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. 2015 Mar;89(5):2575-89.
doi: 10.1128/JVI.02791-14. Epub 2014 Dec 17.

The stress granule protein G3BP1 recruits protein kinase R to promote multiple innate immune antiviral responses

Lucas C Reineke  1 , Richard E Lloyd  2
Affiliations

The stress granule protein G3BP1 recruits protein kinase R to promote multiple innate immune antiviral responses

Lucas C Reineke et al. J Virol. 2015 Mar.

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-κB 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 eIF2α 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.

Importance: Stress 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.

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Figures

FIG 1
FIG 1
G3BP1 promotes antiviral activity. (A) Domain map of G3BP1 indicating the borders of each domain used throughout this study and the 3Cpro cleavage site. RGG represents the RGG domain containing repeats of the arginine-glycine-glycine peptide motif, while RRM represents the RNA recognition motif within G3BP1. (B) HeLa cells were transfected with either GFP or the Q326E mutant of G3BP1, which is not cleaved by poliovirus 3C protease, and cells were infected with poliovirus at an MOI of 10 (10). At 6 h p.i., the cells were fixed and stained with antibodies directed against G3BP1 or poliovirus 3C protease. Standard epifluorescence microscopy was used for analysis. Arrows mark cells that show both G3BP1 and 3Cpro expression, but do not contain granules. (C) G3BP1 KO MEFs were transfected with the indicated proteins; GFP-G3BP1 and G3BP1-GFP refer to N-terminally and C-terminally tagged proteins, respectively. G3BP1ΔNTF2-GFP expression (G3BP1ΔNTF2) does not form SGs. The cells were then infected with the indicated viruses at an MOI of either 10 or 0.1. Supernatants were collected at 24 or 48 h, respectively, and virus titers were determined using TCID50 assays. The error bars indicate standard deviations.
FIG 2
FIG 2
NF-κB and JNK transcription are activated by G3BP1-GFP expression. (A) Luciferase assays were conducted in HeLa cells transfected with either GFP, wild-type G3BP1-GFP, or G3BP1ΔNTF2-GFP (G3BP1ΔNTF2) with reporter constructs driven by the indicated transcriptional response promoters. Fluc, firefly luciferase; RLuc, Renilla luciferase; RLU, relative light units. (B) The NF-κB-luciferase reporter was transfected with increasing concentrations of either G3BP1-GFP or GFP control expression plasmids, as indicated, followed by luciferase assays. (C) Dot blot analysis of 62 cytokine proteins was conducted on conditioned medium from G3BP1 KO MEFs expressing either GFP, G3BP1-GFP, GFP-G3BP1, or G3BP1ΔNTF2-GFP (G3BP1ΔNTF2), as indicated in Fig. 1. A summary of the cytokines elevated during G3BP1 expression is shown. Arb., arbitrary. (D) Cytokines elevated when either G3BP1 or the G3BP1ΔNTF2-GFP (G3BP1ΔNTF2) mutant was expressed. The error bars indicate standard deviations.
FIG 3
FIG 3
Innate immune proteins localize to G3BP1-induced SGs in a cell line-dependent manner. HeLa and U2OS cells and G3BP1 KO MEFs were transfected with plasmids expressing G3BP1, and the cells were fixed and stained with antibodies against either OAS2 or RNase L. In HeLa cells, G3BP1 (red) was expressed from pcDNA 3.1 HisC-G3BP1 and detected with anti-T7 antibodies. In KO MEFs and U2OS cells, G3BP1-GFP was expressed (green). DAPI (4′,6-diamidino-2-phenylindole) is shown in blue. The insets represent the boxed areas.
FIG 4
FIG 4
PKR colocalizes with G3BP1-induced stress granules. (A) HeLa cells, G3BP1 KO MEFs, and U2OS cells were transfected with G3BP1-GFP (green) constructs and stained for PKR (red). DAPI-stained nuclei are visible in blue. (B) PKR-mCherry (red) was expressed in the indicated cells, together with G3BP1-GFP (green). The images were captured using deconvolution microscopy. The insets represent the boxed areas.
FIG 5
FIG 5
PKR antibodies variably report PKR in G3BP1-induced SGs. (A) G3BP1 was expressed from either the pcDNA 3.1 HisC-G3BP1 (SC) construct or pG3BP1-GFP (PSc and BD), and PKR was stained with three different antibodies (SC, PSc, and BD, as indicated) in either HeLa or G3BP1 KO cells as represented in the indicated colors. The antibody epitopes on PKR are as follows: BD, second RNA-binding domain; SC, kinase domain; PSc, N-terminal RNA-binding domain. The BD antibody does not recognize mouse PKR protein. The insets represent the boxed areas. (B) Antibody specificities for the PSc and BD antibodies were demonstrated in IFA after control or PKR-specific siRNAs were used to deplete PKR signal using IF in HeLa cells. (C) Western blot analysis was performed on HeLa cells treated with either control siRNA or PKR-specific siRNA.
FIG 6
FIG 6
PKR-G3BP1 proximity PLA foci increase with stress and PKR activation. Proximity ligation assays were used to assess close colocalization and/or interaction of G3BP1 and PKR in cells. (A and B) HeLa cells (A) or U2OS cells stably expressing GFP-G3BP1 (green) (B) were untreated or stressed with 500 μM arsenite (Ars) for 30 min before processing for PLA. (C and D) Untransfected HeLa cells and U2OS cells stably expressing GFP-G3BP1 (green) (D) were transfected with poly(I·C) to activate PKR, and PLA was performed. (E) PLA was performed on HeLa cells expressing G3BP1-GFP using antibodies against G3BP1 and GFP. (F) PLA was conducted on endogenous G3BP1 and Tia1, and foci per cell were quantified under each condition. (A to E) Red, PLA signal; blue, DAPI. (A to D and F) The graphs depict quantification of PLA as described in Materials and Methods. The error bars represent standard errors. *, P ≤ 0.05; **, P < 0.01; ***P < 0.001; Student's t test.
FIG 7
FIG 7
Determinants of PKR recruitment to SGs. (A) Immunofluorescence microscopy with PKR phospho-T446-specific antibody. Cells were transfected with GFP or G3BP1-GFP, as indicated, and GFP localization was detected directly by fluorescence. T, transfected cells; U, untransfected cell. Pearson's correlation coefficient (PCC) for colocalization of P-PKR (red) with G3BP1-GFP (green), along with the relevant P value, are indicated on the right. (B) Domain map of human PKR with mutated amino acids indicated. RBD, RNA-binding domain. K60/64 and K150/154 are residues implicated in RNA binding; K296 is a key residue within the catalytic site of PKR. (C) G3BP1 expressed from pcDNA 3.1 HisC-G3BP1 (green) and hPKR-mCherry (red) were coexpressed in HeLa cells to investigate colocalization. Colocalization of the wild type, a K60/64/150/154A mutant deficient in RNA binding, and the K296W catalytically inactive PKR mutant was examined, as indicated. G3BP1 transgene expression was detected with anti-T7 antibodies. The PCC and P value for colocalization of each PKR mutant with G3BP1 are indicated.
FIG 8
FIG 8
G3BP1ΔPXXP-induced granules uncouple G3BP1 aggregation from eIF2α phosphorylation. (A) Stress granules were induced with either wild-type G3BP1 or G3BP1ΔPXXP (green) expression in HeLa and U2OS cells and G3BP1 KO MEFs, as indicated. The cells were fixed and stained for PKR (red). The PCC and P value are indicated for each condition. The insets represent the boxed areas. (B) HeLa, U2OS, and G3BP1 KO cells were transfected with wild-type G3BP1 or G3BP1ΔPXXP and stained for phospho-eIF2α as previously described (15). As a control, untransfected cells were treated with sodium arsenite at 500 μM for 30 min. eIF2α phosphorylation was quantified as described in Materials and Methods. Standard errors were calculated for HeLa cells in three experiments, and representative experiments with U2OS cells and MEFs are shown. The y axis represents the percentage of cells with large G3BP1-induced SGs that scored positive for eIF2α phosphorylation, as previously described (15).
FIG 9
FIG 9
Enteroviruses replicate in cells with G3BP1ΔPXXP granules. (A) HeLa, U2OS, and G3BP1 KO cells were transfected with wild-type G3BP1-GFP or G3BP1ΔPXXP (green) and counterstained using antibodies against eIF4G (red). (B) HeLa and U2OS cells were transfected with wild-type G3BP1 or G3BP1ΔPXXP (green) and stained with antibodies against PABP (red). The insets represent the boxed areas. (C) HeLa cells were transfected with the indicated transgene and infected 16 h posttransfection with CVB5 at an MOI of 0.1, and the kinetics of virus in the supernatant were measured with TCID50 assays. Transfection efficiency approached 90% in the experiment. (D) At each time point in panel C, HeLa cells were harvested and IFN-β mRNA was measured using qPCR. IFN-β mRNA levels are expressed relative to a β-actin internal-control mRNA. (E) HeLa cells expressing the indicated GFP-tagged transgenes (green; G3BP1ΔNTF2-GFP [G3BP1ΔNTF2] and G3BP1ΔPXXP-GFP [G3BP1ΔPXXP]) were infected at an MOI of 5 for 7.5 h with CVB3-dsRed (red). Cells were harvested and imaged by epifluorescence microscopy. Mock-infected cells showed no red signal and are therefore not shown. Cells expressing transgenes are marked with a T. All other cells were untransfected. (F) Transfected HeLa cells from panel E were quantified as described in Materials and Methods for the presence of red signal indicative of CVB3 infection. The values represent the percentages of transgene-expressing cells that were red. (D and E) The error bars represent standard errors. *, P < 0.05; **, P < 0.01; ***P < 0.001; Student's t test.
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