Journal: PLoS Pathogens

Article Title: Picornavirus 2A protease regulates stress granule formation to facilitate viral translation

doi: 10.1371/journal.ppat.1006901

Figure Lengend Snippet: (A-B) The location of Sam68, TIA-1, and G3BP in AS-treated (200 μM, 1 h), EV71-infected (MOI = 10, 5 hpi) (A) or 2A-expressing (24 hpt) (B) HeLa cells. “+” indicates EV71-infected or 2A-expressing cells. (C) Dynamics of TIA-1 in AS-, EV71-, or 2A-induced foci. FRAP assays were used to evaluate the averaged recovery of GFP-TIA-1 in the indicated foci. Representative images of fluorescence intensities observed in foci at indicated times during FRAP assays are shown in pseudo-colors (glowdark) in the top panels. (D) Effects of eIF2α phosphorylation on TIA-1 foci. Following transfection with HA-eIF2α S51A for 24 h, HeLa cells were treated with AS, infected with EV71 for 5 h, or transfected with 2A for 24 h and then fixed and stained as indicated. White and yellow arrows show the comparison of TIA-1 foci assembly in the presence and absence of eIF2α S51A . (E) Quantitative analysis of the cells (in D) with TIA-1 foci among HA-positive or HA-negative cells. n = 3, 240 cells/condition were counted, mean±SD; n.s., no statistical significance, ***p<0.001. (F) CHX effects on TIA-1 foci. HeLa cells were treated with AS (0.5 h), EV71 (5 h), or 2A (24 h), with or without CHX for 1 h (CHX+ or CHX-) and then fixed and stained with TIA-1(green) and DAPI (blue). (G) Quantitative analysis of cells with TIA-1 foci in F. n = 3, 240 cells/condition were counted, mean±SD; n.s., no statistical significance, ***p<0.001. Scale bars in B, 2 μm; scale bars in others, 10 μm. See also and Figs.

Article Snippet: Human RD (Rhabdomyosarcoma cells and were obtained from China Center for Type Culture Collection), HEK293T (Human embryonic kidney 293 cells and were obtained from China Center for Type Culture Collection), HeLa cells (Human cervical cancer epithelial cells and were obtained from China Center for Type Culture Collection), and stably expressing cells (GFP-G3BP-HeLa/RD, GFP-G3BP Q326E -HeLa/RD, GFP-TIA-1-HeLa) derived from HeLa or RD cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 U/ml penicillin/streptomycin (Gibco) at 37°C and 5% CO 2 .

Techniques: Infection, Expressing, Fluorescence, Phospho-proteomics, Transfection, Staining, Comparison

Journal: PLoS Pathogens

Article Title: Picornavirus 2A protease regulates stress granule formation to facilitate viral translation

doi: 10.1371/journal.ppat.1006901

Figure Lengend Snippet: (A-C) Effects of protease activity of 2A on aSG formation. HeLa cells were transiently transfected with empty vector, Myc-tagged 2A, or 2A C110S for 24 h and then subjected to WB; GAPDH was the sample loading control. Arrow indicates the N-terminal cleavage products of eIF4G (A). IF assay of aSG formation in 2A C110S -expressing cells (B) and quantitative analysis of the 2A- (in ) or 2A C110S -expressing cells with aSGs in B. N.D., not detected. n = 3, 240 cells/condition were counted, mean±SD; ***p<0.001 (C). (D-F) Effects of cleavage of eIF4GI on aSG formation. HeLa cells were transfected with HA-tagged eIF4GI or eIF4GI cleavage-resistant mutant (eIF4GI G689E ) for 24 h, followed by EV71 infection or 2A transfection. The cleavage of eIF4GI and eIF4GI G689E was analyzed by WB assay, arrow indicates the C-terminal cleavage products of eIF4GI (D), and the formation of aSGs was viewed by IF assay (E). Quantitation analysis and comparison of eIF4GI- or eIF4GI G689E -expressing cells with aSGs (TIA-1 foci) among EV71-infected or 2A-expressing cells in E. n = 3, 240 cells/condition were counted, mean±SD; ***p<0.001 (F). “+” indicates the 2A/2A C110S -expressing or EV71-infected cells, and yellow arrows indicate the HA-tagged eIF4GI/eIF4GI G689E -expressing cells. Scale bars, 10 μm. See also .

Article Snippet: Human RD (Rhabdomyosarcoma cells and were obtained from China Center for Type Culture Collection), HEK293T (Human embryonic kidney 293 cells and were obtained from China Center for Type Culture Collection), HeLa cells (Human cervical cancer epithelial cells and were obtained from China Center for Type Culture Collection), and stably expressing cells (GFP-G3BP-HeLa/RD, GFP-G3BP Q326E -HeLa/RD, GFP-TIA-1-HeLa) derived from HeLa or RD cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 U/ml penicillin/streptomycin (Gibco) at 37°C and 5% CO 2 .

Techniques: Activity Assay, Transfection, Plasmid Preparation, Control, Expressing, Mutagenesis, Infection, Quantitation Assay, Comparison

Journal: PLoS Pathogens

Article Title: Picornavirus 2A protease regulates stress granule formation to facilitate viral translation

doi: 10.1371/journal.ppat.1006901

Figure Lengend Snippet: (A) EV71 effects on AS- and HS-induced tSG formation. HeLa cells were mock-infected or infected with EV71 (MOI = 10) for consecutive times and treated with AS or HS for 1 h prior to fixation. Cells were then stained with Sam68 (magenta), TIA-1 (green), G3BP (red), or HSP27 (red) as indicated. (B) Quantitative analysis of cells with tSGs in A. tSGs were marked by G3BP or HSP27. n = 3, 300 cells/condition were counted, mean±SD. (C) GFP-G3BP- or GFP-G3BP Q326E -HeLa cells were infected with EV71 as in A. Cells were harvested and analyzed via WB. VP1 served as an EV71 marker, and β-actin was the loading control. Full-length GFP-G3BP and N-terminal cleavage products of GFP-G3BP were detected by antibody against GFP, and arrow indicates the N-terminal cleavage products of GFP-G3BP. (D) GFP-G3BP- or GFP-G3BP Q326E -HeLa cells were treated and harvested as in A. Cells were then stained with EV71 (red), GFP-G3BP and GFP-G3BP Q326E (green) were markers of tSGs. (E) Quantitative analysis of EV71-infected cells with tSGs in D. n = 3, 300 cells/condition were counted, mean±SD; ***p<0.001. N.D., not detected. (F) HeLa cells were mock-infected or infected with EV71 in the presence or absence of GuHCl for 6 h as indicated and subjected to WB. Endogenous eIF4G and G3BP were detected by antibodies against eIF4G and G3BP. 3C and VP1 expression served as markers of EV71; β-actin was the sample loading control. (G) HeLa cells were mock-infected or infected with EV71 in the presence of GuHCl for 6 h and treated with AS for 1 h before harvest. Cells were then stained with eIF4G (magenta), G3BP (red), and TIA-1 (green). (H) Quantitative analysis of EV71-infected cells with tSGs (marked by G3BP) in G. n = 3, 240 cells/condition were counted, mean±SD; ***p<0.001. “+” indicates the EV71-infected cells, and yellow arrows indicate the uninfected cells. Scale bars, 10 μm. See also .

Article Snippet: Human RD (Rhabdomyosarcoma cells and were obtained from China Center for Type Culture Collection), HEK293T (Human embryonic kidney 293 cells and were obtained from China Center for Type Culture Collection), HeLa cells (Human cervical cancer epithelial cells and were obtained from China Center for Type Culture Collection), and stably expressing cells (GFP-G3BP-HeLa/RD, GFP-G3BP Q326E -HeLa/RD, GFP-TIA-1-HeLa) derived from HeLa or RD cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 U/ml penicillin/streptomycin (Gibco) at 37°C and 5% CO 2 .

Techniques: Infection, Staining, Marker, Control, Expressing

Journal: PLoS Pathogens

Article Title: Picornavirus 2A protease regulates stress granule formation to facilitate viral translation

doi: 10.1371/journal.ppat.1006901

Figure Lengend Snippet: (A and B) HeLa cells were transfected with 2A for 12 h and 24 h or transfected with empty vector for 24 h as a control, followed by 1 h treatment with AS or HS, and stained with Sam68 (magenta), TIA-1 (green), G3BP (red), or HSP27 (red) as indicated. (C) Quantitative analysis of cells with tSGs in A and B as marked by G3BP or HSP27. n = 3, 240 cells/condition were counted, mean±SD. (D-E) Effects of protease activity of 2A on tSG blockage. HeLa cells were transfected with Myc-tagged 2A C110S for 24 h and treated with AS for 1 h. tSG formation was viewed by IF assay (D). Quantitation analysis of 2A-expressing cells (in A, 24 hpt) or 2A C110S -expressing cells (in D) with SGs. n = 3, 240 cells/condition were counted, mean±SD; ***p<0.001 (E). “+” indicates the 2A/2A C110S -expressing cells, and yellow arrows indicate the cells without 2A/2A C110S expression. Scale bars, 10 μm. See also .

Article Snippet: Human RD (Rhabdomyosarcoma cells and were obtained from China Center for Type Culture Collection), HEK293T (Human embryonic kidney 293 cells and were obtained from China Center for Type Culture Collection), HeLa cells (Human cervical cancer epithelial cells and were obtained from China Center for Type Culture Collection), and stably expressing cells (GFP-G3BP-HeLa/RD, GFP-G3BP Q326E -HeLa/RD, GFP-TIA-1-HeLa) derived from HeLa or RD cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 U/ml penicillin/streptomycin (Gibco) at 37°C and 5% CO 2 .

Techniques: Transfection, Plasmid Preparation, Control, Staining, Activity Assay, Quantitation Assay, Expressing

Journal: PLoS Pathogens

Article Title: Picornavirus 2A protease regulates stress granule formation to facilitate viral translation

doi: 10.1371/journal.ppat.1006901

Figure Lengend Snippet: (A) Confirmation of 2A protease activity ablation in EV71-2A C110S . HeLa cells were mock-infected or infected with EV71 (MOI = 0.5) or recombinant virus encoding 2A C110S (EV71-2A C110S , MOI = 3) and subjected to WB for detection of eIF4G and G3BP cleavage. 3C indicated EV71 replication, and β-actin was the sample loading control. Arrow indicates eIF4G products cleaved by 2A, and “*” indicates eIF4G products cleaved by caspases [ , ]. (B) HeLa cells were infected with EV71 or EV71-2A C110S as in A, and then mock-treated or treated with CHX or AS for 1 h before fixation. Cells were then stained with 3C (magenta), TIA-1 (green), HSP27 (red), or G3BP (red) as indicated. “+” indicates the infected cells, and yellow arrows indicate the uninfected cells. (C) Quantitative analysis of infected cells with TIA-1 foci in B (mock panel and CHX panel). n = 3, 300 cells/condition were counted, mean±SD; ***p<0.001. (D) Quantitative analysis of infected cells with tSGs (G3BP) in B (mock panel and AS panel). n = 3, 300 cells/condition were counted, mean±SD; ***p<0.001.Scale bars, 10 μm. See also .

Article Snippet: Human RD (Rhabdomyosarcoma cells and were obtained from China Center for Type Culture Collection), HEK293T (Human embryonic kidney 293 cells and were obtained from China Center for Type Culture Collection), HeLa cells (Human cervical cancer epithelial cells and were obtained from China Center for Type Culture Collection), and stably expressing cells (GFP-G3BP-HeLa/RD, GFP-G3BP Q326E -HeLa/RD, GFP-TIA-1-HeLa) derived from HeLa or RD cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 U/ml penicillin/streptomycin (Gibco) at 37°C and 5% CO 2 .

Techniques: Activity Assay, Infection, Recombinant, Virus, Control, Staining

Journal: PLoS Pathogens

Article Title: Picornavirus 2A protease regulates stress granule formation to facilitate viral translation

doi: 10.1371/journal.ppat.1006901

Figure Lengend Snippet: (A) KD of PKR effects on EV71-2A C110S -induced phosphorylation of PKR and eIF2α. shNC- and shPKR-HeLa cells were infected with EV71 (MOI = 1, as control) or EV71-2A C110S (MOI = 3) for 12 h and subjected to WB. Tubulin served as a loading control. (B) KD of PKR effects on EV71-2A C110S -induced tSG formation. Cells were infected as in A and subjected to IF assay. “+” indicates the infected cells, and yellow arrows indicate the uninfected cells.(C) Quantitative analysis of cells with TIA-1 foci in B. n = 3, 300 cells/condition were counted, mean±SD; n.s., no statistical significance, ***p<0.001. (D) Phosphorylation of eIF2α effects on EV71-2A C110S -induced tSG formation. HeLa cells were transfected with HA-tagged eIF2α S51A for 24 h, and then infected with EV71-2A C110S (MOI = 3) for 12 h. “+” indicates the infected cells, and yellow arrows indicate the eIF2α S51A -HA-expressing cells. (E) Quantitative analysis of the cells (in D) with tSGs among HA-positive or HA-negative cells. n = 3, 240 cells/condition were counted, mean±SD; ***p<0.001. Scale bars, 10 μm.

Article Snippet: Human RD (Rhabdomyosarcoma cells and were obtained from China Center for Type Culture Collection), HEK293T (Human embryonic kidney 293 cells and were obtained from China Center for Type Culture Collection), HeLa cells (Human cervical cancer epithelial cells and were obtained from China Center for Type Culture Collection), and stably expressing cells (GFP-G3BP-HeLa/RD, GFP-G3BP Q326E -HeLa/RD, GFP-TIA-1-HeLa) derived from HeLa or RD cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 U/ml penicillin/streptomycin (Gibco) at 37°C and 5% CO 2 .

Techniques: Phospho-proteomics, Infection, Control, Transfection, Expressing

Journal: PLoS Pathogens

Article Title: Picornavirus 2A protease regulates stress granule formation to facilitate viral translation

doi: 10.1371/journal.ppat.1006901

Figure Lengend Snippet: (A-C) Effects of EV71-induced aSGs and EV71 C110S -induced tSGs on viral translation. Graphic description of EV71-UTR driven Renilla luciferase reporter expression (A) and reporter assays in HeLa cells (B and C). Cells were infected with EV71 for 3h to induce aSG formation or infected with EV71-2A C110S for 6h to induce tSG formation before transfection with UTR EV71 -Rluc mRNAs, and the luciferase activity was measured at indicated time post-transfection. The EV71-2A C110S -infected eIF4GI/eIF4GI G689E -HA-HeLa cells and EV71-infected shNC/shPKR-HeLa cells were negative controls. n = 3, mean±SD, n.s., no statistical significance; *p<0.05; **p<0.01; ***p<0.001. (D) FISH assays of total polyA mRNA colocalized to TIA-1 foci in GFP-TIA-1-HeLa cells after mock treatment or treatment with AS, EV71, or 2A. GFP-TIA-1 (green), polyA mRNAs (red), and nuclei (blue) are shown. The fluorescence intensity profile of GFP-TIA-1 (green) and polyA mRNA (red) was measured along the line drawn on a 2× zoom panel by Leica Application Suite Advanced Fluorescence Lite. (E) FISH assays of GFP-TIA-1-HeLa cells infected with EV71 or EV71-2A C110S . PABPC1 mRNA (left panel) and EV71 mRNA (+vRNA; right panel) were monitored. The fluorescence intensity profile of GFP-TIA-1 (green) and +vRNA/PABPC1 mRNA (red) was measured along the line drawn on a 2× zoom panel by Leica Application Suite Advanced Fluorescence Lite. Scale bars, 10 μm. See also .

Article Snippet: Human RD (Rhabdomyosarcoma cells and were obtained from China Center for Type Culture Collection), HEK293T (Human embryonic kidney 293 cells and were obtained from China Center for Type Culture Collection), HeLa cells (Human cervical cancer epithelial cells and were obtained from China Center for Type Culture Collection), and stably expressing cells (GFP-G3BP-HeLa/RD, GFP-G3BP Q326E -HeLa/RD, GFP-TIA-1-HeLa) derived from HeLa or RD cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 U/ml penicillin/streptomycin (Gibco) at 37°C and 5% CO 2 .

Techniques: Luciferase, Expressing, Infection, Transfection, Activity Assay, Fluorescence

Journal: PLoS Pathogens

Article Title: Picornavirus 2A protease regulates stress granule formation to facilitate viral translation

doi: 10.1371/journal.ppat.1006901

Figure Lengend Snippet: (A) HeLa cells were transiently transfected with 2A of EV71-BrCr, CVA, or PV for 24 h and then mock-treated or treated with CHX (top panel), AS, or HS (bottom panel). Cells were stained with Sam68 (indicator of 2A expression and aSGs), G3BP (marker of tSGs), and TIA-1. “+” indicates the 2A-expressing cells, and yellow arrows indicate cells without 2A expression. (B) Quantitative analysis of cells with aSGs (marked by TIA-1) upon mock treatment (top and left panel in A). n = 3, 240 cells/condition were counted, mean±SD. (C-D) Quantitative analysis of cells with tSGs marked by G3BP upon AS (C) and HS treatment (D). n = 3, 240 cells/condition were counted, mean±SD; ***p<0.001. Scale bars, 10 μm. See also .

Article Snippet: Human RD (Rhabdomyosarcoma cells and were obtained from China Center for Type Culture Collection), HEK293T (Human embryonic kidney 293 cells and were obtained from China Center for Type Culture Collection), HeLa cells (Human cervical cancer epithelial cells and were obtained from China Center for Type Culture Collection), and stably expressing cells (GFP-G3BP-HeLa/RD, GFP-G3BP Q326E -HeLa/RD, GFP-TIA-1-HeLa) derived from HeLa or RD cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100 U/ml penicillin/streptomycin (Gibco) at 37°C and 5% CO 2 .

Techniques: Transfection, Staining, Expressing, Marker