Figure 1.
CVB3 infection induces SG formation at ∼3 hrs and disassembly at ∼5 hrs post-infection.
(A, B) Representative confocal images of intracellular localization of G3BP1, TIA1 and HuR following CVB3 infection of HeLa cells. HeLa cells were transiently co-transfected with pEGFP-G3BP1 and either pDsRed-TIA1 (A) or pDsRed-HuR (B) for 48 hrs, followed by sham- or CVB3 infection at an MOI of 10 for various time points as indicated. Arsenite treatment at a dose of 50 mM for 1 hr was used as positive controls for inducing SG formation. Cell nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI). (C) Representative confocal images of co-localization of poly-A-mRNA with G3BP1 during CVB3 infection. HeLa cells were transiently transfected with pEGFP-G3BP1 for 48 hrs, followed by sham- or CVB3 infection at an MOI of 10 for different time points as indicated. Poly-A-mRNA was detected by in situ hybridization using an oligo-dT probe synthetically conjugated to Cy3, followed by immunostaining for G3BP1. Cell nuclei were counterstained with DAPI. (D) Quantitation of G3BP1-SG formation from (A, B). Percent of cells expressing SGs was quantified as described in Materials and Methods (mean ± SD, n = 10 images), #p<0.001. (E) Quantitation of poly-A granules from (C). Percent of cells expressing poly-A punctates was quantified as above. N.D., No statistical difference, *p<0.01. (F, G) Representative transmission (F) and immune-electron (G) microscope images. HeLa cells were either sham- or CVB3-infected at an MOI of 10 for 4 hrs. N and M indicate the nucleus and mitochondria, respectively. Arrow heads in (F) indicate ribosomal-like structures. Arrows in (G) indicate cytoplasmic aggregates that were stained positive for endogenous G3BP1.
Figure 2.
Coxsackieviral protease 2A cleaves eIF4G and promotes SG formation.
(A) HeLa cells were either sham- or CVB3-infected at an MOI of 10 for various time points as indicated. Western blotting was performed to assess protein expression of eIF4G, phopho-eIF2α (Ser 51), and total eIF2α. Protein level of viral capsid protein (VP1) and β-actin was also examined as an infection and loading control, respectively. (B) HeLa cells were transfected with pIRES-2A and/or pIRES-3C for 48 hrs. Western blotting was performed to assess protein expression of eIF4G and β-actin. (C) Representative confocal images of viral protease 2Apro inducing G3BP1-SG formation. HeLa cells were co-transfected with pIRES-2Apro or pIRES-3Cpro together with GFP-G3BP1 for 24hrs, followed by confocal microscopy analysis.
Figure 3.
G3BP1-SGs are disassembled while poly-A-SGs remain persistent in CVB3-infected cells.
HeLa cells were infected with CVB3 at an MOI of 10 for 3 or 5-infection (pi) as indicated. Cells treated with arsenite alone for 3 or 5 hrs were used as negative controls. Cells were fixed and stained for endogenous G3BP1, poly-A mRNA, and nuclei. Percent of cells expressing G3BP1-SGs and poly-A granules was quantified as described in Materials and Methods (mean ± SD, n = 5 images), N.D., No statistical difference, #p<0.001.
Figure 4.
G3BP1 is cleaved at ∼5 hrs post-infection while TIA1 remains unchanged during the course of CVB3 infection.
(A) HeLa cells stably expressing exogenous GFP-G3BP1 or (B) regular HeLa cells were either sham- or CVB3-infected at an MOI of 10 for various time points as indicated. Western blotting was performed to assess protein expression of G3BP1 using anti-G3BP1 antibody (A) and TIA1 using anti-TIA1 antibody (B). VP1 expression was used as an infection control and β-actin level was examined as a loading control.
Figure 5.
HeLa cell lysates were incubated with (A) purified 3Cpro for 0, 5, 15, 30, and 60 mins or 2Apro for 60 mins, or (B) purified 2Apro or 2Amutpro for 60 mins. In vitro cleavage assay was performed as described in Materials and Methods and expression of G3BP1 and PABP was detected by western blotting.
Figure 6.
G3BP1 is cleaved at amino acid Q325 and a cleavage-resistant G3BP1 mutant restores SG formation at ∼5 hrs post-infection.
(A) HeLa cells were transfected with FLAG-G3BP1 or FLAG-G3BP1Q325E mutant for 48 hrs, followed by CVB3 infection at an MOI of 10 for 7 hrs. Western blotting was performed to examine G3BP1 cleavage using an anti-FLAG antibody. Protein expression of VP1 and β-actin was examined as an infection and loading control, respectively. (B) Schematic diagram of full length G3BP1 and the cleavage site. The arrow indicates the cleavage point at amino acid Q325 of G3BP1. NTF2-like, Nuclear Transport Factor 2-like; PXXP, SH3-domain binding domain of Ras-GAP; RRM, RNA Recognition Motif; RGG, Arginine-Glycine-rich region. (C) HeLa cells stably expressing GFP-G3BP1Q325E (upper panels) or GFP-G3BP1 (lower panels) were sham- or CVB3-infected at an MOI of 10 for different times as indicated. Intracellular distribution of G3BP1 was examined using confocal microscopy. Cell nuclei were counterstained with DAPI. Cells treated with 50 mM arsenite for 1 hr were used as positive controls. (D) Quantitation of G3BP1-SG formation from (C). Results are presented as mean ± SD (n = 10 images), #p<0.001, N.D., No statistical difference.
Figure 7.
Overexpression of G3BP1 inhibits CVB3 replication.
HeLa cells were transiently transfected with pEGFP-G3BP1 for 24 hrs, followed by CVB3 infection at an MOI of 1 for 16 hrs. (A) Western blotting (left) and confocal microscopy (right) were performed to examine protein level and intracellular distribution of GFP-G3BP1. (B) Western Blotting was performed to assess protein expression of VP1 and β-actin after CVB3 infection. (C) Densitometric analysis using ImageJ was performed on VP1 intensities relative to β-actin intensities of three independent experiments from (B). The value of sham was arbitrarily set as 1. (D) Quantitative RT-PCR was performed to examine viral transcript levels using primers specific to the IRES promoter of the CVB3 RNA transcript. Data is presented as transcript copy number relative to its empty vector control. The value of the empty vector control was arbitrarily set as 1. (E) Plaque assay was performed to examine the effect of G3BP1 overexpression on viral replication and results are presented as relative PFU/ml. The value of the empty vector control was arbitrarily set as 1. Results are presented as mean ± SD (n = 3), #p<0.001, *p<0.01.
Figure 8.
Knockdown of G3BP1 enhances CVB3 replication.
HeLa cells were transfected with either control siRNA (siCon) or G3BP1-targeted siRNA (siG3BP1), followed by sham- or CVB3-infection at an MOI of 1 for 16 hrs. (A) Western blotting (left) and confocal microscopy (right) were performed to assess protein expression and intracellular distribution of GFP-G3BP1. (B) Western blotting was performed to examine VP1 and β-actin protein expression. (C) Densitometric analysis was performed on VP1 intensities of three independent experiments from (B) as described above. (D) Quantitative RT-PCR was performed to examine viral transcript levels as described above. Data is presented as transcript copy number relative to its empty vector control. The value of the siCon control was arbitrarily set as 1. (E) Plaque assay was performed to assess the effect of G3BP1 knockdown on viral replication and results are presented as relative PFU/ml. The value of the siCon control was arbitrarily set as 1. Results are presented as mean ± SD (n = 3), #p<0.001, *p<0.01.
Figure 9.
Figure 9. G3BP1-Cterm fragment reduces SG formation and enhances CVB3 replication.
(A, B) HeLa cells were co-transfected with pEGFP-G3BP1 and either FLAG-G3BP1-Nterm (see Figure S3) or FLAG-G3BP1-Cterm for 48 hrs, followed by sham- or CVB3-infection at an MOI of 10 for the indicated time points. G3BP1-Cterm and G3BP1-Nterm were stained using an anti-FLAG antibody. Arrow heads indicate cells expressing high levels of G3BP1-Cterm but lacking distinct G3BP1(+)-SGs or poly-(A) granules. Cells treated with arsenite (50 mM) for 1 hr were used as positive controls. (C) Quantitation of GFP-G3BP1 foci and poly-A granules from (A and S3A) and (B and S3B) at 3 hr pi, respectively. Quantification was performed by counting cells expressing, empty vector, GFP-G3BP1-SGs or poly-A granules together with FLAG, and dividing by the total number of cells expressing FLAG. Results are presented as mean ± SD (n = 5 images), #p<0.001. N.D., No statistical difference. (D) HeLa cells were co-transfected with pEGFP-G3BP1 and FLAG-G3BP1-Cterm for 48 hrs, and then treated with 50 mM arsenite for 1 hr. Immunostaining was conducted and representative images are displayed. (E) Quantitation of GFP-G3BP1 SGs from (D). The data is presented as mean ± SD (n = 5 images), #p<0.001. (F) Plaque assay was performed to assess the effect of G3BP1-Cterm expression on CVB3 replication. Results are presented as relative PFU/ml. The value of the empty vector control was arbitrarily set as 1. The data is presented as mean ± SD (n = 3), #p<0.001.
Figure 10.
G3BP1-SGs do not localize with CVB3 sense-strand.
HeLa cells were infected with CVB3 at an MOI of 10 for the indicated time points, followed by in situ hybridization and immunostating as described in Materials and Methods. Endogenous G3BP1 was detected using an Alexa-488 secondary antibody. CVB3 sense-strand was detected by the synthetically conjugated Cy5 fluorophore.