g3bp1 gfp  (Addgene inc)

Journal: bioRxiv

Article Title: Alphaviral nonstructural protein-host RBP co-condensation as a mechanism to sustain virus replication

doi: 10.1101/2025.11.30.691455

Figure Lengend Snippet: (A) Representative image of gfp-nsP3 and mCherry-nsP3 expressed in U2OS cells. Scale bar, 10 μm. (B) FRAP experiment of gfp-nsP3 expressed in cells. Quantification curve on the right. Scale bar, 10 μm. (C) Representative images of 1,6-HD treatment of gfp-nsP3 expressing cells. Scale bar, 10 μm. (D) Representative images of co-localization between gfp-nsP3 and Dil labeled vesicles. Two insets are shown on the right. Scale bar, 10 μm. (E) Representative images of co-localization between gfp-SFV infected cells and Dil-labeled vesicles. Two patterns are detected: type I is membrane vesicle attached; type II is distinct from membrane structures. Scale bar, 10 μm. (F) FRAP experiments on gfp-nsP3 condensates in SFV-infected cells. Quantification curve on the right. Scale bar, 10 μm. (G and H) Representative EM images of APEX2-nsP3 stable expressing cells (G) and APEX2-SFV infected cells with or without DAB treatment (H). Two nsP3 patterns are observed: type I is membrane attached; type II is distinct from membrane structures. Scale bar, 500 nm. (I and J) Schematic diagram of biochemical fractionation assay (I) and WB results of the nsP3 fractions expressed in cells and during SFV infection (J). nsP3 was enriched in the pellet fraction of post-20,000 g centrifugation, and its pelleted distribution was unaffected by Triton X-100, a membrane-solubilizing detergent. In contrast, the membranous protein Calnexin was predominantly found in the supernatant upon treatment with Triton X-100.

Article Snippet: For sorting TetOn-gfp-TurboID-Control/G3BP1 inducible U2OS cell lines, the transduced cells were treated with 1 μg/ml doxycycline (Solarbio) for 24 hours to induce the expression of gfp-TurboID-Control/G3BP1 proteins before being subjected to FACS.

Techniques: Expressing, Labeling, Infection, Membrane, Fractionation, Centrifugation

Journal: bioRxiv

Article Title: Alphaviral nonstructural protein-host RBP co-condensation as a mechanism to sustain virus replication

doi: 10.1101/2025.11.30.691455

Figure Lengend Snippet: (A) Diagram of full-length SFV nsP3 protein and deletion or point mutations used in the study. (B) Representative images of WT and nsP3 mutants in cells. Scale bar, 10 μm. (C) Representative images of gfp-nsP3 replicon. The gfp is inserted in the HVD domain of nsP3. Scale bar, 10 μm. (D) Representative images of phase separation of recombinant nsP3 full-length and mutant protein in vitro . Scale bar, 20 μm. (E) Quantification of SFV replication by RT-qPCR in WT and G3BP1/2 dKO U2OS cells. Error bars indicate± SEM with n=3 replicates. (F) Representative WB images for nsP3 and G3BP protein expression during SFV replication. (G) Replication of SFV mutants in cells. Data are mean ± SEM; n=3 replicates. (H) Complementation of SFV(2CA) mutant by different replicons containing combinations of nsPs and mutations. SFV(2CA) mutant construct was co-transfected with each replicon construct, and the results were compared to those of FL_SFV. Data are mean ± SEM; n=3 replicates. Statistical significance was assessed in comparison to WT cells (E) or WT SFV (G) and (H) using an unpaired, two-tailed t test.

Article Snippet: For sorting TetOn-gfp-TurboID-Control/G3BP1 inducible U2OS cell lines, the transduced cells were treated with 1 μg/ml doxycycline (Solarbio) for 24 hours to induce the expression of gfp-TurboID-Control/G3BP1 proteins before being subjected to FACS.

Techniques: Recombinant, Mutagenesis, In Vitro, Quantitative RT-PCR, Expressing, Construct, Transfection, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Alphaviral nonstructural protein-host RBP co-condensation as a mechanism to sustain virus replication

doi: 10.1101/2025.11.30.691455

Figure Lengend Snippet: (A) Representative images of localization patterns of SG markers with gfp-nsP3 in cells. Scale bar, 10 μm. (B) Representative images of localization patterns of SG markers with gfp-SFV infected cells. Scale bar, 10 μm. (C and D) Representative two-color super-resolution images of gfp-nsP3 with G3BP1 in individually expressed nsP3 cells (C) or SFV infection (D). Scale bars, 200 nm for (C) and 1 μm for (D). (E) Representative images of nsP3 mutant and nsP3 expression in WT and G3BP1/2 dKO U2OS cells. Scale bar, 10 μm. (F) Quantification of the threshold concentration for each condition in (E). Data are plotted as a minimum to maximum; lines indicate the first quartile (lower), median, and third quartile (upper) with n=10 values in the determined thresholding bin. See Methods for defining the bin. (G) Representative images of localization pattern between dsRNA and virus genome RNA with nsP3 condensates in SFV infected cells. An inset was shown on the right. Scale bar, 10 μm. (H) RNA-IP coupled with RT-qPCR to show the RNA binding feature of nsP3. Error bars are defined as ± SEM with n=3 replicates. (I) Fluorescence polarization assay to measure the RNA binding of G3BP1 and its enhancement by nsP3. Data are mean ± SEM with n=3 replicates. (J) RT-qPCR results show the viral inhibition effect of different RNase tested in cells. Data are mean ± SEM with n=3 replicates. (K) RT-qPCR analysis of the antiviral effects of the WT RNase L, SAMHD1, and their respective RNase-deficient mutants. Data are mean ± SEM with n=3 replicates. (L) RNA protection effect of nsP3 condensate using in vitro phase separation assay. In vitro transcribed SFV nsP3 RNA was labeled with Cy5-UTP, and the RNA signal was used to indicate the decay of RNA and dissolution of condensate after RNase A addition. Scale bar, 5 μm. (M) RNA protection assay of nsP3 mutants as in (L). Scale bar, 5 μm. Statistical significance in (F), (J), and (K) was assessed using an unpaired, two-tailed t test. Significance in (H) was assessed using a one-way analysis of variance (ANOVA) followed by Turkey test.

Article Snippet: For sorting TetOn-gfp-TurboID-Control/G3BP1 inducible U2OS cell lines, the transduced cells were treated with 1 μg/ml doxycycline (Solarbio) for 24 hours to induce the expression of gfp-TurboID-Control/G3BP1 proteins before being subjected to FACS.

Techniques: Infection, Mutagenesis, Expressing, Concentration Assay, Virus, Quantitative RT-PCR, RNA Binding Assay, Fluorescence, Inhibition, In Vitro, Labeling, Dissolution, Two Tailed Test

Journal: bioRxiv

Article Title: Alphaviral nonstructural protein-host RBP co-condensation as a mechanism to sustain virus replication

doi: 10.1101/2025.11.30.691455

Figure Lengend Snippet: (A) Diagram of the luciferase assay for protein translation activity. (B) Dual-luciferase activity measurement of MCP control, MCP-G3BP1, and untagged G3BP1 in HEK293T WT cells. Data are mean ± SEM with n=3 biological replicates. (C) Dual-luciferase activity measurement of MCP control, MCP-nsP3 in HEK293T WT and G3BP1/2 dKO cells. Data are mean ± SEM with n=3 biological replicates. (D) Dual-luciferase activity measurement of MCP control, MCP-nsP3 WT, and MCP-nsP3(2FA) mutant in HEK293T WT cells. Data are mean ± SEM with n=3 biological replicates. (E) Schematic of the recombinant SFV containing 5×SunTag arrays and a firefly luciferase CDS for viral translation imaging. (F) Representative images of SFV-SunTag-Luc translation treated with 100 μg/ml puromycin for 20 min. Scale bar, 10 μm. (G) Representative images of nsP3 antibody staining in scFv-GFP stable U2OS cells at 6 h after SFV-SunTag-Luc infection. Scale bar, 10 μm. (H) Representative images of vRNA FISH in scFv-GFP stable U2OS cells at 6 h after SFV-SunTag-Luc infection. Scale bar, 10 μm. Statistical significance in (B), (C), and (D) was assessed using an unpaired, two-tailed t test.

Article Snippet: For sorting TetOn-gfp-TurboID-Control/G3BP1 inducible U2OS cell lines, the transduced cells were treated with 1 μg/ml doxycycline (Solarbio) for 24 hours to induce the expression of gfp-TurboID-Control/G3BP1 proteins before being subjected to FACS.

Techniques: Luciferase, Activity Assay, Control, Mutagenesis, Recombinant, Imaging, Staining, Infection, Two Tailed Test

Journal: bioRxiv

Article Title: Alphaviral nonstructural protein-host RBP co-condensation as a mechanism to sustain virus replication

doi: 10.1101/2025.11.30.691455

Figure Lengend Snippet: (A) Representative images of FRAP experiments on EEEV/WEEV/VEEV nsP3 proteins in cells. Scale bar, 10 μm and 1 μm for the inset. (B) Representative images of slow fusion of EEEV/WEEV/VEEV nsP3 droplets in cells. Scale bar, 5 μm. (C) Representative images of WT and 2CA mutants of each nsP3 protein in cells. The alignment of the two cysteines is shown on the top. Scale bar, 10 μm. (D) Representative image of IP-WB to show the interaction between the four nsP3 proteins and G3BP1/FMR1. nsP3 construct was transfected into 293T cells, and WB was performed with G3BP1 or FMR1 antibodies. (E) Representative images of SFV/WEEV/EEEV/VEEV nsP3 protein in WT and G3BP1/2 dKO cells. Scale bar, 10 μm. (F) Representative images of localization between nsP3 and G3BP1. Scale bars, 10 μm and 5 μm for the inset. (G) Representative images of localization between nsP3 and G3BP1 with SA treatment. Scale bars, 10 μm and 5 μm for the inset. (H) Representative images of localization between nsP3 and FMR1. Scale bars, 10 μm and 5 μm for the inset. (I) Left: representative images of VEEV(GFP) replication in WT and FXR1/FXR2/FMR1 triple KO (FXR tKO) U2OS cells. The top row shows the fluorescent signal from VEEV(GFP), and the bright field below indicates the cell density. Scale bar, 10 μm. Right: RT-qPCR quantification of VEEV genome in U2OS WT and FXR tKO cells. Data are mean ± SEM with n=3 replicates. (J) Quantification of relative threshold fluorescence for VEEV nsP3. Data are plotted as a minimum to maximum; lines indicate the first quartile (lower), median, and third quartile (upper). n=10 values in the determined thresholding bin. (K) Disruption of VEEV nsP3 condensate formation by FXR-interacting motif peptide from VEEV (VEEV 2×FIM). Scale bar, 10 μm. (L) RT-qPCR analysis of the VEEV replication in HEK293T transfected with the FXR binding peptide VEEV 2×FIM. Data are mean ± SEM with n=3 replicates. Statistical significance in (I), (J), and (L) was assessed using an unpaired, two-tailed t test.

Article Snippet: For sorting TetOn-gfp-TurboID-Control/G3BP1 inducible U2OS cell lines, the transduced cells were treated with 1 μg/ml doxycycline (Solarbio) for 24 hours to induce the expression of gfp-TurboID-Control/G3BP1 proteins before being subjected to FACS.

Techniques: Construct, Transfection, Quantitative RT-PCR, Fluorescence, Disruption, Binding Assay, Two Tailed Test