TDRD3 is an antiviral restriction factor that promotes IFN signaling with G3BP1

Stress granules (SGs) are highly dynamic cytoplasmic foci that form in response to activation of the integrated stress response (ISR) that results in eIF2 alpha phosphorylation and global translation shutdown. Stress granules, which are largely nucleated by G3BP1, serve as hubs for mRNA triage, but there is mounting evidence that they also perform cell signaling functions that are vital to cell survival, particularly during viral infection. We previously showed that SG formation leads to NF kappa B activation and JNK signaling and that this association may be due in part to G3BP1-dependent recruitment of PKR to SGs. Others have reported close associations between G3BP1 and various innate immune PRRs of the type 1 interferon signaling system, including RIG-I. We also reported SG assembly dynamics is dependent on the arginine-methylation status of G3BP1. Another protein that rapidly localizes to SGs, TDRD3, is a methyl reader protein that performs transcriptional activation and adaptor functions within the nucleus, but neither the mechanism nor its function in SGs is clear. Here, we present evidence that TDRD3 localizes to SGs partly based upon methylation potential of G3BP1. We also characterize granules that TDRD3 forms during overexpression and show that these granules can form in the absence of G3BP but also contain translation components found in canonical SGs. We also show for the first time that SGs recruit additional interferon effectors IRF3, IRF7, TBK1, and Sting, and provide evidence that TDRD3 may play a role in recruitment of these factors. We also present evidence that TDRD3 is a novel antiviral protein that is cleaved by enteroviral 2A proteinase. G3BP1 and TDRD3 knockdown in cells results in altered transcriptional regulation of numerous IFN effectors in complex modulatory patterns that are distinctive for G3BP1 and TDRD3. Overall, we describe a novel role of TDRD3 in innate immunity in which G3BP1 and TDRD3 may coordinate to play important roles in regulation of innate antiviral defenses. Author summaryWhen cells are exposed to environmental stresses, such as oxidative stress and viral infection, it induces a cellular response leading to the formation of Stress Granules (SGs) composed of stalled translation initiation complexes (RNA-binding proteins and mRNA) and many other cellular proteins. SGs are also considered to be antiviral structures when they form during viral infection, but viruses can block SG formation to facilitate their survival, often by targeting the essential SG protein G3BP1. Here, we show that a methyl reader protein, TDRD3, localizes to SGs partly based on the methylation potential of G3BP1, and may play a role in the recruitment of innate immune factors to SGs. Further, when overexpressed, TDRD3 can also form SG-like structures independently of G3BP1. We also present evidence that TDRD3 is a novel antiviral protein. Virus replication is enhanced in the absence of both TDRD3 and G3BP1, and virus infection leads to cleavage of TDRD3 by the enterovirus proteinase 2A. Finally, we also show that depletion of TDRD3 and G3BP1 together in cells leads to restriction of transcriptional activation of numerous IFN effectors in response to dsRNA. The patterns of transcriptional activation are distinctive for G3BP1 and TDRD3. We conclude that TDRD3 may play a novel and important role in the regulation of the host antiviral response.