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

Bacterially expressed and purified His-nsP2-459-798, wt or inactive CASA mutant, were 582 incubated with synthetic substrate in 15 µl of reaction buffer (50 mM Tris, pH 8.0, 2 mM TCEP, 583 4 mM MgCl2) for 30, 60 or 120 min at 30°C. As a negative control substrate as well as proteases 584 were incubated alone in reaction buffer for 0 or 120 min at 30°C. The reactions were stopped 585 by the addition of SDS sample buffer. Samples were fractionated by SDS-PAGE and gels 586 subsequently stained with Coomassie blue to visualize the proteins. 587 588 ADP-ribosylation assay with subsequent in vitro protease assay 589 ADP-ribosylation assays were performed in 30 µl reaction buffer (50 mM Tris, pH 8.0, 2 mM 590 TCEP, 4 mM MgCl2) with 50 µM b-NAD + for 30 min at 30°C. Subsequently synthetic substrate 591 was added to the reactions and they were further incubated at 30°C for 30, 60 or 120 min at 592 30°C. As a negative control substrate was incubated alone in reaction buffer for 0 or 120 min 593 . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 at 30°C. The reactions were stopped by the addition of SDS sample buffer. Samples were 594 fractionated by SDS-PAGE and gels subsequently stained with Coomassie blue or subjected to 595 immunoblotting to visualize the proteins. 596 597