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

Although replication of the EGFP-encoding variants was reduced compared to the wt replicon, 210 it remained dependent on functional protease and MAR hydrolase activities (Fig. 2e,f) . As for 211 nsP2, GFP-tagged processed nsP3 was not properly generated from the hydrolase deficient 212 . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 Then we examined whether replication could also be rescued by co-expression of plasmid-237 encoded nsP2 or nsP3 and the respective replicon mutant RNA (CASA or V33E) 238 ( Supplementary Fig. 4 ). Co-expression of the nsP2-459-798 protease domain partially rescued 239 replication of the CASA mutant suggesting that indeed polyprotein synthesis takes place 240 ( Supplementary Fig. 4a-c) . In contrast, co-expression of nsP3 or the isolated nsP3 241 macrodomain was not sufficient to rescue replication of a hydrolase deficient replicon mutant 242 (Supplementary Fig. 4d-f ). To overcome possible differences in the subcellular localization of 243 replication hubs and plasmid-expressed nsP3 macrodomain, we fused the macrodomain to an 244 anti-GFP-nanobody to enhance targeting to sites of replication of the EGFP encoding 245 replicons. We transfected HEK293 cells with plasmids coding for a GFP-nanobody-nsP3-246 macrodomain fusion protein prior to transfection with the 3 EGFP-replicon RNA. We analyzed 247 GFP by flow cytometry (Fig. 3i -k, Supplementary Fig. 5 ). Whereas the overall amount of GFP-248 positive cells when transfected with the V33E mutant was low compared to the wildtype and 249 stayed unaffected by co-expression of GFP-nanobody-nsP3-macrodomain (Fig. 3i) , we 250 observed an increase in GFP intensity dependent on hydrolytic activity of the GFP-nanobody 251 fusion protein (Fig. 3j, k, Supplementary Fig. 5 ). In line with this replication in presence of the 252 wildtype GFP-nanobody-nsP3-macrodomain was slightly but significantly increased (Fig. 3l) . 253