent MARylation impairs the catalytic activity of the kinase GSK3b, which 263 is antagonized by cellular MAR hydrolases [37, 49] . Furthermore, MARylation is reported to 264 affect protein-protein interactions, mRNA stability and translation [1]. Following our 265 hypothesis, we determined the consequences of MARylation on the protease activity of nsP2, 266 which is responsible for polyprotein processing [43] . Therefore, we tested whether nsP2 267 serves as substrate for mono-ARTDs. His6-tagged fusion proteins of CHIKV nsP2 or nsP2-459- We obtained similar results with nsP2 MARylated by full length ARTD10, while no MARylation 282 was obtained with ARTD10-GW (Fig. 4c ). In addition, nsP2 was reversibly MARylated by full 283 length ARTD12 (Fig. 4d ). To complement these in vitro findings, we measured MARylation of 284 . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.01.07.896977 doi: bioRxiv preprint 13 nsP2 expressed in HEK293 cells transfected with the 2 EGFP replicon (Fig. 4e) . The 285 immunoprecipitated nsP2-2 EGFP stained positively with a MAR binding reagent. In support, 286 this staining was reduced upon incubation with the recombinant nsP3 macrodomain, 287 providing evidence that this protein is MARylated in cells (Fig. 4e) . Similarly, upon co-288 transfection of a plasmid expressing GFP-nsP2 and the V33E replicon, nsP2 stained positively 289 for MAR (Fig. 4f) . Taken together, we identified CHIKV nsP2 as a new substrate for MARylation 290 in vitro and in cells in context of a viral infection. 291 292