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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 14 nsP3 reduces ADP-ribosylation associated with the essential stress granule component G3BP1 Next, we examined the possible molecular targets for the ADP-ribosylhydrolase activity of nsP3 that altered SG composition and structural integrity. We focused on G3BP1 as a candidate because it is a direct binding partner of nsP3 [40, 41, 44, [50] [51] [52] , it is an essential component of SGs formed upon treatment with arsenite, clotrimazole and thapsigargin [10] , it plays a critical role in virus infection [40, 62] , and it is required for the formation of SG-like aggregates associated with ALS [63] [64] [65] . Given that the binding of G3BP1 by the nsP3 HVD is proposed to be critical for SG disassembly [35, 36] and that the ADP-ribosylhydrolase mutant of nsP3 has a decreased ability to suppress SG formation (Fig. 2a ), we next tested whether the observed change in colocalization of SG components is due to the differential ability for nsP3 mutants to bind G3BP1 (Fig. 4a) . GFP-tagged WT or mutants representative of three different classes (G32E, G32S, and Y114A) were transfected into cells, immunoprecipitated and probed for G3BP1. No differences were observed for the G3BP1 association with WT nsP3 and mutants tested, suggesting that ADP-ribosylhydrolase activity does not regulate the association between nsP3 and G3BP1.