Alphavirus nsP3 ADP-ribosylhydrolase Activity Disrupts Stress Granule Formation

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

Besides binding to nsP3, G3BP1 and its associated proteins are also ADP-ribosylated [19] and, therefore, might be targets of nsP3 ADP-ribosylhydrolase activity. To test this possibility, we expressed GFP-G3BP1 alone or co-expressed it with increasing amounts of FLAG-tagged nsP3. GFP-G3BP1 was then immunoprecipitated and probed for ADP-ribosylation. With GFP-G3BP1 expression alone, a prominent signal of ADP-ribosylation appeared at and above the expected molecular weight of GFP-G3BP1. The signal appeared as a smear because of the heterogeneous number of ADP-ribose units added onto G3BP1 and its associated proteins. Upon co-expression of WT full-length nsP3, the ADPribosylation signal associated with G3BP1 was reduced in a dose-dependent manner (Fig. 4b) . Such reduction of signals was not observed when the nsP3 G32E mutant was co-expressed (Fig. 4c) , suggesting that the ADP-ribosylhydrolase activity of nsP3 is responsible for reducing ADP-ribosylation associated with the essential SG component G3BP1.