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

Here we identified the interferon inducible mono-ARTDs, ARTD10 and ARTD12, as host factors 128 restricting CHIKV replication. ARTD10 and ARTD12, dependent on their catalytic activity, 129 prevent polyprotein processing. Similarly, the lack of a hydrolytically active macrodomain 130 results in defective polyprotein processing. We identified nsP2 as substrate for MARylation in 131 vitro and in cells. MARylation of nsP2 by ARTD10 inhibits its proteolytic activity, which 132 supports our observation of defective polyprotein processing. We demonstrate that the MAR 133 hydrolase activity associated with nsP3 removes the modification from nsP2, thereby 134 reactivating its proteolytic activity. Together, our data provide a mechanistic explanation for 135 the role of the CHIKV MAR hydrolase activity of nsP3 for viral replication and offers one 136 mechanism of how MARylation functions in host-virus conflicts. 137 138 139 140 . 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 It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the .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 org/10. /2020 Results 141 ARTD10 and ARTD12 are restrictive for CHIKV replication 142 CHIKV relies on a functional macrodomain for replication [45] . This suggests that the capability 143 to reverse MARylation is essential for proper virus replication and that mono-ARTDs function 144 as antiviral host factors. To identify mono-ARTDs that potentially affect CHIKV replication we 145 performed knockdown experiments (Fig. 1a) . HEK293 cells were transfected with siRNA oligo 146 pools targeting IFNa-inducible mono-ARTDs (Supplementary Fig. 1a and [6] ), such as ARTD10, 147 ARTD12, ARTD8, or ARTD7, prior to transfection with CHIKV replicon RNA. This replicon 148 encodes the four non-structural proteins but lacks the open reading frame of the structural 149 proteins. Instead, the subgenomic promotor of the replicon controls the expression of Gaussia 150 luciferase, which we analyzed as surrogate for virus replication (Fig. 1b) [46] . Replication was 151 analyzed at 9, 24 and 30 hours post transfection (hpt) (Fig. 1a) . At the early time point ARTD10 152 and ARTD12 knockdowns showed a significant increase in replication, while the effect 153 decreased at later time points. Knockdowns of ARTD8 or ARTD7 only allowed for minor 154 increases in replication compared to control (Fig. 1a) . Based on these findings we decided to 155 focus on ARTD10 and ARTD12 and their role in CHIKV replication. We employed HEK293 cells 156 stably expressing TAP-tagged ARTD10 or ARTD12, either wildtype (wt) or catalytically inactive 157 mutants (Supplementary Fig 1b, c and [14] ), to test whether overexpression of these proteins 158 interferes with CHIKV replication (Fig. 1c-g) . Protein expression was induced by doxycycline 159 (Dox) treatment prior to transfection with replicon RNA. Doxycycline treatment itself had little 160 effect on CHIKV replication ( Supplementary Fig. 1d ), still control cells were always treated with 161 doxycycline as well. ARTD10 and ARTD12 reduced replication two-fold, while the respective 162 catalytically inactive variants, ARTD10-G888W(GW) or ARTD12-H564Y(HY), showed little 163 effect (Fig. 1c,d) , suggesting that the MARylation activity of these enzymes is required to 164 . 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://d