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

Finally, RNA was resuspended in elution buffer from the High Pure RNA isolation Kit (Roche). 521 . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 Purity was controlled by agarose gel electrophoresis, concentration was measured using a 522 NanoDrop™ 1000 (Thermo Fisher Scientific) and RNA was stored at -80°C until transfection. the detection of the incorporated radioactive label, dried gels were exposed to X-ray films. 566 567 568 569 . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 //doi.org/10. /2020 In vitro ADP-ribosylation assays with immunoprecipitated ARTD10 and ARTD12 570 HEK293 cells were seeded and after 48 h transfected with plasmids encoding HA-ARTD10 or 571 the inactive GW mutant or with plasmids encoding GFP-ARTD12 using the calcium phosphate 572 precipitation technique. 48 hpt cells were lysed in TAP lysis buffer (50 mM Tris, pH 7.5; 150 573 mM NaCl; 1 mM EDTA; 10% glycerol; 1% NP-40; 2 mM TCEP; PIC) and the lysates were 574 centrifuged at 4°C for 30 min. HA-ARTD10 was immunoprecipitated with 1 μl of anti-HA 575 (BioLegend) antibody and protein G beads and GFP-ARTD12 with 5 µl of GFP-Trap magnetic 576 agarose beads (Chromotek) at 4 °C for 1 h. Afterwards the beads were washed in TAP lysis 577 buffer and reaction buffer (50 mM Tris, pH 8.0, 2 mM TCEP, 4 mM MgCl2). ADP-ribosylation 578 assays were carried out as described above (chapter In vitro ADP-ribosylation assays). 579 580 In vitro protease assay 581