The Stress Granule Protein G3BP1 Recruits Protein Kinase R To Promote Multiple Innate Immune Antiviral Responses

Abstract

Stress granules (SGs) are cytoplasmic storage sites containing translationally silenced mRNPs that can be released to resume translation after stress subsides. We previously showed that poliovirus 3C proteinase cleaves the SG-nucleating protein G3BP1, blocking the ability of cells to form SGs late in infection. Many other viruses also target G3BP1 and inhibit SG formation, but the reasons why these functions evolved are unclear. Previously, we also showed a link between G3BP1-induced SGs and protein kinase R (PKR)-mediated translational control, but the mechanism of PKR interplay with SG and the antiviral consequences are unknown. Here, we show that G3BP1 exhibits antiviral activity against several enteroviruses, whereas truncated G3BP1 that cannot form SGs does not. G3BP1-induced SGs are linked to activation of innate immune transcriptional responses through NF-B and JNK. The G3BP1-induced SGs also recruit PKR and other antiviral proteins. We show that the PXXP domain within G3BP1 is essential for the recruitment of PKR to SGs, for eIF2␣ phosphorylation driven by PKR, and for nucleating SGs of normal composition. We also show that deletion of the PXXP domain in G3BP1 compromises its antiviral activity. These findings tie PKR activation to its recruitment to SGs by G3BP1 and indicate that G3BP1 promotes innate immune responses at both the transcriptional and translational levels and integrates cellular stress responses and innate immunity. IMPORTANCEStress granules appear during virus infection, and their importance is not well understood. Previously, it was assumed that they were nonfunctional artifacts associated with cellular stress. PKR is a well-known antiviral protein; however, its regulation in cells is not well understood. Our work links cellular stress granules with activation of PKR and other innate immune pathways through the activity of G3BP1, a critical stress granule component. The ability of stress granules and G3BP1 to activate PKR and other innate immune transcriptional responses indicates that G3BP1 is an antiviral protein. This work helps to refine a longstanding paradigm indicating stress granules are inert structures and explains why G3BP1 is subverted by many viruses to promote a productive infection. Stress granules (SGs) are macromolecular triage centers for mRNAs that contain translationally silenced messenger RNPs (mRNPs). Stress granules contain many translation initiation factors, 40S ribosomal subunits, mRNAs, and RNA-binding proteins, which form in response to cellular stresses that inhibit protein synthesis (1, 2). Stress responses are induced during virus infection and are countered by viruses to maximize replication efficiency. Indeed, several examples of viral SG disruption and viral subversion of SG proteins have been described (3-6), indicating stress granules may play antiviral roles against these viruses. Ras-GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) is a stress granule-resident protein that nucleates stress granule assembly and is also inactiv...

Disruption of Stress Granule Formation by the Multifunctional Cricket Paralysis Virus 1A Protein

“…For example, pattern recognition receptors like RIG-I are activated upon binding to viral RNAs in SGs, which leads to the stimulation of interferon-related genes (42,43,82). Alternatively, recent studies suggest that in the absence of virus infection, SGs can activate innate immune signaling (11,83,84). Overexpression of G3BP, which leads to SG assembly, recruits and activates PKR within SGs in a novel manner whereby PKR dimerization and viral RNA are not required.…”

Casein Kinase 2 Is Linked to Stress Granule Dynamics through Phosphorylation of the Stress Granule Nucleating Protein G3BP1

“…Proximity ligation assays were performed as previously described (55). Briefly, cells were stressed with arsenite, allowed to recover, and then fixed with 4% formaldehyde.…”

“…For experiments in which high resolution was not necessary, a standard epifluorescence Nikon TE2000 microscope was used. Previously published procedures were followed for sample processing (55,58). The antibodies used for microscopy were as follows: goat anti-CK2␣ (Santa Cruz), rabbit anti-CK2␣= (Bethyl), mouse anti-CK2␤ (Santa Cruz), goat anti-TIA1 (Santa Cruz), rabbit anti-G3BP1 (56), rabbit anti-eIF4G (59), rabbit anti-PKR (ProSci), and mouse anti-Flag (Sigma).…”

Herpes Simplex Virus 2 Virion Host Shutoff Endoribonuclease Activity Is Required To Disrupt Stress Granule Formation

“…However, it is important to bear in mind that SGs are being increasingly viewed as more than a mere consequence of a cell's stress response or a temporary storage depot for mRNPs. Two independent research groups have shown that SGs can promote innate immune responses (76)(77)(78)(79). This has led to the proposal that SGs serve as platforms for recognizing and responding to viral infections (80), which may explain why so many viruses destroy or modify SGs during infection and necessitates a better understanding of SG biology.…”