Alphavirus nsP3 ADP-ribosylhydrolase Activity Disrupts Stress Granule Formation

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

Informatics analyses revealed that RNA granule proteins are enriched for low complexity regions and these proteins are preferentially modified by ADP-ribosylation and that PAR-binding proteins are also enriched for low-complexity regions [27, [70] [71] [72] . Compared with other protein modifications that regulate SG dynamics, ADP-ribosylation is unique in that the polymeric form (i.e., PAR) is able to seed the low-complexity region-containing proteins to form non-membranous structures in vitro [25] [26] [27] [28] 70] . Our data suggest that the nsP3 ADP-ribosylhydrolase likely degrades the PAR that seeds SGs in cells, with G3BP1 as a possible ADP-ribosylated substrate (cf. Fig. 4b, c) . The complete removal of PAR from ADP-ribosylated proteins requires two steps: the degradation of the polymeric chain down to single ADP-ribose units conjugated to proteins, followed by the hydrolysis of the final, proximal ADP-ribose groups from proteins [73] [74] [75] . The alphaviral nsP3 MD alone efficiently hydrolyzes single ADP-ribose groups from ADP-ribosylated proteins in vitro but inefficiently removes the PAR chains [38, 45] . However, it is possible that the nsP3 MD may partner with other protein(s) to process the PAR chains in cells. For example, the hepatitis E viral MD hydrolyzes PARylated proteins efficiently in vitro through association with another viral protein that possesses helicase activities [47] . Notably, several host helicases associate with alphaviral nsP3 in infected cells [39] , and future experiments will help address whether or not any of them are involved in partnering with nsP3 to remove PAR.