Alphavirus nsP3 ADP-ribosylhydrolase Activity Disrupts Stress Granule Formation

Aravinth Kumar Jayabalan; Diane E. Griffin; Anthony K. L. Leung

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/629881 doi: bioRxiv preprint Page 10 SGs are condensates of stalled translation initiation complex along with proteins associated with the untranslated mRNAs. Given that the SG proteome is comprised of multiple protein-protein interaction networks [54, 55] , we tested whether ADP-ribosylhydrolase activity of nsP3 alters the colocalization of SG components in addition to G3BP1 and eIF3b in cells expressing either WT nsP3 (hydrolase-positive MD) or the G32E mutant (hydrolase-negative MD). The examined SG components included the RNA-binding proteins G3BP2, TIA1, TIAR, FMRP, HuR, and PABPC1, as well as the translation-related proteins eIF3e, eIF3i, eIF3j, eIF4G1 and RACK1, and RPS6 (Fig. 2d , e, and S3). As for G3BP1, WT nsP3 co-localized with all tested RNA-binding proteins in both untreated and arsenitetreated cells. On the other hand, as in the case of eIF3b, none of the translation-related proteins colocalized with WT nsP3 in either condition. However, the nsP3 mutant G32E co-localized with all tested SG components in arsenite-treated, but not untreated, cells. Taken together, these data suggest that the nsP3 ADP-ribosylhydrolyase activity regulate associations between SG components.